A DNA machine-based magnetic resonance imaging nanoprobe for in vivo microRNA detection.

In situ monitoring microRNA (miRNA) expression in vivo holds immense potential for directly visualizing the occurrence and progression of tumors. However, the significant barrier to developing a probe that can overcome the low abundance of miRNAs while providing an output signal with unlimited tissue penetration depth remains formidable. In this study, we developed a DNA machine-based magnetic resonance imaging nanoprobe (MRINP) for amplified detection of miR-21 in vivo. The MRINP was constructed with superparamagnetic Fe3O4 nanoparticles (NPs), paramagnetic Gd-DOTA complexes, and miR-21-activated DNA machines; the DNA machine was composed of hairpin DNAzyme (HD) strands serving as the DNAzyme walker and hairpin substrate (HS) strands serving as the track. Once uptake into tumor cells, the intracellular miR-21 specifically recognized and hybridized with the HD strand, restoring the activity of DNAzyme. Subsequently, the DNAzyme walker autonomously traveled on the surface of MRINP, and each step movement of the DNAzyme walker resulted in the cleavage of its substrate strands and the ensued release of the Gd-DOTA complex-labeled oligonucleotides, turning on the T1 signal of Gd-DOTA complexes for in situ imaging of miR-21 in tumor-bearing mice. This strategy would offer a promising approach for mapping tumor-specific biomarkers in vivo with unlimited penetration depth.

Direct quantification of N6-methyladenosine fractions at specific site in RNA based on deoxyribozyme mediated CRISPR-Cas12a platform.

As the most abundant modification in eukaryotic messenger RNA (mRNA) and long noncoding RNA (lncRA), N6-methyladenosine (m6A) has been shown to play essential roles in various significant biological processes and attracted growing attention in recent years. To investigate its functions and dynamics, there is a critical need to quantitatively determine the m6A modification fractions at a precise location. Here, we report a deoxyribozyme mediated CRISPR-Cas12a platform (termed "DCAS") that can directly quantify m6A fractions at single-base resolution. DCAS employs a deoxyribozyme (VMC10) to selectively cleave the unmodified adenine (A) in the RNA, allowing only m6A-modified RNA amplified by RT-PCR. Leveraging the CRISPR-Cas12a quantify the PCR amplification products, DCAS can directly determine the presence of m6A at target sites and its fractions. The combination of CRISPR-Cas12a with RT-PCR has greatly improved the sensitivity and accuracy, enabling the detection of m6A-modified RNA as low as 100 aM in 2 fM total target RNA. This robustly represents an improvement of 2-3 orders of magnitude of sensitivity and selectivity compared to traditional standard methods, such as SCARLET and primer extension methods. Therefore, this method can be successfully employed to accurately determine m6A fractions in real biological samples, even in low abundance RNA biomarkers.

Target-promoted activation of DNAzyme walker for in situ assembly of hemin/G-quadruplex nanowires enable ultrasensitive and label-free electrochemical myocardial microRNA assay.

The concentration elevation of myocardial microRNA (miRNA) biomarker is associated with the pathogenic process of acute myocardial infarction (AMI), and sensitive quantification of myocardial miRNA biomarker plays an important role for early AMI diagnosis and its treatment. In response, this work describes an ultrasensitive and non-label electrochemical biosensor for the assay of myocardial miRNA based on cascade signal amplifications integrated by DNAzyme walker and hemin/G-quadruplex nanowires. The DNAzyme walker is activated by presence of target miRNAs to move along the electrode surface to cyclically cleave the substrate hairpins to release G-quadruplex segments, which further trigger the in situ formation of many hemin/G-quadruplex nanowires. The large amounts of hemin intercalated into the DNA nanowires subsequently generate drastically magnified electrochemical current signals for highly sensitive label-free assay of myocardial miRNAs down to 15.7 fM within dynamic range of 100 fM to 10 nM. Such a biosensor also has high selectivity and can monitor myocardial miRNAs in diluted serums at low levels, providing a sensitive and reliable platform for diagnosing infarct-associated cardiovascular diseases.

Concentration and activation biresponsive strategy in one analysis system with simultaneous use of G4 structure-specific signal probe and enzyme-catalyzed reaction.

BACKGROUND: Enzymes with critical effects on life systems are regulated by expression and activation to modulate life processes. However, further insights into enzyme functions and mechanisms in various physiological processes are limited to concentration or activation analysis only. Currently, enzyme analysis has received notable attention, particularly simultaneous analysis of their concentration and activation in one system. Herein, N-methyl mesoporphyrin IX (NMM), a specific dye with notable structural selectivity for parallel G-quadruplex nucleic acid enzyme (G4h DNAzyme), is employed for the analysis of its concentration. In addition, the peroxidase activity of G4h DNAzyme is characterized based on G4h DNAzyme-catalyzed decomposition of H2O2 to continuously consume luminol. Accordingly, an increased fluorescence (FL) response of NMM and a decreased FL response of luminol could be simultaneously employed to analyze the concentration and activation of G4h DNAzyme. RESULT: Herein, a novel concentration and activation biresponsive strategy is proposed using a G4h DNAzyme-based model that simultaneously employs a G4h structure-specific signal probe for enzyme concentration analysis and G4h DNAzyme-catalyzed reactions for enzyme activation analysis. Under optimal conditions, the biresponsive strategy can be effectively used for the simultaneous analysis of G4h DNAzyme concentration and activation, with detection limits of 718.7 pM and 233.4 nM respectively, delivering acceptable performances both in cell and in vitro. SIGNIFICANCE: This strategy can not only be applied to concentration and activation analyses of G4h DNAzyme but can also be easily extended to other enzymes by simultaneously combining concentration analysis via target-induced direct reaction and activation analysis via target-induced catalytic reaction, offering deeper insights into various enzymes and enabling their effective implementation in bioanalysis and biochemistry.

Highly integrated, self-powered and activatable bipedal DNA nanowalker for imaging of base excision repair in living cells.

DNA walkers have attracted considerable attention in biosensing and bioimaging. Compared with the conventional single leg-based DNA walker, the bipedal DNA walker has remarkable advantages, with improved sensitivity and fast kinetics, and can work efficiently in a crowded cellular environment. However, most reported bipedal DNA walkers are powered by exogenous supplementation, and elaborate DNA sequence designs, auxiliary additives or extra carriers are often needed. A highly integrated bipedal DNA walker that can address robustness, sensitivity and consistency issues in a single system is highly desirable but remains a great challenge. We herein report a novel bipedal DNA nanowalker system through simple assembly of a DNA substrate, hairpin functionalized-AuNPs (AuNPs-H2), and a blocked Mn2+-dependent DNAzyme hairpin (H1) on degradable MnO2 nanosheets, which holds great potential for living cell operation. Highly integrated features enable the simultaneous delivery of core components of the bipedal DNA walker, including a walking track (AuNPs-H2), a walking strand (H1 cleaved by APE1), and a driving force (Mn2+-dependent DNAzyme cleavage) as a whole, thereby enhancing the control of the spatiotemporal distribution of these components at the intracellular target sites. The redox reaction between the MnO2 nanosheets and GSH inside the cells not only consumed the intracellular GSH to improve the biostability of the walking track but also generated abundant Mn2+ as a cofactor of the DNAzyme. As a proof of concept, the developed nanowalker was demonstrated to work efficiently for monitoring base excision repair (BER)-related human apurinic/apyrimidinic endonuclease 1 (APE1) in living cells, highlighting the great potential of the bipedal DNA nanowalker in biological systems.

Novel Ternary System for Electrochemiluminescence Biosensor and Application toward Pb2+ Assay.

This study constructed an electrochemiluminescence (ECL) biosensor for ultrasensitive detection of Pb2+ in a ternary system by employing DNAzyme. The ternary system is composed of a potassium-neutralized perylene derivative (K4PTC) as the ECL emitter, K2S2O8 as the coreactant, and neodymium metal-organic frameworks (Nd-MOFs) as the coreaction accelerators. Nd-MOFs immobilize DNAzymes and enhance the luminescence intensity of the K4PTC/K2S2O8 system. As part of this system, K4PTC enhances the ECL signal in solution and supports Pb2+ detection. The sequence of ferrocene (Fc)-linked DNA (DNA-Fc) is catalytically cleaved by DNAzymes in the presence of Pb2+. This causes the removal of DNA1-Fc from the electrode surface to recover the ECL signal. As a result, the as-prepared ECL biosensor can quantify Pb2+ with a detection limit (LOD) of 4.1 fM in the range of 1 µM to 10 fM. The ECL biosensor displays high specificity, good stability, excellent reproducibility, and desirable practicality for Pb2+ detection in tap water. Moreover, by simply changing the sequence of the DNAzyme, new biosensors can be designed for ultrasensitive detection of different heavy metal ions, offering an excellent approach for monitoring water quality safety.

Light-Triggered Plasmonic DNAzyme Walker Enables Precise Subcellular Molecular Imaging with Reduced Off-Mitochondria Signal Leakage.

The development of highly sensitive and precise imaging techniques capable of visualizing crucial molecules at the subcellular level is essential for elucidating mitochondrial functions and uncovering novel mechanisms in biological processes. However, traditional molecular imaging strategies are still limited by off-mitochondria signal leakage because of the "always-active" sensing mode. To address this limitation, we have developed a light-triggered activation sequence activated plasmonic DNAzyme walker (PDW) for accurate subcellular molecular imaging by the combination of an organelle localized strategy, upconversion nanotechnology, and a plasmon enhanced fluorescence (PEF) technique. Exploiting the advantage of light activation enables precise control over when and where to activate the probe's sensing function, effectively reducing off-mitochondria signal leakage as validated by the dynamic monitoring of changes in off-mitochondria signals during the mitochondrial entry process. Furthermore, by leveraging the PEF capability of triangular gold nanoprisms (Au NPRs), the fluorescence intensity can be enhanced by approximately 11.9 times, ensuring highly sensitive and accurate subcellular molecular imaging.

Enhanced CRISPR/Cas12a Fluorimetry via a DNAzyme-Embedded Framework Nucleic Acid Substrate.

CRISPR/Cas12a fluorimetry has been extensively developed in the biosensing arena, on account of its high selectivity, simplicity, and rapidness. However, typical CRISPR/Cas12a fluorimetry suffers from low sensitivity due to the limited trans-cleavage efficiency of Cas12a, necessitating the integration of other preamplification techniques. Herein, we develop an enhanced CRISPR/Cas12a fluorimetry via a DNAzyme-embedded framework nucleic acid (FNAzyme) substrate, which was designed by embedding four CLICK-17 DNAzymes into a rigid tetrahedral scaffold. FNAzyme can not only enhance the trans-cleavage efficiency of CRISPR/Cas12a by facilitating the exposure of trans-substrate to Cas12a but also result in an exceptionally high signal-to-noise ratio by mediating enzymatic click reaction. Combined with a functional nucleic acid recognition module, this method can profile methicillin-resistant Staphylococcus aureus as low as 18 CFU/mL, whose sensitivity is approximately 54-fold higher than that of TaqMan probe-mediated CRISPR/Cas12a fluorimetry. Meanwhile, the method exhibited satisfactory recoveries in food matrices ranging from 80% to 101%. The DNA extraction- and preamplification-free detection format as well as the potent detection performance highlight its tremendous potential as a next-generation analysis tool.

Target-assisted self-cleavage DNAzyme electrochemical biosensor for MicroRNA detection with signal amplification.

In this work, we reported an electrochemical biosensor with target-assisted self-cleavage DNAzyme function for signal amplified detection of miRNA. The target-recycling amplification led to significant signal enhancement and thus offers high detection sensitivity.

Aptamer-Based Electrochemical Biosensing Platform for Analysis of Cardiac Biomarkers.

Monitoring biomarkers secreted by cardiomyocytes is critical to evaluate anticancer drug-induced myocardial injury (MI). Cardiac troponin I (cTnI) is considered the gold standard biomarker for MI. Herein, an electrochemical aptasensor is engineered for cTnI detection based on lanthanide europium metal-organic frameworks (Eu-MOFs) and a hybridization chain reaction-directed DNAzyme strategy. Three types of Eu-MOF morphologies were easily synthesized by changing the solvent, and the Eu-MOF modulated by mixing the solvent of dimethylformamide and H2O (D-Eu-MOF) exhibited the best performance compared to other morphologies of the Eu-MOFs. Multifunctional nanoprobes were constructed from D-Eu-MOF@Pt loaded with natural horseradish peroxidase and combined with an aptamer-initiated nuclear acid hybridization chain reaction to form G-quadruplex/hemin DNAzymes for signal amplification. A novel capture probe is constructed on the basis of DNA nanotetrahedrons modified on screen-printed gold electrodes to enhance the capture of the target and multifunctional nanoprobes for signal amplification. It exhibits a detection limit of 0.17 pg mL-1 and a linear range from 0.5 pg mL-1 to 15 ng mL-1. The practicality of the platform is evaluated by measuring cTnI in real samples and secreted by cardiomyocytes after drug treatment, which provides great potential in drug-induced MI evaluation for clinical application.

Calcium-Dependent Chemiluminescence Catalyzed by a Truncated c-MYC Promoter G-Triplex DNA.

The dynamic landscape of non-canonical DNA G-quadruplex (G4) folding into G-triplex intermediates has led to the study of G-triplex structures and their ability to serve as peroxidase-mimetic DNAzymes. Here we report the formation, stability, and catalytic activity of a 5'-truncated c-MYC promoter region G-triplex, c-MYC-G3. Through circular dichroism, we demonstrated that c-MYC-G3 adopts a stable, parallel-stranded G-triplex conformation. The chemiluminescent oxidation of luminol by the peroxidase mimicking DNAzyme activity of c-MYC-G3 was increased in the presence of Ca2+ ions. We utilized surface plasmon resonance to characterize both c-MYC-G3 G-triplex formation and its interaction with hemin. The detailed study of c-MYC-G3 and its ability to form a G-triplex structure and its DNAzyme activity identifies issues that can be addressed in future G-triplex DNAzyme designs.

Making target sites in large structured RNAs accessible to RNA-cleaving DNAzymes through hybridization with synthetic DNA oligonucleotides.

The 10-23 DNAzyme is one of the most active DNA-based enzymes, and in theory, can be designed to target any purine-pyrimidine junction within an RNA sequence for cleavage. However, purine-pyrimidine junctions within a large, structured RNA (lsRNA) molecule of biological origin are not always accessible to 10-23, negating its general utility as an RNA-cutting molecular scissor. Herein, we report a generalizable strategy that allows 10-23 to access any purine-pyrimidine junction within an lsRNA. Using three large SARS-CoV-2 mRNA sequences of 566, 584 and 831 nucleotides in length as model systems, we show that the use of antisense DNA oligonucleotides (ASOs) that target the upstream and downstream regions flanking the cleavage site can restore the activity (kobs) of previously poorly active 10-23 DNAzyme systems by up to 2000-fold. We corroborated these findings mechanistically using in-line probing to demonstrate that ASOs reduced 10-23 DNAzyme target site structure within the lsRNA substrates. This approach represents a simple, efficient, cost-effective, and generalizable way to improve the accessibility of 10-23 to a chosen target site within an lsRNA molecule, especially where direct access to the genomic RNA target is necessary.

Nucleic acid-functionalized upconversion luminescence biosensor based on strand displacement-mediated signal amplification for the detection of trivalent chromium ions.

BACKGROUND: Rapid industrial development has generated serious pollution, including the presence of toxic and harmful heavy metal ions. Among them, trivalent chromium ion (Cr3+) is a very important element that poses a threat to life and health in our industrial wastewater pollution. Thus, it is important to develop efficient fluorescence methods for Cr3+ detection. In this study, an upconversion luminescence biosensor for detecting Cr3+ was constructed based on a DNAzyme, strand displacement reaction (SDR), and DNA-functionalized upconversion nanoparticles (UCNPs). RESULTS: The sulfonate-rich poly (sodium 4-styrene sulfonate) (PSS) was modified onto the surface of UCNPs, forming UCNPs@PSS. Then, NH2-Capture probe DNA (NH2-Cp) was further modified onto the UCNPs@PSS surface through sulfonylation, resulting in UCNPs@PSS@NH2-Cp. The DNAzyme activated by Cr3+ triggered the release of the primer probe (Pp), which initiated the SDR system cycle, thereby releasing a tetramethylrhodamine (TAMRA)-modified signal probe (TAMRA-Sp). Finally, UCNPs@PSS@NH2-Cp bound to TAMRA-Sp through complementary base pairing, causing UCNPs and TAMRA to approach each other. Because of the luminescence resonance energy transfer (LRET) mechanism, the upconversion luminescence (UCL) signal of the UCNPs was quenched by TAMRA, enabling the detection of Cr3+ by the change of I585/I545 ratio. This biosensor has good stability, selectivity, and sensitivity, with a linear range of 0.5-75 nM and a detection limit of 0.135 nM for Cr3+. SIGNIFICANCE AND NOVELTY: Firstly, based on LRET between UCNPs and TAMRA, the quantitative analysis of Cr3+ is achieved through the changes of ratio fluorescence. Secondly, the specificity of the biosensor is improved by utilizing the specific recognition of DNA enzymes. Thirdly, the signal amplification technology of the SDR cycle greatly improves the sensitivity of biosensor. This biosensor will be useful for future environmental safety monitoring and biopsy of biological fluids.

Endoprotein-activating DNAzyme assay for nucleic acid extraction- and amplification-free detection of viable pathogenic bacteria.

Pathogenic bacteria in food or environment, can pose threats to public health, highlighting the requirement of tools for rapid and accurate detection of viable pathogenic bacteria. Herein, we report a sequential endoprotein RNase H2-activating DNAzyme assay (termed epDNAzyme) that enables nucleic acid extraction- and amplification-free detection of viable Salmonella enterica (S. enterica). The direct detection allows for a rapid detection of viable S. enterica within 25 min. Besides, the assay, based on sequential reporting strategy, circumvents internal modifications in the DNAzyme's active domain and improve its catalytic activity. The multiple-turnover DNAzyme cutting and the enhanced catalytic activity of DNAzyme render the epDNAzyme assay to be highly sensitive, and enables the detection of 190 CFU/mL and 0.1% viable S. enterica. The assay has been utilized to detect S. enterica contamination in food and clinical samples, indicating its potential as a promising tool for monitoring pathogen-associated biosafety.

Short DNAzymes for Efficient Catalysis of "Click" Reactions in Solution and on Surface.

We have systematically investigated and found surprising superior catalytic activities of very short DNAzymes for copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC), both in solution and on surface. As a key reaction of the "click chemistry" class, CuAAC is a highly efficient and specific covalent conjugation tool with demonstrated applications in organic synthesis, bioconjugation, and surface functionalization; however, it requires the presence of the Cu(I) catalyst, which is an unstable species in aqueous solutions. We show here that one ultrashort, 14-nucleotide-truncated fragment of an earlier in vitro selected DNAzyme (CLICK-17) shows a striking and superior catalytic activity toward the in trans CuAAC reaction in solution and on surface in the presence of either Cu(I) or Cu(II), at significantly lowered concentrations. These results obviate the need for long-sequence DNAzymes, selected out of the homogeneous solution phase, for application in complex surface environments.

Functional DNA-Zn2+ coordination nanospheres for sensitive imaging of 8-oxyguanine DNA glycosylase activity in living cells.

Sensitive monitoring of human 8-oxyguanine DNA glycosylase (hOGG1) activity in living cells is helpful to understand its function in damage repair and evaluate its role in disease diagnosis. Herein, a functional DNA-Zn2+ coordination nanospheres was proposed for sensitive imaging of hOGG1 in living cells. The nanospheres were constructed through the coordination-driven self-assembly of the entropy driven reaction (EDR) -deoxyribozyme (DNAzyme) system with Zn2+, where DNAzyme was designed to split structure and assembled into the EDR system. When the nanospheres entered the cell, the competitive coordination between phosphate in the cell and Zn2+ leaded to the disintegration of the nanospheres, releasing DNA and some Zn2+. The released Zn2+ acted as a cofactor of DNAzyme. In the presence of hOGG1, the EDR was completed, accompanied by fluorescence recovery and the generation of a complete DNAzyme. With the assistance of Zn2+, DNAzyme continuously cleaved substrates to produce plenty of fluorescence signals, thus achieving sensitive imaging of hOGG1 activity. The nanospheres successfully achieved sensitive imaging of hOGG1 in human cervical cancer cells (HeLa), human non-small cell lung cancer cells and human normal colonic epithelial cells, and assayed changes in hOGG1 activity in HeLa cells. This nanospheres may provide a new tool for intracellular hOGG1 imaging and related biomedical studies.

Ultrasensitive Electrochemical Biosensor with Powerful Triple Cascade Signal Amplification for Detection of MicroRNA.

In this work, by ingeniously integrating catalytic hairpin assembly (CHA), double-end Mg2+-dependent DNAzyme, and hybridization chain reaction (HCR) as a triple cascade signal amplifier, an efficient concatenated CHA-DNAzyme-HCR (CDH) system was constructed to develop an ultrasensitive electrochemical biosensor with a low-background signal for the detection of microRNA-221 (miRNA-221). In the presence of the target miRNA-221, the CHA cycle was initiated by reacting with hairpins H1 and H2 to form DNAzyme structure H1-H2, which catalyzed the cleavage of the substrate hairpin H0 to release two output DNAs (output 1 and output 2). Subsequently, the double-loop hairpin H fixed on the electrode plate was opened by the output DNAs, to trigger the HCR with the assistance of hairpins Ha and Hb. Finally, methylene blue was intercalated into the long dsDNA polymer of the HCR product, resulting in a significant electrochemical signal. Surprisingly, the double-loop structure of the hairpin H could prominently reduce the background signal for enhancing the signal-to-noise ratio (S/N). As a proof of concept, an ultrasensitive electrochemical biosensor was developed using the CDH system with a detection limit as low as 9.25 aM, achieving favorable application for the detection of miRNA-221 in various cancer cell lysates. Benefiting from its enzyme-free, label-free, low-background, and highly sensitive characteristics, the CDH system showed widespread application potential for analyzing trace amounts of biomarkers in various clinical research studies.

A target-triggered colorimetric sensor for ultrasensitive detection of miRNAs based on self-powered three-dimensional DNA walker.

MicroRNAs (miRNAs) play an important role in the process of heart failure (HF) and are emerging biomarkers that can be used for the auxiliary diagnosis of HF. However, it is very challenging to accurately analyze the expression levels of trace miRNAs in complex clinical samples. Here, we developed an enzyme-free colorimetric sensor for the ultrasensitive detection of miRNA-423-5p (HF-associated miRNA) based on three-dimensional DNA walkers constructed from functional nucleic acids and gold nanoparticles (AuNPs). DNAzyme with cleavage activity was specifically activated by miRNA-423-5p to sustainably cleave the substrate, thereby releasing the trigger sequence to initiate the subsequent mismatched catalytic hairpin assembly (MCHA) cycle. Then, as the MCHA cycle proceeded to continuously expose the G-quadruplex (GQ) sequence, the sequence bound with hemin to form a large amount of GQ/hemin DNAzyme on the surface of the AuNPs, which rapidly catalyzed the chromogenic oxidation of 3,3',5,5'-tetramethylbenzidine to yield an amplified colorimetric signal readout. The colorimetric sensor exhibited an ultralow detection limit (32 fM), showed excellent specificity and performed well in serum samples. The sensor was applied to detect miRNA-423-5p in clinical plasma samples from healthy individuals and HF patients, and the results revealed its good clinical application in HF diagnosis. Thus, the developed colorimetric sensor provides a convenient detection tool for early screening and diagnosis of HF, as well as for pathophysiological studies.

Recent advances in DNAzymes for bioimaging, biosensing and cancer therapy.

DNAzymes, a class of single-stranded catalytic DNA with good stability, high catalytic activity, and easy synthesis, functionalization and modification properties, have garnered significant interest in the realm of biosensing and bioimaging. Their integration with fluorescent dyes or chemiluminescent moieties has led to remarkable bioimaging outcomes, while DNAzyme-based biosensors have demonstrated robust sensitivity and selectivity in detecting metal ions, nucleic acids, proteins, enzyme activities, exosomes, bacteria and microorganisms. In addition, by delivering DNAzymes into tumor cells, the mRNA therein can be cleaved to regulate the expression of corresponding proteins, which has further propelled the application of DNAzymes in cancer gene therapy and synergistic therapy. This paper reviews the strategies for screening attractive DNAzymes such as SELEX and high-throughput sequencing, and briefly describes the amplification strategies of DNAzymes, which mainly include catalytic hairpin assembly (CHA), DNA walker, hybridization chain reaction (HCR), DNA origami, CRISPR-Cas12a, rolling circle amplification (RCA), and aptamers. In addition, applications of DNAzymes in bioimaging, biosensing, and cancer therapy are also highlighted. Subsequently, the possible challenges of these DNAzymes in practical applications are further pointed out, and future research directions are suggested.

Sensitive detection of dipeptidyl peptidase based on DNA-peptide conjugates and double signal amplification of CHA and DNAzymes.

Dipeptidyl peptidase IV (DPPIV) is an enzyme belonging to the type II transmembrane serine protease family that has gained wide interest in the fields of hematology, immunology, and cancer biology. Moreover, DPPIV has emerged as a promising target for therapeutic intervention in type II diabetes. Due to its biological limitations, traditional strategies cannot meet the requirements of low abundance DPPIV analysis in complex environments. In this work, combining the high programmability of DNA and the chemical diversity of peptides, we designed DNA-peptide conjugates that can be specifically recognized, polypeptides as specific substrates for target DPPIV and DNA probes as primers for catalytic hairpin assembly (CHA), recycling a large amount of DNAzymes by triggering CHA amplification. The DNAzyme substrate modified with the FAM fluorescent group was immobilized on the surface of gold nanoparticles by S-Au chemical bonds to form a signal output probe. The DNAzymes enzyme cleaved the substrate of the signal outputs probe, yielding a double-amplified fluorescence signal. This method has a detection limit as low as 0.18 mU mL-1 and a linear range of 0-5 mU mL-1 in serum samples, showing high stability and good potential for practical applications.