Assembly Reaction Research Articles

Membrane-dependent reactions of blood coagulation: classical view and state-of-the-art concepts

The complex mechanism called hemostasis evolved in living organisms to prevent blood loss when a blood vessel is damaged. In this process, two closely interconnected systems are distinguished: platelet-vascular and plasmatic hemostasis. Plasmatic hemostasis is a system of proteolytic reactions, in which blood plasma proteins called coagulation factors are involved. A key feature of this system is the localization of enzymatic reactions on the surface of phospholipid membranes, which increases their rate by up to 5 orders of magnitude. This review describes the basic mechanisms of coagulation factors binding to phospholipid membranes, pathways for complex assembly and activation reactions, and discusses the role of membranes in this process, their composition and sources. The binding of coagulation factors to procoagulant membranes leads not only to the acceleration of coagulation reactions, but also to their selective localization in restricted areas and protection from being washed away by the flow. The efficiency of coagulation reactions is regulated by the composition of the outer layer of the membrane, primarily through a special mechanism of mitochondria-dependent necrotic platelet death.

Modeling compound lipid homeostasis using stable isotope tracing.

Lipids represent the most diverse pool of metabolites found in cells, facilitating compartmentation, signaling, and other functions. Dysregulation of lipid metabolism is linked to disease states such as cancer and neurodegeneration. However, limited tools are available for quantifying metabolic fluxes across the lipidome. To directly measure reaction fluxes encompassing compound lipid homeostasis, we applied stable isotope tracing, high-resolution mass spectrometry, and network-based isotopologue modeling to non-small cell lung cancer (NSCLC) models. Compound lipid metabolic flux analysis (CL-MFA) enables the concurrent quantitation of fatty acid synthesis, elongation, headgroup assembly, and salvage reactions within virtually any biological system. Here, we resolve liver kinase B1 (LKB1)-mediated regulation of sphingolipid recycling in NSCLC cells and precision-cut lung slice cultures. We also demonstrate that widely used tissue culture conditions drive cells to upregulate fatty acid synthase flux to supraphysiological levels. Finally, we identify previously uncharacterized isozyme specificity of ceramide synthase inhibitors, highlighting the molecular detail revealed by CL-MFA.

Highly sensitive detection of DNA methyltransferase1 triggered by methylation protection

Type 2 diabetes is on the rise year by year, and 80 % of patients with type 2 diabetes die from cardiovascular complications. Strict control of blood glucose can significantly reduce the occurrence and development of diabetic microvascular complications, but can not effectively prevent the occurrence of diabetic macrovascular lesions. DNA methyltransferase 1 (DNMT1) plays an important role in developing phenotypic switching and secretion of inflammatory factors in vascular smooth muscle cells(VSMC), leading to the deregulation of vascular function. In this work, a biosensor based on methylation protection was designed. In the presence of the target DNMT 1, the formed methylation sites protect the dsDNA from being digested by the HpaII restriction enzyme. At the same time, a large number of G-quadruplex are produced by the catalyzed hairpin assembly (CHA) reaction cycle, and the peroxidase-like activity of G-tetraplex and hemin is used to continuously catalyze the color change of substrate ABTS, thus realizing dual signal amplification. The binding of magnetic beads to dsDNA effectively reduces the background signal and enables a highly selective and sensitive analysis of the target DNMT 1 in complex biological samples, with a detection limit as low as 0.22 UmL−1, showing a good linear relationship in the range of 0.22–10 UmL−1. A new approach for the study of macrovascular damage is provided in type 2 diabetes.

Highly sensitive β-amyloid1–42 oligomer aptamer-based assay via dual cyclic amplifications of three-way catalytic hairpin assembly and palindrome polymerase reaction

β-amyloid1–42 oligomer (AβO1–42), a neurotoxic protein, is considered the key factor in the progression of Alzheimer's disease (AD). And, high sensitivity detection of AβO1–42 is of considerable importance for the early diagnosis and deterioration prevention of AD. We present here an ultrasensitive fluorescent AβO1–42 detection approach based on aptamer recognition probes and a new dual cyclic signal amplification strategy by combining three-way catalytic hairpin assembly (3W-CHA) with palindromic sequence-based polymerase amplification (PBPA). Once the aptamers bind to target AβO1–42 molecules, ssDNA sequences are released to initiate 3W-CHA between three hairpins to unfold the signal hairpins and to form Y-shaped DNA structures (Y-DNAs). Subsequently, the assistance ssDNAs bind the Y-DNAs to expose the palindromic sequences to yield Y-DNA dimers, which lead to cyclic PBPA reaction with presence of polymerase to further unfold more signal hairpins for yielding considerably magnified fluorescence recovery, enabling sensitive assay of AβO1–42 down to 2.4 pM within a dynamic range of 5 pM∼50 nM. Furthermore, this strategy has been validated for interrogation of low AβO1–42 levels in artificial cerebrospinal fluid (CSF), suggesting its promising potential for application in early diagnosis and prevention of AD.

CRISPR/Cas13a-mediated triple signal amplification strategy for viral RNA detection

CRISPR/Cas13a system has been combined with different isothermal amplification strategies for RNA analysis. Nevertheless, expensive enzymes are usually indispensable in these isothermal amplification methods, which increases the detection cost. In this study, a CRISPR/Cas13a-mediated triple signal amplification strategy has been designed for selective, sensitive and inexpensive detection of viral RNA. SARS-CoV-2 S RNA activated CRISPR/Cas13a to cut hairpin DNA, triggering catalytic hairpin assembly (CHA) and subsequent hybridization chain reaction (HCR) to form a long-stranded Y-type DNA. G-quadruplexes were distributed on the branch of the long-stranded DNA and bound with N-methyl mesoporphyrin IX (NMM) to enhance the fluorescence signal. SARS-CoV-2 S RNA was specifically detected with a limit of detection (LOD) of 285 fM. Notably, no additional expensive enzymes were required in the entire detection process. The CRISPR/Cas13a-mediated triple signal amplification strategy holds great potential for specific and sensitive viral RNA detection with low cost.

Portable Monitoring System for Assessing Therapeutic Efficacy in HER2‐Overexpressing Breast Cancer: One‐Step Simultaneous Detection of Cancer‐Derived Exosomal Proteins and mRNA in Urine

AbstractPoint‐of‐care (POC) monitoring of patient condition is crucial for effective cancer treatment and prognosis. This can be achieved non‐invasively by analyzing exosomes in body fluids. However, the heterogeneity of exosomes and non‐standardized quantification methods may interfere with clearly determining the patient's condition. Therefore, there is a need for technology that can precisely analyze both tumor‐derived exosomes and normal exosomes. Herein, this study presents the exosome multiple‐separation for simultaneous detection (EXO‐MUSSID) platform, which simultaneously isolates different exosomes based on their magnetization properties and monitors therapeutic efficacy of drugs. Using immunoaffinity magnetophoresis technology, HER2‐overexpressing and normal exosomes are collected separately, enabling real‐time monitoring of HER2 (also known as ERBB2) expression by analyzing the mRNA of each exosome based on a catalytic hairpin assembly (CHA) reaction. A portable fluorescence reader customized for the EXO‐MUSSID platform is developed for POC monitoring of HER2‐overexpressing breast cancer (HER2+ BC). The performance of the EXO‐MUSSID platform is validated using urine samples from HER2+ BC mouse models, confirming the progression of HER2+ BC and the changes in HER2 expression due to trastuzumab treatment. It is expected to serve as a valuable tool for exosome‐based liquid biopsy in disease monitoring.

Intelligent Analysis of Avian Influenza Virus Biomarkers Based on Chemiluminescence Resonance Energy Transfer and DNA Logic Analysis.

A one-step logical analysis of multiple targets remains challenging. Herein, we report a one-pot and intelligent DNA logical analysis platform for the diagnosis of avian influenza virus (AIV) biomarkers based on chemiluminescence resonance energy transfer (CRET) and logic operations. On the surface of Lum/PEI/CaCO3 microparticles, the excited state of luminol underwent CRET with fluorescein isothiocyanate or rhodamine B isothiocyanate, producing three well-separated light emissions at 425, 530, and 590 nm, respectively. Taking advantage of the close distance between fluorophores aligned by the catalytic hairpin assembly reaction, the CRET efficiency was greatly enhanced (53.1%). H1N1, H7N9, and H5N1 were detected with limits of detection values as low as 15, 34, and 58 pM, respectively. Three-input logic circuits were simultaneously conducted on the surface of Lum/PEI/CaCO3 microparticles, enabling the rapid and accurate discrimination of multiple AIV biomarkers in one solution. In terms of peak positions and the normalized value of the total peak intensity, three biomarkers can be simultaneously discriminated without any other complex operations. In summary, the CRET-based multiple analytical assay was developed as an intelligent biosensor for identifying AIV biomarkers, having promising application prospects in the field of multiple analysis and precise disease diagnosis.

Synthesis pathways of (HfZrTiCe/La/Y)O2-x nanoparticles via benzyl alcohol route at critical temperature

The controllable synthesis of high-entropy fluorite oxide (HEO) having large ionic radius mismatch remains a challenging due to poor understanding on nucleation. The (HfZrTiLn)-5HEO nanoparticles with 15 % ionic radius mismatch were synthesized via benzyl alcohol route at 220 °C-5 min in presence of PtCl4 and Fe(acac)3, exhibiting novel optical, electrical and magnetic properties. Nucleation pathways of the 5HEO at the critical temperature were elucidated by using a comparison study of conventional heating and microwave irradiation heating. Consistency of XRD patterns and STEM-EDX observation indicate that the resultant Hf-OBn monomers acted as the nucleation center of the 5HEO, determined by diffusion kinetics. The nucleation rate depended on the metal monomers assembly and esterification reaction, which was accelerated by water vapor pressure produced in-situ by 0.5×10−4mol/l PtCl4 catalyst. The Fe-metal organic cages derived from 1.5×10−4mol/l Fe(acac)3 additive served as the structure stabilizer of Zr/Ti monomers, and prevented early hydrothermal reaction route.

A narrow ratio of nucleic acid to SARS-CoV-2 N-protein enables phase separation

SARS-CoV-2 Nucleocapsid protein (N) is a viral structural protein that packages the 30kb genomic RNA inside virions and forms condensates within infected cells through liquid-liquid phase separation (LLPS). In both soluble and condensed forms, N has accessory roles in the viral life cycle including genome replication and immunosuppression. The ability to perform these tasks depends on phase separation and its reversibility. The conditions that stabilize and destabilize N condensates and the role of N-N interactions are poorly understood. We have investigated LLPS formation and dissolution in a minimalist system comprised of N protein and an ssDNA oligomer just long enough to support assembly. The short oligo allows us to focus on the role of N-N interaction. We have developed a sensitive FRET assay to interrogate LLPS assembly reactions from the perspective of the oligonucleotide. We find that N alone can form oligomers but that oligonucleotide enables their assembly into a three-dimensional phase. At a ∼1:1 ratio of N to oligonucleotide, LLPS formation is maximal. We find that a modest excess of N or of nucleic acid causes the LLPS to break down catastrophically. Under the conditions examined here, assembly has a critical concentration of about 1 μM. The responsiveness of N condensates to their environment may have biological consequences. A better understanding of how nucleic acid modulates N-N association will shed light on condensate activity and could inform antiviral strategies targeting LLPS.

Versatile Bovine Serum Albumin as Ingenious Electron Operator-Enhanced Photoelectrochemical Biosensing for Ultrasensitive Detection of miRNA.

Bovine serum albumin (BSA) has been widely used in biosensors as a blocking agent. Herein, conformist BSA was first exploited as an ingenious operator to enhance the photocurrent response of (2Z,2'Z)-2,2'-(1,4-phenylene)bis(3-(4-(bis(4-methoxyphenyl)amino)phenyl)acrylonitrile) (TPDCN)-based photoelectrochemical (PEC) platform via manipulating the electron transfer process of the detection system. Concretely, the presence of target molecules triggered catalytic hairpin assembly reaction and subsequently powered terminal deoxynucleotidyl transferase-mediated signal amplification to produce the AgNP@BSA-DNA dendrimer nanostructure. After being treated with HNO3, a large amount of BSA could be released from the dendrimer nanostructure. When they were transferred to the TPDCN-based PEC platform, the photocurrent response of the biosensor was largely enhanced because BSA can manipulate the electrons of TPDCN via a well-matched energy level to form a new electron transfer track. Meanwhile, tryptophan (Trp) in BSA could be oxidized to quinone Trp-O under photoirradiation, which can facilitate the oxidation of ascorbate and generate more H+ to promote the migration of photogenerated electrons. As a result, the proposed PEC biosensor exhibits excellent analytical performance for detection of miRNA-21 (as a model target) over a wide linear range of 0.01 to 10,000 pM with detection limit as low as 4.7 fM. Overall, this strategy provides a new perspective on constructing efficient PEC biosensors, which expands the potential applications in bioanalysis and clinical diagnosis.

Multiplex Expression Cassette Assembly: A flexible and versatile method for building complex genetic circuits in conventional vectors.

The manipulation of multiple transcription units for simultaneous and coordinated expression is not only key to building complex genetic circuits to accomplish diverse functions in synthetic biology, but is also important in crop breeding for significantly improved productivity and overall performance. However, building constructs with multiple independent transcription units for fine-tuned and coordinated regulation is complicated and time-consuming. Here, we introduce the Multiplex Expression Cassette Assembly (MECA) method, which modifies canonical vectors compatible with Golden Gate Assembly, and then uses them to produce multi-cassette constructs. By embedding the junction syntax in primers that are used to amplify functional elements, MECA is able to make complex constructs using only one intermediate vector and one destination vector via two rounds of one-pot Golden Gate assembly reactions, without the need for dedicated vectors and a coherent library of standardized modules. As a proof-of-concept, we modified eukaryotic and prokaryotic expression vectors to generate constructs for transient expression of green fluorescent protein and β-glucuronidase in Nicotiana benthamiana, genome editing to block monoterpene metabolism in tomato glandular trichomes, production of betanin in tobacco and synthesis of β-carotene in Escherichia coli. Additionally, we engineered the stable production of thymol and carvacrol, bioactive compounds from Lamiaceae family plants, in glandular trichomes of tobacco. These results demonstrate that MECA is a flexible, efficient and versatile method for building complex genetic circuits, which will not only play a critical role in plant synthetic biology, but also facilitate improving agronomic traits and pyramiding traits for the development of next-generation elite crops.

G-quadruplex embedded in semi-CHA reaction combined with invasive reaction for label-free detection of single nucleotide polymorphisms

G-quadruplex/thioflavin T (G4/THT) is one of the ideal label-free fluorescent light-emitting elements in the field of biosensors due to its good programmability and adaptability. However, the unsatisfactory luminous efficiency of single-molecule G4/THT limits its more practical applications. Here, we developed a G4 embedded semi-catalytic hairpin assembly (G4-SCHA) reaction by rationally modifying the traditional CHA reaction, and combined with the invasive reaction, supplemented by magnetic separation technology, for label-free sensitive detection of single nucleotide polymorphisms (SNPs). The invasive reaction enabled specific recognition of single-base mutations in DNA sequences as well as preliminary signal cycle amplification. Then, magnetic separation was used to shield the false positive signals. Finally, the G4-SCHA was created for secondary amplification and label-free output of the signal. This dual-signal amplified label-free biosensor has been shown to detect mutant targets as low as 78.54 fM. What's more, this biosensor could distinguish 0.01 % of the mutant targets from a mixed sample containing a large number of wild-type targets. In addition, the detection of real and complex biological samples also verified the practical application value of this biosensor in the field of molecular design breeding. Therefore, this study improves a label-free fluorescent light-emitting element, and then proposes a simple, efficient and universal label-free SNP biosensing strategy, which also provides an important reference for the development of other G4/THT based biosensors.

Colorimetric detection of furin based on enhanced catalytic activity of G-quadruplex/hemin DNAzyme

BackgroundRapid and sensitive colorimetric detection methods are crucial for diseases diagnosis, particularly those involving proteases like furin, which are implicated in various conditions, including cancer. Traditional detection methods for furin suffer from limitations in sensitivity and practicality for on-site detection, motivating the development of novel detection strategies. Therefore, developing a simple, enzyme-free, and rapid colorimetric analysis method with high sensitivity for furin detection is imperative. ResultsHerein, we have proposed a colorimetric method in this work for the first time to detect furin, leveraging the assembly of G-quadruplex/hemin DNAzyme with enhanced catalytic activity. Specifically, a peptide-DNA conjugate (PDC) comprising a furin-recognition peptide and flanking DNA sequences for signal amplification is designed to facilitate the DNAzyme assembly. Upon furin treatment, PDC cleavage triggers a cyclic catalytic hairpin assembly reaction to form the complementary double-stranded structures by hairpin 1 (HP1) and hairpin 2 (HP2), bringing the G-quadruplex sequence in HP1 closer to hemin on HP2. Moreover, the resulting G-quadruplex/hemin DNAzymes exhibit robust peroxidase-like activity, enabling the catalysis of the colorimetric reaction of ABTS2− for furin detection. Our method demonstrates high sensitivity, rapid response, and compatibility with complex sample matrices, achieving a detection limit as low as 1.1 pM. SignificanceThe DNAzyme reported in this work exhibits robust catalytic activity, enabling high sensitivity and good efficiency for the detection. By eliminating the requirement for exogenous enzymes, our approach enables visual furin detection without expensive instrumentation and reagents, promising significant utility in biomedical and clinical diagnostic applications. Given the various design of peptide sequence and the programmability of DNA, it can be readily applied to analyzing other useful tumor biomarkers.

High‐Performance Cooperative DNA Nanodevice Enables Sensitive Circular RNA Imaging and Precise Tumor Growth Suppression

The utility of circular RNAs (circRNAs) as emerging biomarkers and regulatory factors in medical diagnostics and therapeutics is hampered by the challenges associated with their sensitive detection and precise modulation. Herein, a high‐performance cooperative DNA nanodevice (HCDN) based on DNA tetrahedron‐confined catalytic DNA assembly reaction (DT‐CDA) that enables both imaging and regulation of circRNAs is developed. Activation of the DT‐CDA is contingent upon the presence of the target circRNA, which, together with a replicative fuel probe, catalyzes the sequential opening of additional DT‐CDAs. This cooperative exponential signal amplification with negligible background interference allows HCDN to effectively detect minute quantities of circRNAs. Employing circSATB2 as a model, the HCDN demonstrates substantial downregulation of Cyclin D1 (CCND1) mRNA and protein levels in cellular and in vivo models, thereby inhibiting tumor growth. The innovative design of HCDN sets the stage for a powerful methodology conducive to enhanced clinical diagnostics and biomolecule manipulation, thereby advancing the capabilities and applications of DNA nanotechnology.

An efficient DNAzyme-locked leakless enzyme-free amplification system for alpha-foetoprotein detection in liver cancer and breast cancer.

Alpha-foetoprotein (AFP) is taken as a diagnostic tumor marker for the screening and diagnosis of cancer. Nucleic acid-based isothermal amplification strategies are emerging as a potential technology in early screening and clinical diagnosis of AFP. The leakages between hairpins dramatically increase the background and reduce the sensitivity. Thus, it is necessary to develop some strategies to reduce the leakage for isothermal amplification strategies. A DNAzyme-locked leakless enzyme-free amplification system was developed for AFP detection in liver cancer and breast cancer. AFP could open the apt-hairpin and initiate the catalytic hairpin assembly (CHA) reaction to produce a Y-shaped duplex. Two tails of a Y-shaped duplex cleaved the two kinds of leakless hairpins. Then, the third tail of the Y-shaped duplex catalyzed the second CHA between the cleaved leakless hairpins to recover the fluorescent intensity. The limit of detection reached 5fg/mL by the two levels of signal amplifications. Importantly, the leakless hairpin design effectively reduced leakage between hairpins and weakened the background. In addition, it also showed a great promising potential for AFP detection in early screening and clinical diagnosis.

SERS biosensors based on catalytic hairpin self-assembly and hybridization chain reaction cascade signal amplification strategies for ultrasensitive microRNA-21 detection.

A highly sensitive surface-enhanced Raman scattering (SERS) biosensor has been developedfor the detection of microRNA-21 (miR-21) using an isothermal enzyme-free cascade amplification method involving catalytic hairpin assembly (CHA) and hybridization chain reaction (HCR). The CHA reaction is triggered by the target miR-21, which causes hairpin DNA (C1 and C2) to self-assemble into CHA products. After AgNPs@Capture captures the resulting CHA product, the HCR reaction is started, forming long-stranded DNA on the surface of AgNPs. A strong SERS signal is generated due to the presence of a large amount of the Raman reporter methylene blue (MB) in the vicinity of the SERS "hot spot" on the surface of AgNPs. The monitoring of the SERS signal changes of MB allows for the highly sensitive and specific detection of miR-21. In optimal conditions, the biosensor exhibits a satisfactory linear range and a low detection limit for miR-21 of 42.3 fM. Additionally, this SERS biosensor shows outstanding selectivity and reproducibility. The application of this methodology to clinical blood samples allows for the differentiation of cancer patients from healthy controls. As a result, the CHA-HCR amplification strategy used in this SERS biosensor could be a useful tool for miRNA detection and early cancer screening.

In vivo cloning of PCR product via site-specific recombination in Escherichia coli

Over the past years, several methods have been developed for gene cloning. Choosing a cloning strategy depends on various factors, among which simplicity and affordability have always been considered. The aim of this study, on the one hand, is to simplify gene cloning by skipping in vitro assembly reactions and, on the other hand, to reduce costs by eliminating relatively expensive materials. We investigated a cloning system using Escherichia coli harboring two plasmids, pLP-AmpR and pScissors-CmR. The pLP-AmpR contains a landing pad (LP) consisting of two genes (λ int and λ gam) that allow the replacement of the transformed linear DNA using site-specific recombination. After the replacement process, the inducible expressing SpCas9 and specific sgRNA from the pScissors-CmR (CRISPR/Cas9) vector leads to the removal of non-recombinant pLP-AmpR plasmids. The function of LP was explored by directly transforming PCR products. The pScissors-CmR plasmid was evaluated for curing three vectors, including the origins of pBR322, p15A, and pSC101. Replacing LP with a PCR product and fast-eradicating pSC101 origin-containing vectors was successful. Recombinant colonies were confirmed following gene replacement and plasmid curing processes. The results made us optimistic that this strategy may potentially be a simple and inexpensive cloning method.Key points•The in vivo cloning was performed by replacing the target gene with the landing pad.•Fast eradication of non-recombinant plasmids was possible by adapting key vectors.•This strategy is not dependent on in vitro assembly reactions and expensive materials.

DNAzyme and controllable cholesterol stacking DNA machine integrates dual-target recognition CTCs enable homogeneous liquid biopsy of breast cancer

Although circulating tumor cells (CTCs) have demonstrated considerable importance in liquid biopsy, their detection is limited by low concentrations and complex sample components. Herein, we developed a homogeneous, simple, and high-sensitivity strategy targeting breast cancer cells. This method was based on a non-immunological stepwise centrifugation preprocessing approach to isolate CTCs from whole blood. Precise quantification is achieved through the specific binding of aptamers to the overexpressed mucin 1 (MUC1) and human epidermal growth factor receptor 2 (HER2) proteins of breast cancer cells. Subsequently, DNAzyme cleavage and parallel catalytic hairpin assembly (CHA) reactions on the cholesterol-stacking DNA machine were initiated, which opened the hairpin structures T-Hg2+-T and C–Ag+-C, enabling multiple amplifications. This leads to the fluorescence signal reduction from Hg2+-specific carbon dots (CDs) and CdTe quantum dots (QDs) by released ions. This strategy demonstrated a detection performance with a limit of detection (LOD) of 3 cells/mL and a linear range of 5–100 cells/mL. 42 clinical samples have been validated, confirming their consistency with clinical imaging, pathology findings and the folate receptor (FR)-PCR kit results, exhibiting desirable specificity of 100% and sensitivity of 80.6%. These results highlight the promising applicability of our method for diagnosing and monitoring breast cancer.

An Enzymatically Activated and Catalytic Hairpin Assembly-Driven Intelligent AND-Gated DNA Network for Tumor Molecular Imaging.

Due to the potential off-tumor signal leakage and limited biomarker content, there is an urgent need for stimulus-responsive and amplification-based tumor molecular imaging strategies. Therefore, two tetrahedral framework DNA (tFNA-Hs), tFNA-H1AP, and tFNA-H2, were rationally engineered to form a polymeric tFNA network, termed an intelligent DNA network, in an AND-gated manner. The intelligent DNA network was designed for tumor-specific molecular imaging by leveraging the elevated expression of apurinic/apyrimidinic endonuclease 1 (APE1) in tumor cytoplasm instead of normal cells and the high expression of miRNA-21 in tumor cytoplasm. The activation of tFNA-H1AP can be achieved through specific recognition and cleavage by APE1, targeting the apurinic/apyrimidinic site (AP site) modified within the stem region of hairpin 1 (H1AP). Subsequently, miRNA-21 facilitates the hybridization of activated H1AP on tFNA-H1AP with hairpin 2 (H2) on tFNA-H2, triggering a catalytic hairpin assembly (CHA) reaction that opens the H1AP at the vertices of tFNA-H1AP to bind with H2 at the vertices of tFNA-H2 and generate fluorescence signals. Upon completion of hybridization, miRNA-21 is released, initiating the subsequent cycle of the CHA reaction. The AND-gated intelligent DNA network can achieve specific tumor molecular imaging in vivo and also enables risk stratification of neuroblastoma patients.

New quasi-Co/Cu-MOF nanozyme coupled with entropy-driven DNA amplification cascades for highly sensitive electrochemical aptamer growth differentiation factor 15 assay

BackgroundThe monitoring of concentration variation of the newly developed growth differentiation factor 15 (GDF15) biomarker in human serum is of great significance for diagnosing cardiovascular diseases. Current methods for the detection of the GDF15 protein mainly are based on antibody-assisted immunoassays, which encounter the limitations in terms of sensitivity, complexity and costs. The development of simple and sensitive biosensors for GDF15 can therefore facilitate the diagnosis of cardiovascular diseases. ResultsA new bimetallic quasi-Cu/Co-MOF nanozyme with high catalytic performance for electrochemical reduction of H2O2 is synthesized via simple one-step precipitation and low-temperature calcination method. Such nanozymes are further employed as amplification tags and coupled with cyclic entropy-driven DNA signal enhancement strategies to construct ultrasensitive aptamer-based biosensor for detecting GDF15 in human serums. GDF15 molecules associate with two aptamers and release the ssDNA trigger sequences via target-binding induced displacement reaction. These ssDNAs subsequently initiate cyclic DNA-fueled strand displacement and catalytic hairpin assembly (CHA) reaction cascades for confining many quasi-Cu/Co-MOF nanozymes on sensor electrode, which yield drastically amplified H2O2 reduction current for detecting GDF15 down to 0.12 pg mL−1 with a dynamic range of 0.5 pg mL−1 to 20 ng mL−1. The electrochemical aptasensor also presents good reproducibility and selectivity and exhibits the capability to detect GDF15 in diluent serums. SignificanceOur aptamer-based GDF15 protein electrochemical assay clearly outperforms current existing antibody-based methods and the quasi-Cu/Co-MOF nanozyme/entropy-driven cascaded signal amplification means can be used as a universal strategy for sensitive monitoring of different biomolecular markers for diverse applications.