Hairpin DNA Strands Research Articles

ATP-driven AP endonuclease-active Exo III@zeolitic imidazolate framework for uracil-DNA glycosylase imaging in living cells

In the currently available approaches for intracellular uracil-DNA glycosylase (UDG) detection, their signal outputs usually rely on the cleavage of the residual AP sites in DNA probes by apurinic/apyrimidinic endonuclease 1 (APE1), whose expression level varies across different types of cells. Therefore, reliable visualizing detection of UDG in living cells remains challenging. Herein, a nanoprobe for imaging of intracellular UDG activity has been designed by taking advantages of both zeolitic imidazolate framework-90 (ZIF-90) and endonuclease III. ZIF-90 is used to deliver exonuclease III (Exo III) and hairpin DNA strands into cells. One of the hairpin strands (HP1) contains a deoxyuridine (dU) base in its stem region, while the other (HP2) was modified with FAM molecule and BHQ1 at its 3′ and 5′ ends, respectively. In the presence of UDG, the dU base in HP1 is excised to form an AP site. Exo III can cleave the AP site in HP1 to generate a stem-loop fragment, which can hybridize with HP2 to initiate the recycling digestion of HP2 by Exo III, resulting in amplification of the fluorescence signal and hence sensitive detection of UDG (limit of detection: 2.0×10−6 U/mL). Experimental results have demonstrated that the nanoprobe has merits of good biocompatibility, high sensitivity and selectivity. It realizes in-situ imaging of intracellular UDG without the involvement of APE1, is a promising alternative tool for UDG detection in fields of clinical diagnosis and biomedical research.

Thermodynamic-assisted highly effective target converting strategy for sensitive electrochemical detection of trace lead (II) ion

In the present work, a thermodynamic-assisted target-converting strategy was designed by utilizing new 2D DNA nanoprobes (DNPs) to develop electrochemical biosensors for the sensitive determination of trace Pb2+. Herein, a stable DNP with a special structure was prepared through the hybridization reaction between three single-strand DNA (RS1, RS2, and P). Once the existence of Pb2+, the DNP will crumble and then release RS1 and RS2 due to the strong thermodynamic instability of the RS1/RS2 duplex. since RS1 and RS2 were designed in the same sequence in the free domain, they can be simultaneously used as effective output DNA, enhancing the conversion rate of Pb2+to ssDNA up to 1:2. Following the same principle, ferrocene-labeled bipedal DNPs (Fc-bDNPs) are prepared for stable electrochemical signal output. In the presence of RS1, RS2, and a hairpin DNA strand (H1), the ferrocene-labeled DNA strands (S3) escape from the Fc-bDNPs due to strand-displacement reaction, accompanying release of the two ssDNA outputs that can simultaneously trigger another strand-displacement reaction. Consequently, the one Pb2+input makes multiple Fc-labeled DNA strands far away from the electrode surface, resulting in a dramatic decrease in the response current of Fc. The developed biosensors show a linear relationship between current value and logarithmic concentration of Pb2+ ranging from 1 pM to 40 nM with a detection limit of 0.30 pM.

An electrochemiluminescence sensing platform combining entropy-driven amplification strategy and inverted DNA tetrahedral nanomachines for detecting SARS-CoV-2 specific gene

In this work, we propose a Ti3C2Tx-based electrochemiluminescence (ECL) sensing platform, combined with a programmable entropy-driven cycling strategy and inverted DNA tetrahedral nanomachines to detect the SARS-CoV-2 RdRp gene. The use of Ti3C2Tx in the ECL sensing platform provides unique advantages that improve the assay's sensitivity. Firstly, the SARS-CoV-2 RdRp gene triggers an entropy-driven cyclic amplification reaction to obtain a large number of Output strands. Subsequently, the Output strand reacts with an inverted DNA tetrahedral swing arm strand to start the self-driven process. Finally, the ferrocene (Fc)-labeled hairpin DNA strand of the inverted DNA tetrahedron is severed to achieve the signal change. Under optimal conditions, the ECL platform integrated with entropy-driven cycle amplification reaction and inverted DNA tetrahedral nanomachines obtained a wide range of 100 aM to 10 nM and a limit of detection (LOD) of 48.3 aM for the SARS-CoV-2 RdRp gene. Moreover, the recovery rates of 97% to 103.3% and 98.34% to 103% were obtained in real environments and human pharyngeal swabs, respectively. This ECL sensing platform, which combines an entropy-driven cycle amplification reaction and inverted DNA tetrahedral nanomachines, opens up new opportunities for the detection of other substances.

Two-Dimensional Analysis Method for Highly Sensitive Detection of Dual MicroRNAs in Breast Cancer Cells.

Multiple biomarker detection is crucial for early clinical diagnosis, and it is significant to achieve the simultaneous detection of multiple biomarkers with the same nanomaterial. In this work, the hairpin DNA strands were selectively modified on the surface of gold nanorods (AuNRs) to construct two kinds of nanoprobes by rational design. When in the presence of dual microRNAs, AuNRs were assembled to form end-to-end (ETE) and side-by-side (SBS) dimers. Compared with a single AuNR, the dark-field scattering intensity and red color percentage variation of dimers were extremely distinguished, which could be developed for dual microRNA detection by combining the red color percentage and scattering intensity with the data processing method of principal component analysis to construct a two-dimensional analysis method. Especially, the fraction of AuNR dimers presented a linear relationship with the amount of microRNAs. Based on this, microRNA-21 and microRNA Let-7a in breast cancer cells were detected with the detection limits of 1.72 and 0.53 fM, respectively. This method not only achieved the sensitive detection of dual microRNAs in human serum but also realized the high-resolution intracellular imaging, which developed a new way for the oriented assembly of nanomaterials and biological detection in living cells.

A Self-Made Optical Tweezers Integrated Upconversion Luminescence Confocal Scanning Instrument Enables Stable and Noninvasive Long-Term In Situ Imaging a Single Suspension Cell Under Exceptionally Efficient Luminescent Resonance Energy Transfer Sensing.

It is necessary to explore labeling probes with worthy optical properties and a noninvasive fluorescence imaging manner for stable long-term in situ measuring a single suspension cell. In response to these goals, we herein make a breakthrough on two fronts. On one hand, a co-sensitizer-induced efficient 808 nm near-infrared light-excited luminescence-confined upconversion nanoparticle with a low thermal effect is fabricated by employing a layer-by-layer seed growing approach to develop a sandwich structure, under which the luminescence domain is vastly restricted into an extremely thin inner shell (∼ 2.77 nm) to finally bring about a high-efficiency luminescent resonance energy transfer (LRET) sensing behavior. On the other hand, a self-made optical tweezers integrated upconversion luminescence confocal scanning instrument is applied to enhance the imaging accuracy, after which the liquid viscous force is sufficiently overcome by the resulting single beam gradient force and the analyzed suspension cell is always immobilized to the focal plane to ensure a constant luminescence excitation condition. By making use of a metal ion-dependent DNAzyme and a hairpin DNA strand to design a corresponding LRET sensing system, our nanoprobe shows satisfactory assay performance for two model biomolecules (Ca2+ and TK1 messenger RNA). Following the optical trapping-assisted imaging, this exceptional measurement method is capable of effectively monitoring the intracellular target changes in different physiological states, endowing a powerful toolbox for single cell analysis.

A universal catalytic hairpin assembly system for direct plasma biopsy of exosomal PIWI-interacting RNAs and microRNAs

PIWI-interacting RNAs (piRNAs) are a complex class of small non-coding RNAs which specifically interact with the PIWI protein to play important roles in germline development and somatic tissues. Aberrant expressions of piRNAs have been recently found in a variety of malignant tumors and associated with cancer hallmarks. However, current methods of analyzing piRNAs are limited to reverse transcription quantitative polymerase chain reaction and next generation sequencing. In this study, we have developed a universal catalytic hybridization assembly system (uniCHA) to quantify piRNAs as well as microRNAs. The system simply comprises two universal hairpin DNA strands and one starting hairpin DNA which can be tailored by a simple rule to bind different piRNA and miRNA targets. The uniCHA system was proved to be able to analyze various piRNAs and miRNAs at the same reaction condition with low leakage and high sensitivity of pM level. With this system, we have detected piR-651 and miR-1246 in 106 particles μL−1 MCF-7 cell-secreted exosomes, and successfully performed a direct plasma biopsy to diagnose breast cancer with sensitivity and specificity both at 100% in cohorts of 21 breast cancer patients and 13 healthy controls. This universal biosensing system provides a simple and efficient strategy in analyzing multiple piRNA/miRNA biomarkers in complicated biological samples, indicating its potential of clinical application in cancer diagnostics.

Synthesis of hairpin DNA mediated Au-Ag bimetallic nanomushrooms for antibacterial application

. Precise control of the synthesis of bimetallic nanoparticles with specific morphology and structure is of great significance due to their excellent catalytic, optical and biological property. DNA molecules are considered as a kind of efficient templates to mediate the precise synthesis of bimetallic nanoparticles with homogeneous morphology due to their specific and controllable structure. In this study specific hairpin DNA strands were successfully utilized as templates to mediate the synthesis of special mushroom-like Au-Ag bimetallic nanoparticles with a high yield of > 90%. Several key factors greatly influencing the precise control of the morphology and UV-Vis characteristics of the proposed Au-Ag nanomushrooms during synthesis were investigated and optimized in detail, including the structure of template DNA, loading amounts of DNA, types of reductant agents and surfactants. Then, the formation mechanism of hairpin DNA mediated Au-Ag nanomushrooms was studied. Finally, the proposed Au-Ag nanomushrooms with good biocompatibility were applied for the antibacterial study by the growth curve of E. coli. The proposed Au-Ag nanomushrooms showed the effective inhibition capability for the growth of E. coli. The results suggested that these DNA mediated Au-Ag nanomushrooms possessed great potential applications for biomedical science in future.

Staring at the Naked Goddess: Unraveling the Structure and Reactivity of Artemis Endonuclease Interacting with a DNA Double Strand.

Artemis is an endonuclease responsible for breaking hairpin DNA strands during immune system adaptation and maturation as well as the processing of potentially toxic DNA lesions. Thus, Artemis may be an important target in the development of anticancer therapy, both for the sensitization of radiotherapy and for immunotherapy. Despite its importance, its structure has been resolved only recently, and important questions concerning the arrangement of its active center, the interaction with the DNA substrate, and the catalytic mechanism remain unanswered. In this contribution, by performing extensive molecular dynamic simulations, both classically and at the hybrid quantum mechanics/molecular mechanics level, we evidenced the stable interaction modes of Artemis with a model DNA strand. We also analyzed the catalytic cycle providing the free energy profile and key transition states for the DNA cleavage reaction.

Ratiometric Fluorescent DNA Nanostructure for Mitochondrial ATP Imaging in Living Cells Based on Hybridization Chain Reaction.

For intracellular molecular detection, the appropriate probes should include the abilities to enter target cells noninvasively, target specific sites, and then respond to the analytes reliably. Herein, a ratiometric fluorescent DNA nanostructure (RFDN) was designed for mitochondrial adenosine triphosphate (ATP) imaging in living cells. The DNA nanostructure was constructed by continuous hybridization of two hairpin DNA strands (HS1-Cy3 and HS2-Cy5) under the initiation of the trigger. HS1-Cy3 and HS2-Cy5 contained split aptamer fragments of ATP and are labeled with a fluorescent donor (Cy3) and acceptor (Cy5), respectively. The RFDN integrated multiple split aptamer fragments and increased the local concentration of sensing probes. The binding of ATP to aptamer fragments on the RFDN shortened the distance between Cy3 and Cy5, resulting in obvious ratiometric signals (fluorescence resonance energy transfer). The RFDN showed good biocompatibility and can be internalized into cells in a caveolin-dependent endocytosis pathway. The co-localization imaging results indicated that the DNA nanostructure could target the mitochondria via Cy3 and Cy5. Moreover, the confocal imaging results showed that the intracellular ATP changes stimulated by drugs in living cells could be indicated by the RFDN. In this way, the RFDN is expected to be a simple, flexible, and general platform for chemo/biosensing in living cells.

The Strategy of Constructing DNA Logic Circuits Based on DNA Origami Substrate

The construction of DNA logic circuits is the basis of DNA computers. DNA origami has nanoscale spatial addressability, which provides an ideal framework for the construction of DNA logic circuits. Here, we propose a strategy for constructing DNA logic circuits based on a DNA origami substrate. First, hairpin DNA strands are anchored on a DNA origami substrate to form a DNA wire track. Then, we design three different output ends for different logic circuits (AND, OR and NAND). The operations of the logic circuits are completed on the DNA wire track through the proximal hairpin circuit cascade. Finally, the results of the logical operation are determined by fluorescence detection. We use this strategy to construct two-input AND, OR, and NAND gates on a DNA origami substrate, and then extend this strategy to the construction of large-scale logic circuits and functional combinational logic circuits.

Novel cytosensor for accurate detection of circulating tumor cells based on a dual-recognition strategy and BSA@Ag@Ir metallic-organic nanoclusters

Herein, based on a dual-recognition strategy and BSA@Ag@Ir metallic-organic nanoclusters (BSA@Ag@Ir MONs), a highly specific and sensitive cytosensor was developed for detecting circulating tumor cells (CTCs). To amplify current signal, novel BSA@Ag@Ir MONs with outstanding catalytic activity and huge specific surface area were synthesized, and conjugated with hairpin DNA strands as signal probes. Orion carbon black 40 (Ocb40)//AuNPs were firstly used to modify electrode to increase its conductivity and surface area. Moreover, the dual recognition strategy based on DNA proximity effect was designed to improve the specificity of cytosensor. When two capture probes respectively bound to two adjacent membrane markers of target cells, the probes could form the associative toehold through the proximity effect to capture the signal probes. Only CTCs simultaneously expressing two membrane markers could be captured and generate current responses. The developed cytosensor could detect CTCs in the range of 3 - 3 × 106 cells mL-1 with a detection limit of 1 cell mL-1. Notably, the cytosensor could accurately identify CTCs even in whole blood. Therefore, this cytosensor has great potential for application in biological science, biomedical engineering and personalized medicine.

The Magnetic Bead Computing Model of the 0-1 Integer Programming Problem Based on DNA Cycle Hybridization

Magnetic beads and magnetic Raman technology substrates have good magnetic response ability and surface-enhanced Raman technology (SERS) activity. Therefore, magnetic beads exhibit high sensitivity in SERS detection. In this paper, DNA cycle hybridization and magnetic bead models are combined to solve 0-1 integer programming problems. First, the model maps the variables to DNA strands with hairpin structures and weights them by the number of hairpin DNA strands. This result can be displayed by the specific binding of streptavidin and biotin. Second, the constraint condition of the 0-1 integer programming problem can be accomplished by detecting the signal intensity of the biological barcode to find the optimal solution. Finally, this model can be used to solve the general 0-1 integer programming problem and has more extensive applications than the previous DNA computing model.

Numerous long single-stranded DNAs produced by dual amplification reactions for electrochemical detection of exosomal microRNAs

Exosomal microRNAs (miRNAs) have been explored as an extremely promising biomarker of liquid biopsy for the diagnosis, treatment and prognosis of diseases such as cancer, in which sensitive and selective detection is significant. Herein, we describe the construction and testing of an electrochemical biosensor for the sensitive detection of exosomal miRNAs. It is based on synthetizing numerous long single-stranded DNAs (ssDNAs), which are produced by dual amplification reactions of target-triggered cyclic strand displacement reaction (TCSDR) and primer exchange DNA amplification reaction (PEDAR). In the first signal amplification step, target miRNAs are captured by the hairpin DNA strands (capture probes, Cp) that are immobilized on electrode. After strand unfolding with target capture, primer probes (Pp) enable to hybridize with Cp. And then target miRNAs were displaced for starting the TCSDR process that enable the introduction of numerous primers in Pp. In the second signal amplification step, the primers associated with PEDAR produce copious amounts of elongated ssDNAs. These ssDNAs absorb abundant quantities of methylene blue (MB) that enables the highly sensitive and label-free detection of exosomal miRNAs. This dual amplification process is characterized by a low limit of detection (LOD) of 3.04 aM. In addition, the electrochemical biosensor exhibits good selectivity for miR-21 detection, and shows benefits of simple operation, low cost, portability. Overall, the electrochemical biosensor provides a promising platform for the early diagnosis and screening of tumor biomarkers and the development of devices for point-of-care testing (POCT).

Target-Induced Cascade Amplification for Homogeneous Virus Detection.

Detection of viruses with high sensitivity is critical for the prevention and treatment of the related disease. Two homogeneous target-induced cascade amplification methods were proposed for the detection of enterovirus 71 and coxsackievirus B3. These methods both employ DNAzyme but differ in the way in which the DNAzyme is amplified. In the hybridization chain reaction (HCR)-based strategy, the DNAzyme is assembled by hairpin DNA strands, while in the rolling circle amplification (RCA)-based strategy, the DNAzyme is synthesized by the polymerase. On the basis of the virion structure, we investigated the effects of using only VP1-antibody or VP1-antibody and VP2-antibody on the detection. And the combination of two kinds of antibodies was found to further improve the performance of the detection. Subsequently, the simultaneous detection of EV71 and CVB3 was achieved by the RCA-based strategy. And the proposed methods were also applied in clinical samples analysis with a satisfactory result, showing great potential for applications in virus detection.

Lighting Up MicroRNA in Living Cells by the Disassembly of Lock-Like DNA-Programmed UCNPs-AuNPs through the Target Cycling Amplification Strategy.

Intracellular microRNAs imaging based on upconversion nanoprobes has great potential in cancer diagnostics and treatments. However, the relatively low detection sensitivity limits their application. Herein, a lock-like DNA (LLD) generated by a hairpin DNA (H1) hybridizing with a bolt DNA (bDNA) sequence is designed, which is used to program upconversion nanoparticles (UCNPs, NaYF4 @NaYF4 :Yb, Er@NaYF4 ) and gold nanoparticles (AuNPs). The upconversion emission is quenched through luminescence resonance energy transfer (LRET). The multiple LLD can be repeatedly opened by one copy of target microRNA under the aid of fuel hairpin DNA strands (H2) to trigger disassembly of AuNPs from the UCNP, resulting in the lighting up of UCNPs with a high detection signal gain. This strategy is verified using microRNA-21 as model. The expression level of microRNA-21 in various cells lines can be sensitively measured in vitro, meanwhile cancer cells and normal cells can be easily and accurately distinguished by intracellular microRNA-21 imaging via the nanoprobes. The detection limit is about 1000 times lower than that of the previously reported upconversion nanoprobes without signal amplification. This is the first time a nonenzymatic signal amplification method has been combined with UCNPs for imaging intracellular microRNAs, which has great potential for cancer diagnosis.

Novel 2D-DNA-Nanoprobe-Mediated Enzyme-Free-Target-Recycling Amplification for the Ultrasensitive Electrochemical Detection of MicroRNA.

In this work, on the basis of a new 2D DNA nanoprobe (DNP) and an enzyme-free-target-recycling amplification, an electrochemical biosensor is developed for the ultrasensitive detection of microRNA-21 (miRNA-21). Herein, two ferrocene-labeled bipedal DNPs, which show small steric hindrance and strong stability, are prepared on the basis of the mechanism of the proximity-ligation assay (PLA), improving the space utilization. In the presence of the target, miRNA-21, and a hairpin DNA strand, the DNP will collapse, and then two ferrocene-labeled DNA strands and the miRNA-21 will be simultaneously released from the electrode surface through toehold-mediated strand-displacement reactions (TSDRs), leading to a decrease in the electrochemical signal and realization of enzyme-free target recycling. As a result, the one input target, miRNA-21, could release 2 N ferrocene-labeled DNA strands, achieving a dramatic decrease in the electrochemical signal. Combining DNPs and enzyme-free target recycling, this proposed biosensor showed a linear dependence with miRNA-21 concentration, ranging from 1.0 fM to 10 nM with a detection limit of 0.31 fM. In addition, it is worth mentioning that this biosensor can be regenerated through incubating with three assistant-DNA strands, realizing the reuse of raw materials. Surprisingly, the elaborated biosensor provides a novel strategy for building controllable DNA nanoprobes for the sensitive detection of various biomarkers.

Visualizing miR-155 To Monitor Breast Tumorigenesis and Response to Chemotherapeutic Drugs by a Self-Assembled Photoacoustic Nanoprobe

MicroRNA-155 (miR-155), which facilitates breast tumor growth and invasion by promoting tumor cell proliferation and inhibiting cell apoptosis, is considered an ideal early diagnostic and prognostic marker. Herein, we developed a self-assembled hybridization chain reaction (HCR)-based photoacoustic (PA) nanoprobe for highly sensitive in situ monitoring of dynamic changes in miR-155 expression during breast tumorigenesis and chemotherapy. The PA nanoprobes (Au-H1/PEG and Au-H2/PEG) were constructed by linking poly(ethylene glycol) (PEG) and two hairpin DNA strands (H1 and H2, respectively) to the surface of gold nanoparticles (AuNPs). In the presence of miR-155, the PA nanoprobes self-assembled into Au aggregates via HCR between H1, H2, and miR-155. The decreased interparticle distance in these aggregates enhanced the surface plasmon resonance (SPR) in the AuNPs. Consequently, the absorption peak of the PA nanoprobes red-shifted, and strong PA signals were generated. This strategy enabled the sensitive and quantitative detection of miR-155 with a low detection limit of 0.25 nM. As a result, PA signals of miR-155 were captured on the second day after tumor inoculation when the solid tumor had not yet formed. Dynamic changes in miR-155 during tumor growth and chemotherapy were also monitored in real time to assess the therapeutic effects via PA imaging. By virtue of these advantages, the PA nanoprobes may provide a powerful platform for in situ detection of miR-155 and thus real-time monitoring of tumorigenesis and drug response in breast cancer.

An ultrasensitive and simple method for alkaline phosphatase assay and targeted natural compound screening in vitro.

As an essential phosphate group hydrolase, alkaline phosphatase (ALP), whose level in serum is correlated with bone disease, liver dysfunction, and cancer, could be used as a biomarker for clinical diagnosis and biomedical studies. Hence, developing a convenient and sensitive method for ALP assay has importance in disease diagnosis, drug treatment, and prognosis assessment. In this work, using a hairpin DNA strand as the substrate, we developed an ultrasensitive and simple fluorescence method for quantitative ALP assay based on the binding difference of reduced graphene oxide (rGO) with different DNA strands coupled with λ exonuclease (λ exo) cleavage. Under the optimal conditions, the limit of detection (LOD) of ALP is estimated to be 0.01U/L with the linear region from 0.5U/L to 70U/L. Furthermore, the proposed assay was used to detect ALP in complicated cell-free extracts and evaluate the inhibitory effects of two well-known inhibitors of ALP activity. Finally, the method was used to investigate the effect of natural compounds on ALP activity and five compounds with different inhibitory capability were screened. In summary, we propose that the new method for ALP assay can be applied for therapeutic drug monitoring (TDM) and high-throughput compound screening in combination with multiwell plate technology.

A Sensitive Photoelectrochemical Aptasensor for miRNA‐21 Based on the Sensitization Effect of CdSe Quantum Dots

AbstractA novel photoelectrochemical aptasensor for the sensitive detection of miRNA‐21 was designed based on the sensitization effect of CdSe quantum dots as a signal amplification strategy. The TiO2NTs/RGO/AuNPs electrode was prepared by the electrodeposition of graphene oxide and Au nanoparticles on TiO2 nanotubes with electrochemical reduction. The electrode was prepared as the matrix of the electrochemical aptasensor. The hairpin DNA strands decorated with CdSe quantum dots were immobilized on the surface of the electrode by Au−S bonds. Because of the card‐type structure of the hairpin DNA (HP‐DNA), the CdSe quantum dots were close to the base electrode, which would lead to the sensitization effect and cause the increase of the photocurrent intensity. When a certain concentration of the miRNA‐21 was added, miRNA‐21 hybridized with the complementary part of the HP‐DNA. At the same time, the hairpin structure of HP‐DNA was broken into a straight chain structure, so the CdSe quantum dots on the end of the DNA would move away from the electrode surface, the sensitization effect would be weakened, and the photocurrent intensity would be decreased. The designed photoelectrochemical aptasensor exhibited a low detection limit of 5.6 fM and a wide linear range from 20 fM to 0.2 nM, and it also showed good specificity, reproducibility and stability. The signal amplification strategy was also expected to provide a significant photoelectrochemical biosensor platform for the sensitive detection of biomolecules.

Ultra-Sensitive Colorimetric Assay System Based on the Hybridization Chain Reaction-Triggered Enzyme Cascade Amplification.

A versatile and ultrasensitive colorimetric detection platform has been developed based on the hybridization chain reaction (HCR)-triggered enzyme cascade amplification in this work. The proposal involves the preparation of two different hairpin DNA strands consisting of the H1, modified with glucose oxidase (GOx-H1) and H2, modified with horseradish peroxidase (HRP-H2). The H1 and H2 were composed of complementary sequence of nucleic acid target (T) and interlaced complementary stem-loop sequences. In the nucleic acid detection, the hybridization of T and its complementary sequence induces the autonomous assembly of GOx-H1 and HRP-H2 through the predictable HCR, accompanied by the formation of GOx/HRP enzyme pairs with a multiple enzymatic cascade. In contrast to the crude mixture of free GOx-H1 and HRP-H2, the catalytic performance of enzyme cascade reaction has been significantly enhanced, which can be determined by monitoring the absorbance change of 2, 2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS2-), a typical substrate with hydrogen peroxide for the HRP. Furthermore, this platform can be utilized in the assay of biological substances by the introduction of corresponding aptamer (Apt), complementary strands (Com), and an assistant hairpin DNA strand (HAssist). In view of the signal amplification of HCR and the enhanced catalytic performance of cascaded enzymes, our colorimetric assay system exhibits excellent sensitivity, and the detection limits have been calculated to be 5.2 fM and 0.8 pM for the nucleic acid target (T as a model) and biological substances (ATP as a model), respectively.