CN101609062A - Measure the electrochemical method of toxicity effect of multi-walled carbon nanotubes - Google Patents

Measure the electrochemical method of toxicity effect of multi-walled carbon nanotubes Download PDF

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CN101609062A
CN101609062A CNA2009101816444A CN200910181644A CN101609062A CN 101609062 A CN101609062 A CN 101609062A CN A2009101816444 A CNA2009101816444 A CN A2009101816444A CN 200910181644 A CN200910181644 A CN 200910181644A CN 101609062 A CN101609062 A CN 101609062A
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walled carbon
ldh
mwcnts
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moll
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CN101609062B (en
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毕树平
王娜
章福平
程炯佳
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Nanjing University
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Abstract

The present invention relates to a kind of electrochemical method of measuring toxicity effect of multi-walled carbon nanotubes, simple to operate, equipment is light, helps the multi-walled carbon nano-tubes in the environment is monitored.The side of described mensuration toxicity effect of multi-walled carbon nanotubes is as follows: add lactic dehydrogenase (LDH) in MWCNTs solution, after stirring, add pyruvic acid (Pyr) and reduced form nicotinamide adenine dinucleotide disodium salt (NADH), record product nicotinamide adenine dinucleotide (NAD +) reduction peak current that produces at hanging mercury electrode.MWCNTs solution obtains as follows: MWCNTs is placed three (methylol) aminomethane-HCl (Tris-HCl) buffer solution, sonicated 10min again after stirring fast.The present invention can provide new approaches for making up small-sized, portable bioelectrochemical sensor, all has a good application prospect at aspects such as biological redox system of research and environmental pollution monitoring.

Description

Measure the electrochemical method of toxicity effect of multi-walled carbon nanotubes
Technical field
The present invention relates to a kind of electrochemical method of measuring toxicity effect of multi-walled carbon nanotubes.
Background technology
Nanometer technology is the symbol of 21 century science and technology, because the special nature of nano material makes it have broad application prospects in each field.But had a lot of articles that its negative effect is discussed in succession in recent years, wherein the 44th the 6th phase of volume of Carbon is published in instalments the potential hazard of 6 pieces of articles discussion carbon nano-tube to health.Carbon nano-tube (carbon nanotubes, CNTs) be a kind of nano material of fully artificial one-dimentional structure, found by Iijima as far back as 1991, belong to Fu Le (fullerene) carbon system, be a kind of of carbon nanomaterial, favorable mechanical, electricity and magnetic property are arranged, can be divided into Single Walled Carbon Nanotube (SWCNTs) and multi-walled carbon nano-tubes (MWCNTs).Not processed carbon nano-tube is very light, might be by the lung of air to the intelligent.Therefore, Single Walled Carbon Nanotube is also paid close attention to by people at first to environment and biological security.And, can adsorb more poisonous and harmful substance on its surface, thereby produce more complicated biological effect because the surface effect of carbon nanomaterial promptly has huge surface area.For example use carbon nanomaterial (carbon nano-tube and C in recent years 60) remove trace heavy metal in the water body and organic contaminant the research report quite a few, but the carbon nanomaterial that remains in the water body also can exert an influence to human body and vegeto-animal health, and the carbon nanomaterial that has adsorbed heavy metal or organic contaminant has bigger toxicity after moving, transforming in environment.Existing many about carbon nano-tube biological effect and toxicologic research, research object mostly is mouse, rat and cell, analytical approach also mostly is drip in conjunction with mtt assay, found that experimental subjects reaction free and dose dependent occurs, the also oxidative stresss that belong to of its damage more, its cytotoxicity is relevant with particle size, and its cytotoxicity can increase after the acidification.Above-mentioned analytical approach complicated operation, equipment volume is bigger, is unfavorable for whenever and wherever possible environment being monitored, and measures the intoxicating effect of multi-walled carbon nano-tubes.
Summary of the invention
The invention provides a kind of electrochemical method of measuring toxicity effect of multi-walled carbon nanotubes, simple to operate, equipment is light, helps the multi-walled carbon nano-tubes in the environment is monitored.
The side of described mensuration toxicity effect of multi-walled carbon nanotubes is as follows: add lactic dehydrogenase (LDH) in MWCNTs solution, after stirring, add pyruvic acid (Pyr) and reduced form nicotinamide adenine dinucleotide disodium salt (NADH), record product nicotinamide adenine dinucleotide (NAD +) reduction peak current that produces at hanging mercury electrode.
Those of ordinary skill in the art can be according to the selected concrete condition determination of the characteristic of above-mentioned measurement system.Preferred condition determination is: LDH concentration is 1 * 10 -4-1.5 * 10 -4GL -1, Pyr concentration is 6.0 * 10 -4-10.0 * 10 -4MolL -1, NADH concentration is 1.0 * 10 -4-4.0 * 10 -4MolL -1Preferred condition is to be 6.0～10.2 in the pH value, to measure in the nitrogen atmosphere, adopts 0.15-1.0molL -1KCl is as supporting electrolyte.
MWCNTs solution obtains as follows: MWCNTs is placed three (methylol) aminomethane-HCl (Tris-HCl) buffer solution, sonicated 10-20min again after stirring fast.
LDH is a kind of cytoplasm enzyme, extensively is present in the heart, liver, lung and other the multiple histoorgan.One group of tetramer molecule that LDH is made up of H and two kinds of subunits of M almost is present in the vertebrate all cells, is a key enzyme in the biological anaerobic respiration, is the marker enzyme of cytosol.It has five kinds of isodynamic enzymes, and different enzymes has different dynamicss, and in the presence of coenzyme NAD H, LDH can be converted into lactic acid and NAD+ by the catalysis pyruvic acid, and is significant to the energy metabolism of biology.The present invention directly indicates its influence to the LDH activity with MWCNTs and LDH effect back by the variation of measuring NAD+ reduction peak current on the hanging mercury electrode first.With 0.1molL -1Tris-HCl buffer solution (pH=7.5) 25mL places three-electrode system, supporting electrolyte 0.15molL -1KCl, constant temperature (25 ℃), letting nitrogen in and deoxidizing 10min add 35mg L -1MWCNTs (d＜10nm, long 5-15 μ m), stir behind the 2min sonicated 10min more fast, keep nitrogen atmosphere, inject LDH liquid 30 μ L, stirred adding pyruvic acid (8.0 * 10 5 minutes -4MolL -1) and NADH (2.0 * 10 -4MolL -1), NAD under the record differential responses time +Differential pulse volt-ampere reduction peak current i P, NAD +(in d＜10nm)-LDH reaction system, the material that electrochemical response is arranged on the hanging mercury electrode is pyruvic acid and NAD at MWCNT +Before enzymatic reaction begins, have only the substrate pyruvic acid on the position of-1.38V, a reduction peak (pH=7.5) to be arranged.After the reaction beginning, the prolongation along with the reaction time is positioned at-NAD of 0.89V place +Reduction peak increase (pH=7.5) gradually, and the reduction peak of pyruvic acid reduces (as shown in Figure 1) gradually.After reaction finishes, NAD +Reduction peak no longer increase, and the reduction peak of pyruvic acid also no longer changes.At the 5min of initial reaction, i pThe linear increase with the prolongation in reaction time, i after reaction reaches 19min pSubstantially tend towards stability.By the linear relationship in the 3min, can be by i P, NAD +Value and typical curve comparison, NAD +Concentration, calculate the first speed v of reaction then.Adopt the NADH of variable concentrations to measure NAD when writing down 3min then +Reduction peak current.3min internal reaction speed v, NADH concentration and reaction velocity exist following relational expression [Li Yuanzong, Chang Wenbao. biochemical analysis, Beijing: Higher Education Publishing House, 2003, p.6-14.]:
1 v = K m v max × C NADH + 1 v max
Wherein:
V: first speed (the μ molL of reaction -1Min -1);
K m: Michaelis constant (μ molL -1);
v Max: maximum reaction rate (μ molL -1Min -1);
C NADH: the concentration of NADH (μ molL -1)),
With v -1To C NADH -1Press Lineweaver-Burk method mapping, obtain straight line, the kinetic parameter K that can try to achieve the LDH catalyst system and catalyzing according to the slope and the intercept of straight line mAnd v Max, and then the expression enzymatic activity.
With NAD +The size of reduction peak current indicate the variation of LDH activity.Be product because of it on the one hand, can simply, conveniently represent the variation of enzymatic activity, the jump of the reduction peak current of pyruvic acid is bigger on the other hand, instability.
By investigating, confirmed that a small amount of MWCNTs of dissolving also can have remarkable influence to the LDH activity, and should influence irrelevant with the suction-operated of MWCNTs, but a kind of performance of intoxicating effect.In view of LDH at aspects such as medical science, biological study, environment measurings particularly in the vital role aspect the biology indication, the present invention can provide new approaches for making up small-sized, portable bioelectrochemical sensor, all has a good application prospect at aspects such as biological redox system of research and environmental pollution monitoring.This not only helps to understand the influence of MWCNTs to the enzyme reaction process, also helps to study under the varying environment condition influence to enzyme.
In sum, the present invention is simple to operate, and equipment is light, helps the multi-walled carbon nano-tubes in the environment is monitored.
Description of drawings
Fig. 1 is the differential pulse volt-ampere analysis figure that carries out the MWCNTs-LDH system along with the reaction time, and wherein to represent the reaction time successively be 1,3,5,7,9,15,19 to a → h, the volt-ampere curve during 23min;
Fig. 2 is the cyclic voltammetry scan signal graph;
Fig. 3 is the canonical plotting of Pyr differential pulse volt-ampere scanning;
Fig. 4 is NAD +The canonical plotting of differential pulse volt-ampere scanning;
Fig. 5 injects mixing time behind the LDH solution LDH activity influenced figure-LDH reaction system;
Fig. 6 injects mixing time behind the LDH solution LDH activity influenced figure-MWCNTs-LDH reaction system;
What Fig. 7 was the pH value to the LDH activity influences figure-LDH reaction system;
What Fig. 8 was the pH value to the LDH activity influences figure-MWCNTs-LDH reaction system;
Fig. 9 is the influence figure of MWCNTs concentration to the LDH activity in the MWCNTs-LDH reaction system, and wherein MWCNTs concentration is: a-0; B-24mgL -1C-48mgL -1D-90mgL -1E-160mgL -1
Figure 10 is the influence figure of MWCNTs concentration to the LDH activity in the MWCNTs-LDH reaction system, and wherein MWCNTs concentration is: a-0; B-6mgL -1C-12mgL -1D-20mgL -1
Figure 11 is that the length of MWCNTs influences figure-MWCNTs diameter＜10nm to the LDH activity;
Figure 12 is that the length of MWCNTs influences figure-MWCNTs diameter=10-20nm to the LDH activity;
Figure 13 is that the length of MWCNTs influences figure-MWCNTs diameter=40-60nm to the LDH activity;
Figure 14 is that the diameter of MWCNTs influences the long 1-2 μ of figure-MWCNTs m to the LDH activity;
Figure 15 is that the diameter of MWCNTs influences the long 5-15 μ of figure-MWCNTs m to the LDH activity;
Figure 16 is v -1To C NADH -1Rectilinear, a:MWCNTs-LDH system wherein, b:LDH system.
Embodiment
1 instrument and reagent
(1) reagent
Pyruvic acid (biochemical reagents), purity＞98.5%, China Medicine (Group) Shanghai Chemical Reagent Co..Three (methylol) aminomethane (Tris), China Medicine (Group) Shanghai Chemical Reagent Co..β-reduced form nicotinamide adenine dinucleotide disodium salt (β-NADH), β-nicotinamide adenine dinucleotide (β-NAD +) (biochemical reagents), purity＞90.0% is Shanghai uncle bio tech ltd product difficult to understand.OX-heart lactic dehydrogenase (LDH) (10mgmL -1) be Sigma company product, stand-by after diluting 100 times.(diameter is respectively MWCNTs:＜10nm, 10-20nm, 40-60nm; Length: 1-2 μ m and 5-15 μ m), totally six kinds of specifications, purity 〉=99.5%, ash≤0.02wt%, specific surface area 40-300m 2G -1, agraphitic carbon＜3%, Nanometer Port Co., Ltd., Shenzhen.Experiment forms by the configuration of analytical chemistry handbook with the Tris-HCl damping fluid of pH value, and supporting electrolyte is 0.15molL -1KCl.Used chemical reagent is analysis except that specifying pure.Before using, experimental ware cleaned successively with tap water, distilled water and redistilled water again behind (v/v) nitric acid dousing 24h at 1: 10.Experimental water is redistilled water.
(2) instrument
Three-electrode system: hanging mercury electrode is working electrode (S=0.022cm 2, Jiangsu Electrical Analysis Instrument Factory), the platinized platinum electrode is to electrode, saturated calomel electrode is a contrast electrode.CHI660B electroanalyzer (Shanghai occasion China company).Differential pulse volt-ampere parameter is: sweep fast 20mVs -1, pulse height 50mV, pulse width 50ms.KH2200B type numerical control supersonic washer, Kunshan standing grain wound ultrasonic instrument company limited.79-1 type magnetic force heating stirrer (state China instrument plant).Thunder magnetic PHS-2F digital ph (Shanghai Precision Scientific Apparatus Co., Ltd).501 type ultra thermostats (Shanghai experimental apparatus factory).UV3600 ultraviolet-visible pectrophotometer (day island proper Tianjin company).
2. experimental technique
(1) differential pulse voltammetry (being called for short DPV) is measured the influence of MWCNTs to the LDH activity
0.1molL -1Tris-HCl buffer solution 25mL places three-electrode system, and constant temperature (25 ℃), letting nitrogen in and deoxidizing 10min add a certain amount of MWCNTs, stir fast behind the 2min sonicated 10min again, keep nitrogen atmosphere, inject LDH liquid 30 μ L, stirring reaction left standstill 1 minute, added pyruvic acid (8.0 * 10 -4MolL -1) and NADH, NAD under the record differential responses time +Differential pulse volt-ampere reduction peak current i p
(2) Pyr or NAD +Typical curve
0.10molL -1Tris-HCl buffer solution (pH=7.5) 25mL places three-electrode system, and supporting electrolyte is 0.15molL -1KCl, constant temperature (25 ℃), letting nitrogen in and deoxidizing 10min add MWCNTs and stir 2min fast, ultrasonic again 10min.Keep nitrogen atmosphere, injection Pyr or NAD +Solution behind the electromagnetic agitation 5min, left standstill 1 minute, scanned with DPV, and record differential pulse reduction peak current changes Pyr or NAD +The concentration of solution is with Pyr or NAD +Reduction peak current to its concentration map typical curve, obtain Pyr or NAD under aforementioned adding MWCNTs condition +The solution typical curve as calculated, obtains Pyr or NAD +The typical curve equation.
3. absorption is to the influence of experiment
LDH may have absorption as a kind of biomolecule on hanging mercury electrode, and MWCNTs also has very strong adsorbability to LDH.There are five kinds of materials in the LDH enzymatic reaction system: reactant pyruvic acid (Pyr), NADH, product lactic acid (Lac), NAD +And LDH enzyme.For confirm MWCNTs in this experiment to the influence of LDH activity because of its directly to the influence of LDH rather than because the effect of other material in the reaction is caused that we inquire into the problems referred to above.
(1) absorption of LDH on hanging mercury electrode
1. 0.10molL -1Tris-HCl buffer solution (pH=7.5) 25mL places three-electrode system, and supporting electrolyte is 0.15molL -1KCl, constant temperature (25 ℃), letting nitrogen in and deoxidizing 10min, cyclic voltammetry scanning obtains blank Tris curve; Again hanging mercury electrode is placed the 0.10molL that contains LDH -110min among Tris-HCl buffer solution (pH=7.5) 25mL, taking-up is put into three-electrode system with hanging mercury electrode later with secondary water washing, cyclic voltammetry scanning under similarity condition, obtain the Tris-LDH curve, the results are shown in Figure 2, even illustrate that it also is very weak that LDH has absorption, can not impact the LDH enzymatic activity on hanging mercury electrode.
2. the amount of used LDH is few in this experiment, and (pyruvic acid is 8.0 * 10 with other material -4MolL -1, NADH is 2.0 * 10 -4MolL -1) compare little a lot.Therefore, even LDH has absorption on hanging mercury electrode, amount also can be very little, can not influence the LDH enzymatic activity.
(2) MWCNTs is to the absorption of various components in the LDH enzymatic system
1. action time is to Pyr or NAD +Influence
0.10molL -1Tris-HCl buffer solution (pH=7.5) 25mL places three-electrode system, and supporting electrolyte is 0.15molL -1KCl, constant temperature (25 ℃), letting nitrogen in and deoxidizing 10min add MWCNTs (d=10-20nm, 5-15 μ m) and stir 2min, ultrasonic again 10min fast.Keep nitrogen atmosphere, injection Pyr or NAD +Solution behind the electromagnetic agitation 5min, left standstill 1 minute, scanned with DPV, and record differential pulse reduction peak current changes Pyr or NAD +The concentration of solution is with Pyr or NAD +Reduction peak current to its concentration map typical curve, change to add the concentration of MWCNTs again, obtain the series of standards curve, as Fig. 3,4.The result shows that the typical curve that adds the MWCNT front and back almost overlaps, and this has just illustrated that MWCNT is to Pyr and NAD +Almost be not influence.
2. be further checking The above results, by the Calculation of chemical equilibrium stoichiometric proportion before and after the short reaction of each material basic symbols synthase in the reaction system as can be seen.0.1molL -1Tris-HCl buffer solution 25mL (pH=7.5) places three-electrode system, supporting electrolyte 0.15molL -1KCl, constant temperature (25 ℃), letting nitrogen in and deoxidizing 10min add 35mg L -1MWCNTs (d=10-20nm, long 5-15 μ m), stir behind the 2min sonicated 10min more fast, keep nitrogen atmosphere, inject LDH liquid 30 μ L, stirred adding pyruvic acid (8.0 * 10 5 minutes -4MolL -1) and NADH (2.0 * 10 -4MolL -1), record Pyr differential pulse volt-ampere reduction peak current i P, PyrAnd NAD +Differential pulse volt-ampere reduction peak current i P, NAD +, according to adding 35mg L -1MWCNTs (d=10-20nm, long 5-15 μ m) time Pyr and NAD +The typical curve equation calculates, and obtains Pyr and NAD under the differential responses time +Concentration specifically sees Table 1.Reaction begins to add 8.0 * 10 -4MolL -1Pyr and 2.0 * 10 -4MolL -1NADH, calculating in the solution residue after reaction reaches balance has 6.0 * 10 approximately -4MolL -1Pyr, and generated about 2.0 * 10 -4MolL -1NAD +, and the NAD that generates in the course of reaction +With remaining Pyr and be about 8.0 * 10 -4MolL -1, meet the stoichiometric proportion before and after the enzymatic reaction.Proof MWCNTs almost is not influence in the reaction time to other material in the LDH enzymatic reaction system.Wherein:
NAD +Typical curve: i P, NAD +(μ A)=0.00406+2.559C NAD +(mM);
The typical curve of Pyr: i P, Pyr(μ A)=-0.06591+2.610C Pyr(mM).
Table 1
??t(min) ??i p，NAD +(nA) ?C NAD +(mM) ??i p，Pyr(μA) ??C Pyr(mM) ??C NAD + +C Pyr(residue) (mM)
??1 ??50.14 ??0.018 ??1.949 ??0.772 ??0.79
??3 ??188.8 ??0.072 ??1.913 ??0.758 ??0.83
??5 ??247.3 ??0.095 ??1.778 ??0.707 ??0.80
??7 ??320.3 ??0.124 ??1.694 ??0.674 ??0.80
??9 ??296.9 ??0.1114 ??1.387 ??0.557 ??0.67
??12 ??437.8 ??0.17 ??1.628 ??0.649 ??0.82
??15 ??424.3 ??0.164 ??1.434 ??0.575 ??0.74
??17 ??489.7 ??0.19 ??1.551 ??0.62 ??0.81
??19 ??520.1 ??0.2 ??1.514 ??0.605 ??0.81
??23 ??489.1 ??0.19 ??1.475 ??0.59 ??0.78
3. above-mentioned cause influence not quite mainly can be owing to the dynamics reason.From the dynamics angle, remove in the experiment of poisonous and harmful substance palycyclic aromatic in anhydrating etc. at carbon nanomaterial, 1 in the water is removed in CNTs absorption, needs 40min just can reach balance during the 2-dichloro-benzenes, and removes four kinds of haloform (CHCl in the water 3, CHBrCl 2, CHBr 2Cl, CHBr 3) time, when low concentration, all need 450min just can reach balance approximately, and all need 350min to reach balance approximately during high concentration, in this experiment in MWCNTs and the LDH enzymatic system each component be no more than 10min total action time, even each component materials in so short time internal reaction system has absorption on MWCNTs, also just very a spot of, can not exert an influence to experimental result; Thereby with regard to explanation MWCNTs is because it is not to cause because of its absorption to material in the system to the influence of LDH enzyme to the influence of LDH activity, therefore can be with the influence of nano particles such as MWCNTs in the active indicative for environments of LDH.
Comparative examples 1
0.1molL -1Tris-HCl buffer solution (pH=7.5) 25mL places three-electrode system, supporting electrolyte 0.15molL -1KCl, constant temperature (25 ℃), letting nitrogen in and deoxidizing 10min keep nitrogen atmosphere, inject LDH liquid 30 μ L, stir, and add pyruvic acid (8.0 * 10 -4MolL -1) and NADH (2.0 * 10 -4MolL -1), NAD under the record differential responses time +Differential pulse volt-ampere reduction peak current i P, NAD +, the mixing time that change is injected behind the LDH liquid is carried out serial experiment, and the result is as shown in Figure 5.The result shows, LDH is placed in the damping fluid, and behind the stirring different time, the i under certain reaction time P, NAD +Change not clearly i when placing 5min P, NAD +Be maximum, and along with the prolongation of standing time, the LDH activity change is also not obvious.
Embodiment 1
0.1molL -1Tris-HCl buffer solution (pH=7.5) 25mL places three-electrode system, supporting electrolyte 0.15molL -1KCl, constant temperature (25 ℃), letting nitrogen in and deoxidizing 10min add 35mg L -1MWCNTs (d=10-20nm, long 1-2 μ m), stir behind the 2min sonicated 10min more fast, keep nitrogen atmosphere, inject LDH liquid 30 μ L, stir certain hour, add pyruvic acid (8.0 * 10 -4MolL -1) and NADH (2.0 * 10 -4MolL -1), NAD under the record differential responses time +Differential pulse volt-ampere reduction peak current i P, NAD +, the mixing time of change injecting behind the LDH liquid is carried out serial experiment, and with comparative examples 1 in mixing time be that the LDH reaction system of 5min compares, the result is as shown in Figure 6.As can be seen from the figure i behind LDH and the MWCNT effect 5min P, NAD +Decline with respect to not adding carbon nano-tube clearly.10min almost overlaps with two curves of 15min, and two curves significantly and 5min difference arranged.In conjunction with Fig. 5,6 results, inject LDH after, mixing time be decided to be 5 minutes proper.
Comparative examples 2
0.1molL -1Tris-HCl buffer solution 25mL places three-electrode system, supporting electrolyte 0.15molL -1KCl, constant temperature (25 ℃), letting nitrogen in and deoxidizing 10min keep nitrogen atmosphere, inject LDH liquid 30 μ L, stir 5 minutes, add pyruvic acid (8.0 * 10 -4MolL -1) and NADH (2.0 * 10 -4MolL -1), NAD under the record differential responses time +Differential pulse volt-ampere reduction peak current i P, NAD +, the pH value of change buffer solution is carried out serial experiment, and the result is as shown in Figure 7.
Embodiment 2
0.1molL -1Tris-HCl buffer solution 25mL places three-electrode system, supporting electrolyte 0.15molL -1KCl, constant temperature (25 ℃), letting nitrogen in and deoxidizing 10min add 35mg L -1MWCNTs (d=10-20nm, long 1-2 μ m), stir behind the 2min sonicated 10min more fast, keep nitrogen atmosphere, inject LDH liquid 30 μ L, stirred adding pyruvic acid (8.0 * 10 5 minutes -4MolL -1) and NADH (2.0 * 10 -4MolL -1), NAD under the record differential responses time +Differential pulse volt-ampere reduction peak current i P, NAD +, the pH value of change buffer solution is carried out serial experiment, and the result is as shown in Figure 8.
By Fig. 7,8 as can be known, the damping fluid of two kinds of enzymatic systems, close pH, i under the same reaction time P, NAD +Very approaching, this may be because acidity does not have influence or the very little cause of influence to the solubleness of MWCNTs.The pH value is lower than at 6.0 o'clock, and NADH can not stable existence, thereby can't use i P, NAD +The variation of indicative of enzyme activity, and the reduction peak of pyruvic acid also divides at this moment; PH is too high, and afterreaction speed is very slow, reaches the balance time lengthening.So only selected pH to experimentize in the experiment at the Tris-HCl of 6.0-10.2 damping fluid.
Embodiment 3
0.1molL -1Tris-HCl buffer solution 25mL (pH=7.5) places three-electrode system, supporting electrolyte 0.15molL -1KCl, constant temperature (25 ℃), letting nitrogen in and deoxidizing 10min add MWCNTs (d=10-20nm, long 1-2 μ m), stir behind the 2min sonicated 10min more fast, keep nitrogen atmosphere, inject LDH liquid 30 μ L, stir adding pyruvic acid (8.0 * 10 5 minutes -4MolL -1) and NADH (2.0 * 10 -4MolL -1), NAD under the record differential responses time +Differential pulse volt-ampere reduction peak current i P, NAD +, the addition of change MWCNTs is carried out serial experiment, and the result is shown in Fig. 9,10.The result shows: add i after a spot of MWCNT aqueous solution P, NAD +Peak current changes apparent in view, i P, NAD +Just begin to have significant difference to the 12min after-current, may be because the very little cause of the solubleness of MWCNT in water, but only finely dispersed MWCNT also can exert an influence to the LDH activity, and MWCNT (d=10-20nm) the aqueous solution after-current that adds more amount changes obvious relatively.Two groups of data all illustrate with the big more influence to the LDH activity of MWCNT concentration obvious more.
Embodiment 4
0.1molL -1Tris-HCl buffer solution 25mL (pH=7.5) places three-electrode system, supporting electrolyte 0.15molL -1KCl, constant temperature (25 ℃), letting nitrogen in and deoxidizing 10min add 35mg L -1MWCNTs, stir behind the 2min sonicated 10min more fast, keep nitrogen atmosphere, inject LDH liquid 30 μ L, stirred adding pyruvic acid (8.0 * 10 5 minutes -4MolL -1) and NADH (2.0 * 10 -4MolL -1), NAD under the record differential responses time +Differential pulse volt-ampere reduction peak current i P, NAD +, change diameter and the length of MWCNTs, carry out serial experiment, and with equal experiment condition under, the LDH reaction system that does not add MWCNTs compares, the result is shown in Figure 11-15.
By Figure 11-13 as can be known, the MWCNT of length 1-2 μ m is bigger to the influence of LDH activity than the MWCNT of length 5-15 μ m to the influence of LDH activity, and that diameter is the MWCNTs of two kinds of length of 40-60nm is relatively very little to the difference of LDH activity influence.
By Figure 14,15 as can be known, for the carbon nano-tube of length 1-2 μ m, the short more MWCNTs of diameter is big more to the influence of LDH activity; And for the carbon nano-tube of length 5-15 μ m, the MWCNTs of three different-diameters is similar to the influence of LDH activity.Under this experiment condition the MWCNTs of short (the 1-2 μ m) of length, different-diameter to the LDH activity to influence difference bigger.
Embodiment 5
0.1molL -1Tris-HCl buffer solution 25mL (pH=7.5) places three-electrode system, supporting electrolyte 0.15molL -1KCl, constant temperature (25 ℃), letting nitrogen in and deoxidizing 10min add 35mg L -1MWCNTs (d=10-20nm, long 5-15 μ m), stir behind the 2min sonicated 10min more fast, keep nitrogen atmosphere, inject LDH liquid 30 μ L, stirred adding pyruvic acid (8.0 * 10 5 minutes -4MolL -1) and NADH, NAD during record reaction 3min +Differential pulse volt-ampere reduction peak current i P, NAD +, the concentration of change NADH is carried out serial experiment.By i P, NAD +Compare with typical curve, obtain C NAD +,, obtain reacting speed just divided by reaction time 3min.With v -1To C NADH -1Press Lineweaver-Burk method mapping, obtain straight line (shown in Figure 16 cathetus a), the kinetic parameter K that can try to achieve the MWCNTs-LDH system according to the slope and the intercept of straight line mAnd v MaxSame condition does not add MWCNTs, experimentizes, and maps (shown in Figure 16 cathetus b), calculates the kinetic parameter K of LDH system mAnd v MaxThe results are shown in Table 2.
Table 2
Figure G2009101816444D00081
As can be seen from Figure 16, pH=7.5, MWCNT meets state of conflict inhibition mechanism to the inhibition mechanism of LDH in the time of 25 ℃.Although experiment condition is not quite similar, from K that data are tried to achieve mAnd v MaxThe order of magnitude and document [Yuan Qinsheng. modern zymetology, Shanghai: East China publishing house of University of Science and Technology, 2001, p.31-44.] result in is the same, thereby the experimental result that we are described is rational.As can be seen from Table 2, add MWCNT to v 0, v MaxAlmost not influence, but K mRelatively change greatly, increase to 125 μ M by 97, because K mBe dissociation constant, the activity of hour enzyme is big more more for its value, that is to say to add the activity that has influenced LDH behind the MWCNT, and its activity has been reduced.

Claims (6)

1. electrochemical method of measuring toxicity effect of multi-walled carbon nanotubes, it is characterized in that in multi-walled carbon nano-tubes solution, adding lactic dehydrogenase, after stirring, add pyruvic acid and reduced form nicotinamide adenine dinucleotide disodium salt, the reduction peak current that record product nicotinamide adenine dinucleotide produces at hanging mercury electrode.
2. the electrochemical method of mensuration toxicity effect of multi-walled carbon nanotubes as claimed in claim 1 is characterized in that according to the i in the 3min P, NAD +Linear with the time, by i P, NAD +Value and typical curve comparison after, calculate the first speed v of reaction, according to 3min internal reaction speed v, there are following relational expression in NADH concentration and reaction velocity:
1 v = K m v max × C NADH + 1 v max
Wherein:
V: first speed (the μ molL of reaction -1Min -1);
K m: Michaelis constant (μ molL -1);
v Max: maximum reaction rate (μ molL -1Min -1);
C NADH: concentration (the μ molL of reduced form nicotinamide adenine dinucleotide disodium salt -1)),
With v -1To C NADH -1Mapping obtains straight line, tries to achieve K mAnd v Max, the activity of expression lactic dehydrogenase.
3. the electrochemical method of mensuration toxicity effect of multi-walled carbon nanotubes as claimed in claim 1 is characterized in that condition determination is: the lactic dehydrogenase enzyme concentration is 1 * 10 -4-1.5 * 10 -4GL -1, pyruvic acid concentration is 6.0 * 10 -4-10.0 * 10 -4MolL -1, reduced form nicotinamide adenine dinucleotide disodium salt concentration is 1.0 * 10 -4-4.0 * 10 -4MolL -1
4. the electrochemical method of mensuration toxicity effect of multi-walled carbon nanotubes as claimed in claim 1 is characterized in that being 6.0～10.2 in the pH value, measures in the nitrogen atmosphere, adopts 0.15-1.0molL -1KCl is as supporting electrolyte.
5. the electrochemical method of mensuration toxicity effect of multi-walled carbon nanotubes as claimed in claim 1 stirs 5-15min after it is characterized in that adding lactic dehydrogenase in multi-walled carbon nano-tubes solution.
6. as the electrochemical method of each described mensuration toxicity effect of multi-walled carbon nanotubes among the claim 1-5, it is characterized in that multi-walled carbon nano-tubes solution obtains as follows: the multi-walled carbon nano-tubes mother liquor is placed three (methylol) aminomethane-HCl buffer solution, sonicated 10-20min again after stirring fast.
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