KR20100027831A - Biosensor having screen-printed electrode based on catalytic activity of hydrazine for detecting biomolecule and preparation method thereof - Google Patents

Abstract

PURPOSE: A biosensor for detecting biomolecule using screen print electrode based on hydrazine catalytic activity is provided to reproducibly and sensitively detect biomolecule. CONSTITUTION: A biosensor for detecting biomolecule comprises: a screen print electrode in which surface is coated with electrical conductivity polymer; a dendrimer layer in which the electrical conductivity polymer and amide are bound; a probe fixed on an Au nanoparticle layer; a biotin target probe conjugated with biomolecule; and an avidin target hydrazine conjugated with the biotin.

Description

Biosensor having screen-printed electrode based on catalytic activity of hydrazine for detecting biomolecule and preparation method according to hydrazine catalytic activity

The present invention relates to a biosensor for detecting biomolecules and a method of manufacturing the same, which can detect biomolecules such as DNA and protein easily, sensitively and reproducibly using screen printing electrodes based on the catalytic activity of hydrazine.

Methods of very sensitive detection of DNA and proteins are important in biological assays. To this end, interest in transduction of DNA and protein recognition events has increased. In order to increase the sensitivity of DNA detection, various methods are known using ferrocene-functional cationic polythiophene, functional-liposomes, nanoparticles, structural modification of modified double-stranded DNA, and the like.

On the other hand, in order to increase the sensitivity of protein detection, a number of immunosensor systems using DNA and enzyme markers as amplification materials have been developed. This includes assays based on bio barcodes, immuno-PCR, liposome-PCR, carbon nanotube-induced amplification, paramagnetic beads, gold markers and polymer beads.

Although most of these methods can detect DNA and protein in a very sensitive manner, there are some limitations in practical use due to problems of manufacturing complexity, multi-step reactions, detection procedures, and the like.

For example, specific and sensitive detection methods of proteins using bio barcodes, immuno PCR and liposome PCR have a problem of using complex experimental techniques such as PCR. Therefore, the development of a simple, reliable and high sensitivity detection method is required.

In order to solve the problems of the prior art, the inventors coat an electrically conductive polymer on the surface of the screen printed electrode, and modify the electrode by using a carrier for a hydrazine-labeled unit, thereby simplifying and sensitive DNA and protein based on the catalytic activity of hydrazine. A new biosensor for the detection was produced.

In order to achieve the above object, the present invention is a screen printed electrode surface-coated with an electrically conductive polymer; A dendrimer layer having an amide bond with the electrically conductive polymer; Au nanoparticle layer adsorbed unreacted amine group of the dendrimer layer; A probe fixed to the Au nanoparticle layer; A biomolecule coupled to the probe; A biotin marker probe bound to the biomolecule; And it provides a biosensor for detecting biomolecules, characterized in that consisting of avidin-labeled hydrazine coupled to the biotin.

The electrically conductive polymer used in the biosensor of the present invention is a poly-5,2 ': 5', 2 "-terthiophene-3'-carboxyl having a carboxyl group or an amine group activated with carbodiimide or N-hydroxysuccinimide. Acids are preferred.

In addition, the dendrimer is an amine-terminated dendrimer, and the dendrimer having surface functionality is a spherical nanostructure having a large number of branches, which can immobilize a large amount of Au nanoparticles symmetrically, and have a spherical shape of the dendrimer. Due to the linear amine, unlike the linear polyamine, it provides a completely different environment at the center and periphery of the dendrimer, and compared to the linear polyamine, a large number of amine groups of the densified functional dendrimer can stably fix the Au nanoparticles on the surface of the biosensor. have.

The probe used in the biosensor of the present invention is any one selected from a DNA probe having a thiol group or an antibody probe, and the biomolecule is a protein labeled with biotin or a DNA labeled with biotin.

Hereinafter, with reference to the drawings will be described in more detail the biosensor according to the present invention.

1 is a conceptual diagram of a biosensor for detecting DNA according to the present invention. First, an electrically conductive polymer, for example, poly-5,2 ': 5', 2 "-terthiophene-3'-carboxylic acid (hereinafter, pTTCA) is formed on the screen printed electrode (SPE) by the potential cycle method, and then the carboxylic acid group of the pTTCA is used as an activator, for example, 1-ethyl-3- (3-dimethylaminopropyl) carbodi Activate with mid or N-hydroxysuccinimide.

A pTTCA / DEN modified electrode is prepared by immobilizing an amine-terminated dendrimer on the pTTCA surface by an amide bond between the carboxyl group on the activated pTTCA surface and the amine group of the amine-terminated dendrimer (DEN).

After stably fixing the dendrimer, the pTTCA / DEN modified electrode was immersed in the Au nanoparticle (AuNP) solution to adsorb the unreacted amine group site of the dendrimer with AuNP to prepare a pTTCA / DEN / AuNP electrode combination.

After adsorbing a probe having a thiol group to the AuNP layer of the surface-modified pTTCA / DEN / AuNP electrode combination, each treated with 1-decanethiol solution to block the remaining exposed AuNP surface to prevent nonspecific adsorption.

The target DNA and biotin-labeled probe are sequentially added to the probe having the thiol group adsorbed to the pTTCA / DEN / AuNP electrode combination to hybridize or bind.

The resulting electrode is reacted with a hydrazine label and the electrocatalytic reduction potential of H ₂ O ₂ is varied by a differential pulse voltammetry (DPV) in the potential range of 100 to -350 mV (Ag / AgCl equivalent). Measure

As shown in FIG. 2, a reduction peak with respect to 10 mM H ₂ O ₂ in the presence of 50 pM target DNA was observed at ˜-150 mV, which is indicated by the Av-Hyd marker binding to the complementary duplex and the electrocatalyst of H ₂ O ₂ . It is found to induce redox (2c). The prepared pTTCA / DEN / AuNP electrode combination can be used to detect DNA very sensitively without using an enzyme.

On the other hand, in the presence of a 100-fold excess of non-complementary DNA (5nM, 2b) or untreated DNA (2a), no significant catalytic potential appears in the biosensor. However, in the presence of a 100-fold excess of complementary DNA (5 nM, 2d), a distinct reduction potential appears at -150 mV.

In addition, as shown in FIG. 3A, the peak potential of 10.5 ± 0.7nA with a relative standard deviation of 4.6% in the presence of single-base fault-binding DNA is 6 times lower than that of 58.5 ± 0.9nA, which is the peak potential in the presence of the target DNA. A small amount of single base misbinding DNA hybridizes with a thiol group and a biotin-labeled probe to weaken binding to Av-Hyd.

In addition, as a result of examining the catalytic reduction effect of H ₂ O ₂ by AuNP, the probe-modified electrode having a thiol group was treated with 1-decanethiol before hybridization with the target DNA to block the exposed surface of AuNP (FIG. 3B). No significant change in peak is observed even if not blocked (solid line in FIG. 3B).

Figure 4 relates to the dependence of the electrocatalyst reduction current with the concentration of the target DNA solution, showing a voltammetric hybridization reaction for very low concentrations of target DNA ranging from 50 fM to 7.5 nM.

A wide dynamic range was observed between 50 fM and 7.5 nM, which covers a 6-fold concentration. The detection limit of the target DNA is 30 fM, indicating a 10-fold lower detection limit compared to the enzyme free method used previously.

In addition, Figure 5 shows a conceptual diagram of the protein detection biosensor according to the present invention, by using such a protein detection biosensor can be detected very sensitive antigen antigen.

The anti-human IgG antibody was adsorbed onto the AuNP layer of the pTTCA / DEN / AuNP electrode with the screen-modified electrode prepared in the same manner as the biosensor for DNA detection, and then treated with bovine serum albumin (BSA) to expose the remaining AuNP surface. Blocking prevents nonspecific adsorption.

Anti-human IgG antibodies adsorbed to the pTTCA / DEN / AuNP electrode combinations are subsequently hybridized or bound by adding the desired protein and biotin-labeled probe.

The resulting electrode is reacted with a hydrazine label and the electrocatalytic reduction potential of H ₂ O ₂ is varied by a differential pulse voltammetry (DPV) in the potential range of 100 to -350 mV (Ag / AgCl equivalent). Measure

6A relates to DPV signals measured for solutions containing 5 pg / ml human IgG and those obtained in control experiments. The clear electrocatalytic reduction peak at about -200 mV obtained from 5 pg / ml human IgG indicates successful binding of the Av-Hyd marker to the immunosensor layer (curve b).

If the concentration of human IgG is zero, no background peak is observed between 50 and -450 mV (curve a), so nonspecific binding is negligible.

The effect was examined using thrombin instead of human IgG as a control to investigate nonspecific binding. As a result, when 500 pg / ml thrombin was used instead of 5 pg / ml human IgG as shown in FIG. 6B, no catalytic current was observed (curve i), indicating that nonspecific binding of thrombin was negligible.

In addition, anti-IgG can be covalently bound to the pTTCA layer without the DNE / AuNP layer by forming an amide bond between the carboxyl group of the polymer and the amine group of the antibody. Therefore, the effect of the DEN / AuNP layer on the sensitivity of the strained electrode was examined.

As shown in FIG. 6B, the use of pTTCA / DEN / AuNP electrode combinations (curve iii) instead of the anti-IgG immobilized pTTCA layer (curve ii) resulted in over 50-fold improvement in sensitivity. The improvement of this signal is due to the large amount of AuNP loading provided by the pTTCA / DEN layer, so that a large amount of protein binding Av-Hyd is combined with AuNP, resulting in an improved current.

7A and 7B relate to the dependence of the electrocatalytic reduction current on the concentration of human IgG, wherein the reduction peak increases rapidly, although not linearly, with the concentration of the desired human IgG. A linear dynamic range can be observed at 40 fg / ml to 2.5 ng / ml, with a detection limit of 25 fg / ml.

The detection limit was at least three times lower than that obtained with the conventional method based on AuNP tracer or the most sensitive protein detection method based on conventional methods based on recovery of electroactive products.

In addition, the present invention comprises the steps of coating an electrically conductive polymer having a carboxyl group or an amine group activated on the surface of the screen printed electrode; Fixing an amine-terminated dendrimer to the surface of the electrically conductive polymer; Adsorbing Au nanoparticles to the unreacted amine of the dendrimer; Adsorbing a probe having a thiol group on the surface of the Au nanoparticles; Sequentially combining a biomolecule and a biotin-labeled probe with the probe to prepare a three-component conjugate; Reacting the three-component conjugate with avidin-labeled hydrazine; And it provides a method for producing a biosensor for detecting biomolecules comprising the step of measuring the electrocatalytic reduction potential of hydrogen peroxide by the hydrazine.

The biosensor for detecting biomolecules according to the present invention can detect biomolecules such as DNA and protein easily, sensitively and reproducibly, and the production of biosensors is simple for the development of practical devices such as infection diagnosis, genetic modification confirmation, and forensic investigation. It can be very useful.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the present invention is not limited by these examples.

Example 1 AuNP and Av-Hyd Conjugate Preparation

AuNPs were synthesized by known methods ( Anal. Chem. , 79, 3724, 2007). In other words, were added while 2.5X10 ^-4 M HAuCl ₄ and 2.5X10 ^-4 M tree after a 20ml aqueous solution containing sodium citrate prepared in an Erlenmeyer flask, stirring the fresh 0.1M _{NaBH 4 (ice-cold) 0.6ml} . This solution formed pink particles.

In the UV-visible spectrum and TEM image, the absorption band at 521 nm confirmed that the particle size was ˜3.5 nm (concentration, ˜188 nM).

Hydrazine was labeled with avidin by glutaraldehyde crosslinking. Hydrazine-avidin (Av-Hyd) conjugates were prepared as follows. A mixed solution of hydrazine sulfate [4 mg in 200 μl of 100 mM PBS pH 7.4] and glutaraldehyde solution (25%, 200 μl) was incubated at 25 ° C. for 18 hours.

At this time, the Sephadex G-25 column was equilibrated with 0.9% NaCl to remove excess glutaraldehyde. Then, a solution obtained by dissolving 4 mg of avidin (from egg white) in 400 µl of 0.5 M sodium carbonate buffer (pH 9.5) was added to the mixed solution.

The mixed solution thus obtained was incubated at 4 ° C. for 24 hours, treated with 0.1 ml of 1.0M lysine (neutralized to pH 7.4) to block the remaining active groups, and then dialyzed overnight with PBS (pH 7.4). Finally, the conjugate was filtered using a sterile millipore membrane (0.2 μm), and then the filtrate was stored and used at -20 ° C.

Example 2 pTTCA / DEN / AuNP-electrode assembly preparation

First, the manufacturing process of the screen printed electrode (SPE) used in the present invention is as follows. An Ag conducting layer was printed on a polyethylene base film (10 mm × 30 mm). Each film consisted of 20 strips, each strip defined by an individual electrode.

The silver conductive layer was printed and then cured at 60 ° C. for 20 minutes. Thereafter, the mixed carbon ink and the cellulose-dsDNA working electrode layer (in the control group, only the carbon ink layer was printed on the film) were printed on the base film on which the silver conductive layer was printed.

At this time, the method of printing the carbon ink using a patterned stencils printing technique, the printed carbon ink was printed on the silver conductive layer with a diameter of 1.25 mm. After the carbon ink was printed, it was cured at 60 ° C. for 20 minutes.

PTTCA (poly 5,2 ': 5', 2 "-terthiophene-3'-carboxylic acid), a dendrimer, and an AuNP layer were formed on the prepared screen printed electrode surface as follows.

In the process of surface coating with conductive polymer (pTTCA), di (propylene glycol) methyl ether and tri (propylene glycol) methyl ether are 1: A 1 mM TTCA monomer solution including a solvent mixed at a ratio of 1 was prepared, and the TTCA monomer solution was dropped on the surface of the screen printed electrode, thereby polymerizing and coating the surface of the electrode.

At this time, the TTCA monomer used was synthesized according to a previously known method ( Synth. Met. , 126 , 105, 2002).

The electrode coated with pTTCA was treated with 0.1M phosphate buffer (pH) containing a mixed solution of 10.0 mM 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide [EDC] and 10 mM NHS (N-hydroxysuccinimide) for 6 hours. 7.0) to activate the carboxylic acid group of pTTCA.

Thereafter, incubated in 500 µl of 20% (w / w) dendrimer solution (1: 1) dissolved in methanol at 4 ° C for 12 hours. In this case, an amine-terminated G3 poly (amidoamine) dendrimer [dendri PAMAM (NH ₂ ) ₃₂ ] was used as the dendrimer.

Then, incubated in 500 μl AuNP solution (1:10 in PBS) for 12 hours at room temperature to form a pTTCA / DEN / AuNP layer on the electrode. Thereafter, the electrode was thoroughly washed with 0.1 M phosphate buffer solution (pH 7.0) and dried under nitrogen.

Example 3 Fabrication of DNA Sensor Biosensor

For DNA detection, the previously prepared pTTCA / DEN / AuNP-electrode combination was subjected to a DNA probe (base sequence consisting of 5′-HS (CH ₂ ) ₆ -SEQ ID NO: 1) having 1.0 μM thiol group prepared in 10 mM PBS. Soaked. In order to block the electrode surface that was not treated with AuNP, the probe modified electrode with thiol group was treated with 1-decanethiol solution.

Then, the pTTCA / DEN / AuNP electrode combination in which the DNA probe is immobilized is a target DNA represented by SEQ ID NO: 2, a DNA mismatched by one base represented by SEQ ID NO: 3, and a non-complementary DNA represented by SEQ ID NO: 4 After soaking at 28 ° C. for 4 hours in a solution of an appropriate concentration of each DNA including 50 ° C. and a DNA probe having a thiol group, hybridization was performed with each DNA, and then washed three times with PBS.

The probe solution was labeled with biotin for the cleaning electrode (base sequence consisting of SEQ ID NO: _{5 - (CH 2) 6 -biotin} -3 '; 1.0μM) a three-component combination electrode obtained soaked for a further 28 ℃, 4 hours at It was soaked for 30 minutes at room temperature in the Av-Hyd solution (500 μl) prepared previously. After washing three times with PBS solution, the resulting biosensor was stored at 4 ° C. Electrode surface drying under nitrogen was performed between each step.

The buffer used in the washing and culturing step of the experiment was a solution consisting of 10 mM NaH ₂ PO ₄ , 10 mM Na ₂ HPO ₄ and 275 mM NaCl (10 mM PBS, pH 7.4).

The target DNA was detected in a 5.0 ml cell containing the prepared biosensor, Ag / AgCl standard electrode and platinum-wire counter electrode. DPV was performed by scanning at a potential of 100 to -350 mV compared to Ag / AgCl at a scanning rate of 5 mV / s. Other conditions were as follows: pulse amplitude 100 mV; Sample width 2 ms; Pulse width 50 ms; And pulse period 1000 ms. All electrochemical experiments were performed in degassed 10 mM PBS for 40 minutes by removing nitrogen gas. At this time, the PBS used contained 10 mM H ₂ O ₂ .

As a result, as shown in Figs. 4A and 4B, the detection limit of the target DNA was found to be 30 fM (S / N = 3).

Example 4 Fabrication of Protein Detection Biosensor

For protein detection, the pTTCA / DEN / AuNP-electrode combinations were incubated for 12 hours at 4 ° C. in an anti-human IgG solution (produced in mouse) at 3.5 mg / ml, washed with PBS, and then the surface was washed with 0.1%. Soak in bovine serum albumin (BSA) solution was soaked at 4 ° C. for 30 minutes to minimize nonspecific adsorption. BSA treated electrodes were incubated for 1 hour at 4 ° C. in human IgG solution (450 μl) at various concentrations (40 fg / ml to 2.5 ng / ml).

The washed electrode was immersed in a biotin-labeled anti-human IgG in solution (3.5 mg / ml) at 4 ° C. for 12 hours, and the three-component combination electrode was prepared in the previously prepared Av-Hyd solution (500 μl). Soak for 30 minutes at room temperature. After washing three times with PBS solution, the resulting biosensor was stored at 4 ° C. Electrode surface drying under nitrogen was performed between each step.

The buffer used in the washing and culturing step of the experiment was a solution consisting of 10 mM NaH ₂ PO ₄ , 10 mM Na ₂ HPO ₄ and 275 mM NaCl (10 mM PBS, pH 7.4).

The target protein was detected in a 5.0 ml cell containing the biosensor, Ag / AgCl standard electrode and platinum-wire counter electrode. DPV was performed by scanning at a potential of 0 to -250 mV compared to Ag / AgCl at a scanning rate of 5 mV / s. Other conditions were as follows: pulse amplitude 100 mV; Sample width 2 ms; Pulse width 50 ms; And pulse period 1000 ms. All electrochemical experiments were performed in degassed 10 mM PBS for 40 minutes by removing nitrogen gas. In this case, the PBS used includes 10 mM H ₂ O ₂ .

As a result, as shown in Figs. 7A and 7B, the detection limit of the target protein was found to be 25fg / ml (S / N = 3).

For comparison, the use of 500 ng / ml of thrombin instead of 5 pg / ml of human IgG confirmed that the sensitivity of the biosensor of the present invention was not affected by nonspecific binding of thrombin as shown in FIG. 6A.

On the other hand, in order to compare the sensitivity of the pTTCA / IgG / Hyd electrode and pTTCA / DEN / AuNP / IgG / Hyd electrode on the electrochemical properties of the target biological material, a mixed solution of 10.0 mM EDC and 10 mM NHS The carboxylic acid group was activated by soaking in 0.1 M phosphate buffer (pH 7.0) for 6 hours.

Thereafter, the EDC-treated pTTCA-coated electrode was washed with PBS, and then incubated in 10 mM phosphate buffer solution (pH 7.4) containing 3.5 mg / ml anti-IgG at 4 ° C. for 12 hours. During this step, the amide bond formation between the amine group of the anti-IgG and the carboxyl group of pTTCA causes the anti-IgG to covalently bond with pTTCA. Then 500 μl of Av-Hyd solution was treated for 30 minutes at room temperature.

As a result, using a pTTCA / DEN / AuNP / IgG / Hyd electrode (see iii of FIG. 6B) instead of an anti-IgG fixed pTTCA / IgG / Hyd electrode (see ii of FIG. 6B) resulted in a 50-fold improvement in sensitivity. Was observed.

1 shows a conceptual diagram of a biosensor for detecting DNA according to the present invention,

Figure 2 shows the DPV obtained by analyzing the target DNA and a series of control experiments (a: target DNA untreated, b: 5nM non-complementary DNA treatment, c: 50pM target DNA treatment, d: 5nM target DNA treatment),

Figure 3a shows the DPV obtained by analyzing 5nM target DNA in the presence or absence of single base misbinding DNA (i: untreated DNA, ii: presence of single base misbinding DNA; iii: absence of single base misbinding DNA),

Figure 3b shows a 10pM target DNA analysis DPV using a DNA biosensor (dotted line) after the 1-decanthiol adsorption process (dotted line) and a DNA biosensor (solid line) without the adsorption process, respectively,

Figure 4 shows the DPV analysis of the target DNA of various concentrations using the biosensor for DNA detection according to the present invention (a: 50fM, b: 200fM, c: 750fM, d: 10pM, e: 100pM, f: 1 nM, g: 7.5 nM and h: 100 nM), inset shows detection of 50 fM target DNA,

5 shows a conceptual diagram of a biosensor for protein detection according to the present invention,

6A shows DPV following human IgG untreated (a) and 5 pg / ml human IgG treated (b) on pTTCA / DEN / AuNP / IgG / Hyd electrodes,

6B shows DPV according to 500 pg / ml thrombin treatment (i) instead of human IgG (i), assay using pTTCA / IgG / Hyd electrodes (ii), assay using pTTCA / DEN / AuNP / IgG / Hyd electrodes (iii) Will,

Figure 7a shows a DPV analysis of human IgG at various concentrations using a biosensor for protein detection according to the present invention (a: 0, b: 40fg / ml, c: 250fg / ml, d: 750fg / ml, e: 5 pg / ml, f: 750 pg / ml, g: 2.5 ng / ml, h: 50 ng / ml),

7b shows the dependence of peak current on human IgG concentration.

<110> Institute for research & industry cooperation, Pusan National University <120> Biosensor having screen-printed electrode based on catalytic          activity of hydrazine for detecting biomolecule and preparation          method <130> sp-2008-0027 <160> 5 <170> KopatentIn 1.71 <210> 1 <211> 14 <212> DNA <213> Artificial Sequence <220> <223> capture probe having thiol group <400> 1 gcgcgaaccg tata 14 <210> 2 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> target DNA <400> 2 agcgtaggat agatatagat atacggttcg cgc 33 <210> 3 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> one-base mismatch DNA <400> 3 agcgtaggat agatatagat ataccgttcg cgc 33 <210> 4 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> noncomplementary DNA <400> 4 ttgagcatgc gcattatctg agccagtacc gaatcg 36 <210> 5 <211> 12 <212> DNA <213> Artificial Sequence <220> <223> biotinylated probe <400> 5 tctatcctac gc 12  

Claims (6)

Screen printed electrodes surface-coated with an electrically conductive polymer; A dendrimer layer having an amide bond with the electrically conductive polymer; Au nanoparticle layer adsorbed unreacted amine group of the dendrimer layer; A probe fixed to the Au nanoparticle layer; A biomolecule coupled to the probe; A biotin marker probe bound to the biomolecule; And an avidin-labeled hydrazine coupled to the biotin. The poly-5,2 ': 5', 2 "-terthiophene-3'-carboxyl according to claim 1, wherein the electrically conductive polymer is a carbodiimide or an N-hydroxysuccinimide. Biosensor for detecting biomolecules, characterized in that the acid. The biosensor for detecting biomolecules according to claim 1 or 2, wherein the dendrimer is an amine-terminated dendrimer. The biosensor for detecting a biomolecule according to claim 1 or 2, wherein the probe is any one selected from a DNA probe having a thiol group or an antibody probe. The biosensor for detecting a biomolecule according to claim 1 or 2, wherein the biomolecule is DNA or protein. Coating an electrically conductive polymer having a carboxyl group or an amine group activated on the surface of the screen printed electrode; Fixing an amine-terminated dendrimer to the surface of the electrically conductive polymer; Adsorbing Au nanoparticles to the unreacted amine of the dendrimer; Adsorbing a probe having a thiol group on the surface of the Au nanoparticles; Sequentially combining a biomolecule and a biotin-labeled probe with the probe to prepare a three-component conjugate; Reacting the three-component conjugate with avidin-labeled hydrazine; And Method for producing a biosensor for detecting biomolecules, characterized in that comprising the step of measuring the electrocatalytic reduction potential of hydrogen peroxide by the hydrazine.

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