A dual-amplification aptasensor for highly sensitive detection of thrombin based on the functionalized graphene-Pd nanoparticles composites and the hemin/G-quadruplex

In this work, an advanced sandwich-type electrochemical aptasensor for thrombin was proposed by integrating hemin/G-quadruplex with functionalized graphene-Pd nanoparticles composites (PdNPs-RGs). The hemin/G-quadruplex formed by intercalating hemin into thrombin binding aptamer (TBA), firstly acted as a NADH oxidase, assisting the oxidation of NADH to NAD⁺ accompanying with the generation of H₂O₂ in the presence of dissolved O₂. Subsequently, the hemin/G-quadruplex acted as HRP-mimicking DNAzyme that rapidly bioelectrocatalyze the reduction of the produced H₂O₂. At the same time, the Pd nanoparticles supported on p-iodoaniline functionalized graphene were also adopted to catalyze the reduction of H₂O₂. Thus, with the dual catalysis, a dramatically amplified electrochemical signal could be obtained. Besides, the avidin–biotin system for binding aptamer sequences on electrodes not only improved the sensitivity of thrombin analysis but also obtained an acceptable repeatability of the aptasensor. With several factors mentioned above, a wide linear ranged from 0.1 pM to 50 nM was acquired with a relatively low detection limit of 0.03 pM (defined as S/N = 3). These excellent performances provided our approach a promising way for ultrasensitive assay in electrochemical aptasensors.     

A pseudo triple-enzyme electrochemical aptasensor based on the amplification of Pt–Pd nanowires and hemin/G-quadruplex

Our present work aimed at developing a pseudo triple-enzyme cascade electrocatalytic electrochemical aptasensor for determination of thrombin with the amplification of alcohol dehydrogenase (ADH)-Pt–Pd nanowires bionanocomposite and hemin/G-quadruplex structure that simultaneously acted as NADH oxidase and HRP-mimicking DNAzyme. With the addition of ethanol to the electrolyte, the ADH immobilized on the Pt–Pd nanowires catalyzed ethanol to acetaldehyde accompanied by NAD⁺ being converted to NADH. Then the hemin/G-quadruplex firstly served as NADH oxidase, converting the produced NADH to NAD⁺ with the concomitant local formation of high concentration of H₂O₂. Subsequently, the hemin/G-quadruplex acted as HRP-mimicking DNAzyme, bioelectrocatalyzing the produced H₂O₂. At the same time, the Pt–Pd nanowires employed in our strategy not only provided a large surface area for immobilizing thrombin binding aptamer (TBA) and ADH, but also served as HRP-mimicking DNAzyme which rapidly bioelectrocatalyzed the reduction of the produced H₂O₂. Thus, such a pseudo triple-enzyme cascade electrochemical aptasensor could greatly promote the electron transfer of hemin and resulted in the dramatic enhancement of electrochemical signal. As a result, a wide dynamic concentration linear range from 0.2 pM to 20 nM with a low detection limit of 0.067 pM for thrombin (TB) determination was obtained. The excellent performance indicated that our strategy was a promising way for ultrasensitive assays in electrochemical aptasensors.     

基于酶辅助靶标循环的适体传感器灵敏检测中药中黄曲霉毒素B1

该文基于酶辅助靶标循环信号放大策略构建了用于黄曲霉毒素B1(AFB1)高灵敏检测的化学发光适体传感器。以G-四链体/氯化血红素DNA酶为信号分子设计了免标记的适体探针H1-S1和发夹探针H2。适体探针结合目标AFB1,在核酸外切酶I辅助下,触发靶标循环反应产生发夹H1。发夹H1与H2杂交,释放出完整的G-四链体序列,并进一步与氯化血红素结合形成G-四链体/氯化血红素DNA酶。DNA酶通过催化氧化鲁米诺-H2O2化学发光体系产生化学发光信号,实现AFB1的放大检测。在最优实验条件下,化学发光强度与AFB1质量浓度的对数在0.001~100 ng/mL范围内呈良好的线性关系,相关系数(r2)为0.9955,检出限为0.93 pg/mL,回收率为93.7%~107%。该适体传感器操作简单、灵敏度高、特异性好,在黄曲霉毒素污染检测方面具有良好的应用前景。     

Amplified electrochemiluminescent aptasensor using mimicking bi-enzyme nanocomplexes as signal enhancement

In this work, a sandwich-type electrochemiluminescence (ECL) aptasensor for ultrasensitive detection of thrombin (TB) was designed based on mimicking bi-enzyme cascade catalysis to in situ generate coreactant of dissolved oxygen (O₂) for signal amplification. We utilized hollow Au nanoparticles (HAuNPs) as carriers to immobilize glucose oxidase nanoparticles (GOxNPs) and Pt nanoparticles (PtNPs) by electrostatic adsorption. Then, the detection aptamer of thrombin (TBA 2) was immobilized on the PtNPs/GOxNPs/HAuNPs nanocomplexes. Finally, hemin was intercalated into the TBA 2 to obtain the hemin/G-quadruplex structure. The hemin/G-quadruplex was an interesting DNAzyme that commonly mimiced horseradish peroxidase (HRP). Herein, GOxNPs, hemin/G-quadruplex and PtNPs could form mimicking bi-enzyme cascade catalysis system to in situ generate dissolved O₂ as coreactant in peroxydisulfate solution when the testing buffer contained proper amounts of glucose. This method had successfully overcome the disadvantage of difficulty to label the dissolved O₂ and realized the ECL signal amplification. The experiment proved that the aptasensor had good linear relationship on low concentration of TB. The linear range was 1 × 10⁻⁶–10 nM, with a detection limit of 0.3 fM.     

Label-free and amplified electrochemical detection of cytokine based on hairpin aptamer and catalytic DNAzyme

In this work, by incorporating a specific DNAzyme sequence into a hairpin aptamer probe, we describe a label-free and sensitive method for electrochemical detection of cytokines using recombinant human IFN-γ as the model analyte. The hairpin aptamer probes are immobilized on a gold electrode through self-assembly. The presence of IFN-γ opens the hairpin structure and forms the hemin/G-quadruplex peroxidase-mimicking DNAzyme with subsequent addition of hemin. The peroxidase-mimicking DNAzyme catalyzes the electro-reduction of H(2)O(2) and amplifies the current response for IFN-γ detection, which enables the monitoring of IFN-γ at the sub-nanomolar level. The proposed sensor also shows high selectivity towards the target analyte. Our strategy thus opens new opportunities for label-free and amplified detection of different types of cytokines.     

A novel electrochemical aptasensor for highly sensitive detection of thrombin based on the autonomous assembly of hemin/G-quadruplex horseradish peroxidase-mimicking DNAzyme nanowires

In this work, a new signal amplified strategy was constructed based on isothermal exponential amplification reaction (EXPAR) and hybridization chain reaction (HCR) generating the hemin/G-quadruplex horseradish peroxidase-mimicking DNAzyme (HRP-mimicking DNAzyme) nanowires as signal output component for the sensitive detection of thrombin (TB). We employed EXPAR’s ultra-high amplification efficiency to produce a large amount of two hairpin helper DNAs within a minutes. And then the resultant two hairpin helper DNAs could autonomously assemble the hemin/G-quadruplex HRP-mimicking DNAzymes nanowires as the redox-active reporter units on the electrode surface via hybridization chain reaction (HCR). The hemin/G-quadruplex structures simultaneously served as electron transfer medium and electrocatalyst to amplify the signal in the presence of H₂O₂. Specifically, only when the EXPAR reaction process has occurred, the HCR could be achieved and the hemin/G-quadruplex complexes could be formed on the surface of an electrode to give a detectable signal. The proposed strategy combines the amplification power of the EXPAR, HCR, and the inherent high sensitivity of the electrochemical detection. With such design, the proposed assay showed a good linear relationship within the range of 0.1 pM–50 nM with a detection limit of 33 fM (defined as S/N = 3) for TB.     

A repeatable assembling and disassembling electrochemical aptamer cytosensor for ultrasensitive and highly selective detection of human liver cancer cells

In this work, a repeatable assembling and disassembling electrochemical aptamer cytosensor was proposed for the sensitive detection of human liver hepatocellular carcinoma cells (HepG2) based on a dual recognition and signal amplification strategy. A high-affinity thiolated TLS11a aptamer, covalently attached to a gold electrode through Au–thiol interactions, was adopted to recognize and capture the target HepG2 cells. Meanwhile, the G-quadruplex/hemin/aptamer and horseradish peroxidase (HRP) modified gold nanoparticles (G-quadruplex/hemin/aptamer–AuNPs–HRP) nanoprobe was designed. It could be used for electrochemical cytosensing with specific recognition and enzymatic signal amplification of HRP and G-quadruplex/hemin HRP-mimicking DNAzyme. With the nanoprobes as recognizing probes, the HepG2 cancer cells were captured to fabricate an aptamer-cell-nanoprobes sandwich-like superstructure on a gold electrode surface. The proposed electrochemical cytosensor delivered a wide detection range from 1 × 10² to 1 × 10⁷ cells mL⁻¹ and high sensitivity with a low detection limit of 30 cells mL⁻¹. Furthermore, after the electrochemical detection, the activation potential of −0.9 to −1.7 V was performed to break Au–thiol bond and regenerate a bare gold electrode surface, while maintaining the good characteristic of being used repeatedly. The changes of gold electrode behavior after assembling and desorption processes were investigated by electrochemical impedance spectroscopy and cyclic voltammetry techniques. These results indicate that the cytosensor has great potential in disease diagnostic of cancers and opens new insight into the reusable gold electrode with repeatable assembling and disassembling in the electrochemical sensing.     

Multiplexed and amplified chemiluminescence resonance energy transfer (CRET) detection of genes and microRNAs using dye-loaded hemin/G-quadruplex-modified UiO-66 metal–organic framework nanoparticles

Dye-loaded UiO-66 metal–organic framework nanoparticles (NMOFs) modified with catalytic hemin/G-quadruplex DNAzyme labels act as functional hybrid modules for the chemiluminescence resonance energy transfer (CRET) analysis of miRNAs (miRNA-155 or miRNA-21) or genes (p53 or BRCA1). The dye-loaded NMOFs (dye = fluorescein (Fl) or rhodamine 6G (Rh 6G)) are modified with hairpin probes that are engineered to include in their loop domains recognition sequences for the miRNAs or genes, and in their stem regions caged G-quadruplex domains. In the presence of the analytes miRNAs or genes, the hairpin structures are opened, leading, in the presence of hemin, to the self-assembly of hemin/G-quadruplex DNAzyme labels linked to the dye-loaded NMOFs. In the presence of luminol and H₂O₂, the hemin/G-quadruplex DNAzyme labels catalyze the generation of chemiluminescence that provides radiative energy to stimulate the process of CRET to the dye loaded in the NMOFs, resulting in the luminescence of the loaded dye without external excitation. The resulting CRET signals relate to the concentrations of the miRNAs or the genes and allow the sensitive analysis of miRNAs and genes. In addition, the DNA hairpin-functionalized dye-loaded NMOF sensing modules were further applied to develop amplified miRNA or gene CRET-based sensing platforms. The dye-loaded NMOFs were modified with hairpin probes that include in their loop domain the recognition sequences for miRNA-155 or miRNA-21 or the recognition sequences for the p53 or BRCA1 genes. Subjecting the hairpin-modified NMOFs to the respective miRNAs or genes, in the presence of two hairpins H_i and H_j that include in their stem regions caged G-quadruplex subunit domains, results in the analyte-triggered opening of the probe hairpin linked to the NMOFs, and the opened hairpin tethers induce the cross-opening of the hairpins H_i and H_j by the hybridization chain reaction, HCR, resulting in the assembly of G-quadruplex wires tethered to the NMOFs. The binding of hemin to the HCR-generated chains yields hemin/G-quadruplex DNAzyme wires that enhance, in the presence of luminol/H₂O₂, the CRET processes in the hybrid nanostructures. These amplification platforms lead to the amplified sensing of miRNAs and genes. By mixing the Fl- and Rh 6G-loaded hairpin-functionalized UiO NMOFs, the multiplexed CRET detection of miRNA-155, miRNA-21 and the p53 and BRCA1 genes is demonstrated.

Hemin/G-quadruplex DNAzyme-modified metal–organic framework nanoparticles act as functional hybrids for the catalyzed oxidation of luminol by H₂O₂, causing chemiluminescence and activation of chemiluminescence resonance energy transfer to the dye loads.     

Sensitive pseudobienzyme electrocatalytic DNA biosensor for mercury(II) ion by using the autonomously assembled hemin/G-quadruplex DNAzyme nanowires for signal amplification

Herein, a novel sensitive pseudobienzyme electrocatalytic DNA biosensor was proposed for mercury ion (Hg²⁺) detection by using autonomously assembled hemin/G-quadruplex DNAzyme nanowires for signal amplification. Thiol functionalized capture DNA was firstly immobilized on a nano-Au modified glass carbon electrode (GCE). In presence of Hg²⁺, the specific coordination between Hg²⁺ and T could result in the assembly of primer DNA on the electrode, which successfully triggered the HCR to form the hemin/G-quadruplex DNAzyme nanowires with substantial redox probe thionine (Thi). In the electrolyte of PBS containing NADH, the hemin/G-quadruplex nanowires firstly acted as an NADH oxidase to assist the concomitant formation of H₂O₂ in the presence of dissolved O₂. Then, with the redox probe Thi as electron mediator, the hemin/G-quadruplex nanowires acted as an HRP-mimicking DNAzyme that quickly bioelectrocatalyzed the reduction of produced H₂O₂, which finally led to a dramatically amplified electrochemical signal. This method has demonstrated a high sensitivity of Hg²⁺ detection with the dynamic concentration range spanning from 1.0 ng L⁻¹ to 10 mg L⁻¹ Hg²⁺ and a detection limit of 0.5 ng L⁻¹ (2.5 pM) at the 3S_blank level, and it also demonstrated excellent selectivity against other interferential metal ions.     

A sensitive electrochemical aptasensor for ATP detection based on exonuclease III-assisted signal amplification strategy

A target-induced structure-switching electrochemical aptasensor for sensitive detection of ATP was successfully constructed which was based on exonuclease III-catalyzed target recycling for signal amplification. With the existence of ATP, methylene blue (MB) labeled hairpin DNA formed G-quadruplex with ATP, which led to conformational changes of the hairpin DNA and created catalytic cleavage sites for exonuclease III (Exo III). Then the structure-switching DNA hybridized with capture DNA which made MB close to electrode surface. Meanwhile, Exo III selectively digested aptamer from its 3′-end, thus G-quadruplex structure was destroyed and ATP was released for target recycling. The Exo III-assisted target recycling amplified electrochemical signal significantly. Fluorescence experiment was performed to confirm the structure-switching process of the hairpin DNA. In fluorescence experiment, AuNPs–aptamer conjugates were synthesized, AuNPs quenched fluorescence of MB, the target-induced structure-switching made Exo III digested aptamer, which restored fluorescence. Under optimized conditions, the proposed aptasensor showed a linear range of 0.1–20 nM with a detection limit of 34 pM. In addition, the proposed aptasensor had good stability and selectivity, offered promising choice for the detection of other small molecules.     

Label-free and fluorescence turn-on aptasensor for protein detection via target-induced silver nanoclusters formation

A simple turn-on and homogeneous aptasensor, which relies on target induced formation of silver nanoclusters (Ag NCs), was developed for the determination of platelet-derived growth factor B-chain homodimer (PDGF-BB). The aptasensor contains two hairpin DNA probes termed as P1 and P2. P1 consists of the aptamer sequence of PDGF-BB. Meanwhile, P2 contains the Ag NCs nucleation sequence, which is blocked by the hairpin stem region. P1 and P2 can co-exist metastably in the absence of PDGF-BB and maintain hairpin structure. However, in the presence of PDGF-BB, the binding of PDGF-BB with aptamer will result in the hybridization between P1 and P2, and release the Ag NCs nucleation sequence. In this case, Ag NCs can be formed via the reduction of Ag⁺ by NaBH₄. By monitoring the increase in fluorescence intensity, we could detect the target protein with high sensitivity. The detection limit of this aptasensor is 0.37 nM, which is comparable with that of other reported aptasensors. Furthermore, this proposed aptasensor shows high selectivity toward its target protein. Thus, the proposed aptasensor based on target induced formation of Ag NCs could be used as a sensitive and selective platform for the detection of target protein.     

Chemiluminescent and chemiluminescence resonance energy transfer (CRET) detection of DNA, metal ions, and aptamer-substrate complexes using hemin/G-quadruplexes and CdSe/ZnS quantum dots

Nucleic acid subunits consisting of fragments of the horseradish peroxidase (HRP)-mimicking DNAzyme and aptamer domains against ATP or sequences recognizing Hg(2+) ions self-assemble, in the presence of ATP or Hg(2+), into the active hemin-G-quadruplex DNAzyme structure. The DNAzyme-generated chemiluminescence provides the optical readout for the sensing events. In addition, the DNAzyme-stimulated chemiluminescence resonance energy transfer (CRET) to CdSe/ZnS quantum dots (QDs) is implemented to develop aptamer or DNA sensing platforms. The self-assembly of the ATP-aptamer subunits/hemin-G-quadruplex DNAzyme, where one of the aptamer subunits is functionalized with CdSe/ZnS QDs, leads to the CRET signal. Also, the functionalization of QDs with a hairpin nucleic acid that includes the G-quadruplex sequence in a 'caged' configuration is used to analyze DNA. The opening of the hairpin structure by the target DNA assembles the hemin-G-quadruplex DNAzyme that stimulates the CRET signal. By the application of three different sized QDs functionalized with different hairpins, the multiplexed analysis of three different DNA targets is demonstrated by the generation of three different CRET luminescence signals.     

Hemin/G-quadruplex simultaneously acts as NADH oxidase and HRP-mimicking DNAzyme for simple, sensitive pseudobienzyme electrochemical detection of thrombin

For the first time, hemin/G-quadruplex was employed to simultaneously serve as NADH oxidase and an HRP-mimicking DNAzyme for constructing a simple and sensitive pseudobienzyme-amplifying electrochemical aptasensor for thrombin detection.     

Aptamer-based label-free method for hemin recognition and DNA assay by capillary electrophoresis with chemiluminescence detection

An aptamer-based label-free approach to hemin recognition and DNA assay using capillary electrophoresis with chemiluminescence detection is introduced here. Two guanine-rich DNA aptamers were used as the recognition element and target DNA, respectively. In the presence of potassium ions, the two aptamers folded into the G-quartet structures, binding hemin with high specificity and affinity. Based on the G-quartet–hemin interactions, the ligand molecule was specifically recognized with a K _d ≈ 73 nM, and the target DNA could be detected at 0.1 μM. In phosphate buffer of pH 11.0, hemin catalyzed the H₂O₂-mediated oxidation of luminol to generate strong chemiluminescence signal; thus the target molecule itself served as an indicator for the molecule–aptamer interaction, which made the labeling and/or modification of aptamers or target molecules unnecessary. This label-free method for molecular recognition and DNA detection is therefore simple, easy, and effective. Figure A label-free approach to aptamer-based hemin recognition and DNA detection is introduced, which gives great potential for using a small molecule itself as the indicator for molecular recognition and DNA detection thereby avoiding any labeling or modification step     

A sensitive electrochemical aptasensor based on palladium nanoparticles decorated graphene–molybdenum disulfide flower-like nanocomposites and enzymatic signal amplification

In the present study, with the aggregated advantages of graphene and molybdenum disulfide (MoS₂), we prepared poly(diallyldimethylammonium chloride)–graphene/molybdenum disulfide (PDDA–G–MoS₂) nanocomposites with flower-like structure, large surface area and excellent conductivity. Furthermore, an advanced sandwich-type electrochemical assay for sensitive detection of thrombin (TB) was fabricated using palladium nanoparticles decorated PDDA–G–MoS₂ (PdNPs/PDDA–G–MoS₂) as nanocarriers, which were functionalized by hemin/G-quadruplex, glucose oxidase (GOD), and toluidine blue (Tb) as redox probes. The signal amplification strategy was achieved as follows: Firstly, the immobilized GOD could effectively catalyze the oxidation of glucose to gluconolactone, coupling with the reduction of the dissolved oxygen to H₂O₂. Then, both PdNPs and hemin/G-quadruplex acting as hydrogen peroxide (HRP)-mimicking enzyme could further catalyze the reduction of H₂O₂, resulting in significant electrochemical signal amplification. So the proposed aptasensor showed high sensitivity with a wide dynamic linear range of 0.0001 to 40 nM and a relatively low detection limit of 0.062 pM for TB determination. The strategy showed huge potential of application in protein detection and disease diagnosis.     

Highly effective colorimetric and visual detection of nucleic acids using an asymmetrically split peroxidase DNAzyme

G-quadruplex containing peroxidase DNAzyme is a complex of hemin and a single-stranded guanine-rich DNA (hemin-binding DNA aptamer), which is used as an attractive catalytic label for biosensing recently. Therein, the hemin-binding DNA aptamer contains four GGG repeats and can fold into a G-quadruplex structure. In this paper, we have developed a new split mode to divide the hemin-binding DNA aptamer into two parts: one possesses three GGG repeats, and another part possesses one GGG repeat, namely, the 3:1 split mode. The combination of G-quadruplex and hemin binding could be used as a sensitive probe for the identification of single nucleotide polymorphisms by giving a color signal, visible to the naked eye at room temperature. The G-quadruplex containing peroxidase DNAzyme utilizes the 3:1 split mode and can be directly used for the identification of SNPs with a detection limit in the nM range when the matching length of the probe is short enough. When the matching length of the probe is relatively long, another method adding competition sequences to the probe could also operate effectively for the identification of SNPs. The results also suggested that we could detect the signal when the mutation sample was only 5% in the total target DNA with a competition strategy.     

Label-free colorimetric aptasensor for sensitive detection of ochratoxin A utilizing hybridization chain reaction

The combination of high selectivity of aptamer with the peroxidase-mimicking property of DNAzyme has presented considerable opportunities for designing colorimetric aptasensor for detection of ochratoxin A (OTA). The activities of both aptamer (as biorecognition element) and DNAzyme (as signal amplification element) are blocked via base pairing in the hairpin structure. Hybridization chain reaction (HCR) between two hairpin DNAs was employed to further improve the sensitivity of this method. The presence of OTA triggers the opening of the hairpin structure and the beginning of HCR, which results in the release of many DNAzyme, and generates enhanced colorimetric signals, which is correlated to the amounts of OTA with linear range between 0.01 to 0.32 nM, and the limit of detection is 0.01 nM under optimal conditions. OTA in yellow rice wine and wheat flour samples was also detected using this method. We demonstrate that a new colorimetric method for the detection of OTA has been established, which is simple, easy to conduct, label-free, sensitive, high throughput, and cost-saving.     

A label-free DNA reduced graphene oxide-based fluorescent sensor for highly sensitive and selective detection of hemin

G-quadruplex structure aptamer (PS2.M) can capture acridine orange (AO) from reduced graphene oxide (rGO). When the AO-PS2.M/rGO mixture is incubated with hemin, the specific binding of hemin with PS2.M results in a release of AO from PS2.M and return of AO back to rGO. Based on the quenching of fluorescence, the target hemin was detected sensitively and selectively, giving a detection limit of 50 nM.     

Synthetic multivalent DNAzymes for enhanced hydrogen peroxide catalysis and sensitive colorimetric glucose detection

A peroxidase-mimic DNAzyme is a G-quadruplex (G₄) DNA–hemin complex, in which the G₄-DNA resembles an apoenzyme, and hemin is the cofactor for hydrogen peroxide (H₂O₂) catalysis. Twenty-one-mer CatG4 is a well-proven G₄-DNA as well as a hemin-binding aptamer for constituting a DNAzyme. This work studied if a multivalent DNAzyme with accelerated catalysis could be constructed using a multimeric CatG4 with hemin. We compared CatG4 monomer, dimer, trimer, and tetramer, which were prepared by custom oligo synthesis, for G₄ structure formation. According to circular dichroism (CD) analysis, we found that a CatG4 multimer exhibited more active G₄ conformation than the sum effect of equal-number CatG4 monomers. However, the DNAzyme kinetics was not improved monotonically along with the subunit number of a multimeric CatG4. It was the trivalent DNAzyme, trimeric CatG4:hemin, resulting in the rapidest H₂O₂ catalysis instead of a tetravalent one. We discovered that the trivalent DNAzyme’s highest catalytic rate was correlated to its most stable hemin-binding G₄ structure, evidenced by CD melting temperature analysis. Finally, a trivalent DNAzyme-based colorimetric glucose assay with a detection limit as low as 10 μM was demonstrated, and this assay did not need adenosine 5′-tri-phosphate disodium salt hydrate (ATP) as a DNAzyme boosting agent.     

Characterization of G-quadruplex/hemin peroxidase: substrate specificity and inactivation kinetics

Recently, G-quadruplex/hemin (G4/hemin) complexes have been found to exhibit peroxidase activity, and this feature has been extensively exploited for colorimetric detection of various targets. To further understand and characterize this important DNAzyme, its substrate specificity, inactivation mechanism, and kinetics have been examined by comparison with horseradish peroxidase (HRP). G4/hemin DNAzyme exhibits broader substrate specificity and much higher inactivation rate than HRP because of the exposure of the catalytic hemin center. The inactivation of G4/hemin DNAzyme is mainly attributed to the degradation of hemin by H(2)O(2) rather than the destruction of G4. Both the inactivation rate and catalytic oxidation rate of G4/hemin DNAzyme depend on the concentration of H(2)O(2), which suggests that active intermediates formed by G4/hemin and H(2)O(2) are the branch point of catalysis and inactivation. Reducing substrates greatly inhibit the inactivation of G4/hemin DNAzyme by rapidly reacting with the active intermediates. A possible catalytic and inactivation process of G4/hemin has been proposed. These results imply a potential cause for the hemin-mediated cellular injury and provide insightful information for the future application of G4/hemin DNAzyme.