CN110373448A - A kind of new method monitoring nano material bacteriostatic activity - Google Patents

A kind of new method monitoring nano material bacteriostatic activity Download PDF

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CN110373448A
CN110373448A CN201910744170.3A CN201910744170A CN110373448A CN 110373448 A CN110373448 A CN 110373448A CN 201910744170 A CN201910744170 A CN 201910744170A CN 110373448 A CN110373448 A CN 110373448A
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cuprous oxide
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廖艳娟
黄在银
汤焕丰
覃小芳
韦慧
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Guangxi University for Nationalities
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Abstract

The invention discloses a kind of new methods for monitoring nano material bacteriostatic activity, pass through the heat of micro- calorimeter real time on-line monitoring Escherichia coli Growth metabolic process, dynamic information, heat, dynamics data based on reaction process realize cuprous oxide to the fungistatic effect and Cu of Escherichia coli2The research of influence and mechanism of the O dimensional effect to fungistatic effect provides a kind of new method to study the antifungal mechanism of nano material.

Description

A kind of new method monitoring nano material bacteriostatic activity
Technical field
The present invention relates to nano material bacteriostatic activity research field, and in particular to a kind of monitoring nano material bacteriostatic activity New method.
Background technique
Nano cuprous oxide (Cu2O) due to its Strong oxdiative ability, high catalytic activity, excellent stability and strong absorption The advantages such as the ability of molecular oxygen become one of the hot spot material on the ground that receives much attention, and are widely used in organic catalysis synthesis, light is urged Change, CO oxidation, sensing, antibacterial etc..The chemical reactivity of usual nano material is directly decided by that its pattern, size and surface are former The factors such as son reconstruct.That is, the different crystal faces of nano material, various sizes of surface can have differences, and its surface energy Size determine nano material reactivity.Wang etc. is found to have the octahedra nano cuprous oxide of eight { 111 } crystal faces Tool is apparently higher than there are six the cube nano cuprous oxide in { 100 } face to Escherichia coli inhibitory effect, this is octahedral shape The result of stronger electrostatic force interaction between looks and Escherichia coli.Padmavathy etc. compares copper oxide and cuprous oxide pair Escherichia coli antifungal mechanism, discovery be different from copper oxide formed living radical mechanism, cuprous oxide be due to monovalence copper from It is sub to cause Escherichia coli lethal with amino acids formed copper (I) peptide complex.Although the antibacterial pattern effect of cuprous oxide and Its mechanism obtains greater advance, but less for the research of its antibacterial dimensional effect.Since the size of cuprous nano crystalline substance is got over Small, surface can be higher, causes surface chemical reaction activity higher.Therefore, nano material Cu is studied2The scale of O and antibacterial effect The structure answered is imitated to the great meaning of research for understanding cuprous oxide antifungal mechanism.
The method of microorganisms growth metabolism at present, such as measuring the UV-VIS spectrophotometry of metabolite With the high performance liquid chromatography or Liquid Chromatography-Mass Spectrometry analyzed for intermediate product, a certain moment, a certain is mostly detected The measuring method of the discontinuous of state, off-line type, it is difficult in real time, the growth metabolism of on-line monitoring microorganism.Modern micro hot skill Art directly acquires the change procedure due to having the variation of high-precision, the micro heat of real-time monitoring system in high sensitivity In-situ heat, dynamic information.And the difference of the metabolite, energy variation in different microbial metabolisms, it determines Every kind of microorganism has it specifically to grow finger-print.Therefore, Microcalorimetric method is widely used in measuring chemical reaction process Thermodynamics and kinetics, monitor microorganism growth metabolism process.Nevertheless, nano cuprous oxide is to Escherichia coli size The heat of effect, dynamics thermometric analysis have no more report.
Summary of the invention
To solve the above problems, the present invention provides a kind of new methods for monitoring nano material bacteriostatic activity.
To achieve the above object, the technical scheme adopted by the invention is as follows:
A kind of new method monitoring nano material bacteriostatic activity, using micro- calorimeter real-time monitoring cuprous oxide to large intestine bar The fungistatic effect of bacterium, specifically comprises the following steps:
S1, in the sample cell of the micro- calorimeter of RD 496-CK 2000, respectively by 1.34mL culture medium, 160 μ L difference rulers The Cu of very little various concentration2O is placed in the 2mL glass small casing on micro- calorimeter sample cell upper layer, by the Liquid Culture of 0.1mL bacterium solution Base is placed in the big glass bushing of the 15mL of lower layer, then is put into togerther in stainless steel tube, and stainless steel lid is covered, by stainless steel sample Product pond is carefully placed into micro- calorimeter；
S2,1.6mL LB culture medium is added in reference cell as control, corresponding response parameter, constant temperature to 35 is set DEG C, after baseline stability, being broken with cable release makes LB culture medium in the tubule of upper layer and cuprous oxide solution under in glass tube E.coli cell in the big pipe of layer, which contacts, to be mixed, the heat of micro- automatic real-time perfoming reaction process of calorimeter, dynamics data acquisition, Heat production power (P-t) curve of E.coli cell growth metabolism, time 34546s when being successively recorded in 35 DEG C；
The S3, (C by calculating compared under different conditionI, 50) measure cube cuprous oxide fungistatic effect；
S4, by comparing E.coli cell and cube cuprous oxide (k-c), (tG- c) and (Pmax- c) relationship, obtain Influence of the dimensional effect of cube cuprous oxide to fungistatic effect.
It is worth noting that, measuring bacterium solution OD value before measurement every time, bacterial clump is determined by standard curve fit equation Number, so that bacterium solution bacterial population used is about 10 every time8CFU/mL。
Further, cuprous oxide is best antibacterial having a size of 42nm, and the optimum allowable concentrations of cuprous oxide are 120 μ g mL-1。
The invention has the following advantages:
Real-time monitoring, heat, power based on reaction process are carried out to the antibacterial process of cuprous oxide by micro- calorimeter technology Data are learned, realize cuprous oxide to the fungistatic effect and Cu of Escherichia coli2Influence and mechanism of the O dimensional effect to fungistatic effect Research, provide a kind of new method to study the antifungal mechanism of nano material.
Detailed description of the invention
Fig. 1 is four size cube Cu2What SEM spectrum of the XRD spectrum of O.
In figure: (a) four size cube Cu2The XRD spectrum of O, (b) four size cube Cu2The SEM spectrum of O.
Fig. 2 is thermography (P-t) curve of 35 DEG C of Escherichia coli.
Fig. 3 is four size various concentration size cube nanometer Cu at 35 DEG C2Thermography of the O to E.coli cytosis Figure；In figure: (a) 42nm；(b)55nm；(c)67nm；(d) 116nm, wherein (arrange) be corresponding in turn to by wave crest from top to bottom blank；40μg·mL-1；60μg·mL-1；80μg·mL-1；100μg·mL-1；120μg·mL-1。
Fig. 4 is E.coli cell growth rate constant k and cube nanometer Cu2The relationship of O concentration c.
Fig. 5 is E.coli cell generation time tGWith cube nanometer Cu2The relationship of O concentration c.
Fig. 6 is E.coli cell maximum heating power PmaxWith cube nanometer Cu2The relationship of O concentration c.
Fig. 7 is 35 DEG C of four kinds of size various concentration cube nanometer Cu2Inhibiting rate curve of the O to Escherichia coli.
Specific embodiment
The present invention is described in detail combined with specific embodiments below.Following embodiment will be helpful to the technology of this field Personnel further understand the present invention, but the invention is not limited in any way.It should be pointed out that the ordinary skill of this field For personnel, without departing from the inventive concept of the premise, various modifications and improvements can be made.These belong to the present invention Protection scope.
Embodiment
1.1 reagents and instrument
Strain: Escherichia coli (test by Escherichia coli, Guangxi Universities microorganism and plant resources utilization emphasis Room) culture medium (LB culture medium): the NaCl of the tryptone of 10g, the yeast extract of 5g, 10g are dissolved in 1000mL distilled water In, solution ph is adjusted to 7；In 121 DEG C, 1.034 × 105Pa sterilizing 30min.
Salzburg vitriol crystal (CuSO4·5H2O, AR, Sinopharm Chemical Reagent Co., Ltd.), ascorbic acid (C6H8O6, AR, Guangdong Xilong Chemical Co., Ltd), trisodium citrate (C6H5Na3O7·2H2O, AR, the western Gansu Province chemical industry in Guangdong Limited liability company), sodium hydroxide (NaOH, AR, Sinopharm Chemical Reagent Co., Ltd.), potassium chloride (KCl, 99.99%, Aladdin Reagent Company)
X-ray diffractometer (XRD, Ultima IV type, Rigaku Co., Ltd.), field emission scanning electron microscope (FE-SEM, JEOL JSM-6700F, Jeol Ltd.), ultraviolet-visible spectrophotometer (UV-Vis, Cary 60, Anjelen Sci. & Tech. Inc, the U.S.), micro- calorimeter (RD 496-CK 2000, Mianyang Zhong Wu thermal-analysis instrumentation Co., Ltd).
The preparation of 1.2 cube nano cuprous oxides
The present embodiment prepares four various sizes of cube nano cuprous oxides using solwution method, in 30 DEG C of water bath conditions Under, by the amount (respectively 0.5ml 0.3M, 0.5ml 0.6M, 0.5ml 0.9M, the 0.5ml that control trisodium citrate 1.2M), 200ML deionized water and 0.5mL, X molL are being filled-1(X=0.3,0.6,0.9 and 1.2) trisodium citrate 0.5mL, 1.2molL are sequentially added in beaker-1 CuSO4Solution, 0.5mL, 4.8molL-1NaOH solution and 0.5mL, 1.2mol·L-1Ascorbic acid, standing after reacting and carrying out 30min settles precipitating, then uses deionized water and dehydrated alcohol respectively Centrifuge washing each 3 times, single point that 42nm, 55nm, 67nm and 116nm size were prepared at 60 DEG C of vacuum ovens dry 6 hours Dissipate cube nano cuprous oxide
The microcalorimetric method bacteriostatic experiment of 1.3 cube cuprous oxide
Before carrying out the experiment of low-grade fever amount, by joule heating effect and measurement KCl water in dissolution enthalpy (1:1110, mKCl: Mde-water calorimeter) is calibrated, (17.792 ± 0.029) kJmol-1 and standard value (17.524 ± 0.028) kJ are measured mol-1It is close, illustrate that instrument is accurate and reliable.
In the sample cell of the micro- calorimeter of RD 496-CK 2000, respectively by 1.34mL culture medium, 160 each concentration of μ L Cu2O(Cu2O final concentration is respectively 40,60,80,100,120 μ gmL-1) it is placed in the 2mL glass on micro- calorimeter sample cell upper layer In small casing, the fluid nutrient medium of 0.1mL bacterium solution (is measured bacterium solution OD value before measurement every time, passes through standard curve fit equation Bacteria colony count is determined, so that bacterium solution bacterial population used is about 10 every time8CFU/mL) it is placed in the big glass bushing of the 15mL of lower layer In, then be put into togerther in stainless steel tube, stainless steel lid is covered, stainless steel sample pond is carefully placed into micro- calorimeter；Joining It is used as control than 1.6mL LB culture medium is added in pond, corresponding response parameter is set, constant temperature is to 35 DEG C, after baseline stability, The E.coli made in the culture medium of the LB in the tubule of upper layer and cuprous oxide solution and the big pipe of lower layer in glass tube is broken with cable release Cell contact mixes, the heat of micro- automatic real-time perfoming reaction process of calorimeter, dynamics data acquisition, when being successively recorded in 35 DEG C Heat production power (P-t) curve of E.coli cell growth metabolism, time 34546s.
Results and discussion
The characterization of 2.1 cuprous oxide
Fig. 1 (a) is Cu2X-ray diffractometer (X-ray Powder diffractometer, XRD) map of O, diffraction Peak and cube Cu2O standard card JPCDS card (No.05-0667) unanimously, 2 θ be 29.9 °, 37.0 °, 42.6 °, 62.4 ° and Diffraction maximum at 74.4 corresponds respectively to Cu2(110), (111), (200), (220) and (311) crystal face of O；Each diffraction maximum point It is sharp and have no impurity peaks, show that crystal form is single, crystal is purer.Fig. 1 (b) is Cu2Scanning electron microscope (the scanning of O Electron microscope, SEM) figure.As seen from the figure, Cu2O is in four various sizes of cubics, uniform particle diameter, list Dispersion, morphological rules, surface is smooth and corner angle are clearly demarcated.By Nano Measurer to nanometer Cu2The electron microscope of O is counted, and is led to It crosses size distribution histogram and shows corresponding Cu2O distribution concentrate and epigranular, average-size be followed successively by (42 ± 6), (55 ± 7), (67 ± 8) and (116 ± 17) nm.
The Thermogram of 2.2 Bacillus coli cells normal growths
Fig. 2 is thermography (P-t) curve of Bacillus coli cells under the conditions of 35 DEG C, as shown, the curve first stage For lag phase (I), after baseline is walked surely, the small glass bushing for breaking sample cell upper layer is equipped with E.coli by culture medium injection lower layer The big glass bushing of cell, the phase bacterial slow growth, quantity has no too big variation, therefore thermal change is basically unchanged.Second Stage is the first logarithmic growth phase (II), and bacterium is entering logarithmic growth phase after the preparation of the substance of lag phase, and with two Index increase bacterial number, bacterium component intracellular largely synthesizes at this time, constantly releasing heat.Phase III is raw for the second logarithm (III) for a long time, as seen from the figure, since nutriment is gradually consumed, and bacterium is still constantly increasing, and leads to the second growth logarithm It is slow that the appearance of phase and its speed of growth grow logarithmic phase compared with first, thus the heat released also accordingly is reduced.Therefore, 2 are showed A logarithmic growth phase.With the variation of nutrient digestion and growing environment condition, culture medium no longer supports bacterium schizogamy, Rate of bacterial growth is zero and enters stationary phase (IV), and bacterial growth rate is equal to the death rate at this time.Decline phase (V) is finally entered, With nutriment exhausts, toxic metabolic products accumulation, bacterial metabolism activity reduce, aging simultaneously there is self-dissolving, dead bacterium number with 2nMode increases, and the heat of releasing gradually decreases, until close to 0 microwatt (μ W).
The growth rate constant k and generation time t of E.coli cellG
Escherichia coli Growth is balanced growth in logarithmic phase, and each components in certain proportion of cell increases, and cell quantity is at 2nIncrease Add.Therefore the growth of E.coli cell meets mathematical model in logarithmic phase:
Nt=N0·2n (1)
logNt=logN0+n·log2 (2)
In formula: N0For starting when culture medium in cell number, NtBacterial number when for t, n are to increase after time t Generation number.Assuming that the thermal power perseverance that single bacterium discharges during growth metabolism is W, then
NtW=N0·W·2n (3)
Enable Pt=NtW, P0=N0W, then
Pt=P0·2n (4)
Both sides take logarithm to obtain simultaneously:
ln(Pt)=ln (P0·2n) (5)
Data derivation in Fig. 2 is deformed into (lnP-t) curve, wherein the exponential phase of growth of E.coli cell is linear increment Area, slope are the generation time t of the exponential phase of growth of E.coli cell under respective conditionsGInverse.E.coli cell Generation time tGDirectly determine its rate of rise, inverse is exponential phase of growth average growth rate, and detailed process is such as Under:
ln(Pt)=ln (P0)+nln2 (6)
Fitting a straight line slope are as follows:
Known average growth rate is increased generation number in the bacterium unit time, is indicated with k, then
Pass through 5 repetition measuring Escherichia coli normal growth rate constant k=(0.053838 ± 0.000192) min-1, coefficient R is all larger than 0.997, illustrates that experimental result has good reproducibility and correlation.As shown in table 1.Passage Time is shown in Table 2.
1 microcalorimetric method of table measures Escherichia coli normal growth rate constant (n=5)
Influence of the 2.3 different size cube cuprous oxide to Escherichia coli
Fig. 3 (a-d) is cube Cu of 35 DEG C of Escherichia coli in four kinds of size various concentrations2(P-t) under O effect is bent Line.In the cube nanometer Cu of four size various concentrations2Under O effect, the heat score-curve trend of E.coli cell is consistent, only One logarithmic growth phase, and thermal power P peak value (Pmax) is below the bacterium of normal growth, in the slope of logarithmic growth phase (k) different, it shows in identical size cube Cu2Under O effect, PmaxIncrease with k with concentration and reduces.By Fig. 3 (a) as it can be seen that Work as Cu2When the partial size of O is 42nm, with the raising of concentration, the power of E.coli cell institute heat production is smaller, until when concentration increases To 120
μg·mL-1When, when the maximum thermal power released is 30.987 μ W, only normal growth after system reaction about 6000s 1/8 times of maximum thermal power, growth rate constant is only 0.024, is 1/1.5 times of normal growth speed per hour rate constant, shows Cuprous oxide bactericidal agent restrained effectively the growth of E.coli cell in system.In Fig. 3 (b-c), as cube aoxidizes The increase of cuprous size inhibits the activity of E.coli cell growth lower, and concentration is got in the cuprous oxide bactericidal agent of same size Greatly, sterilizing activity is better.
2 35 DEG C of Escherichia coli of table are in four kinds of size various concentration cube nanometer Cu2The lower thermodynamics ginseng grown of O effect Number
In conjunction with formula (8) and (2), growth rate constant k/ln2 is ln (Pt/P0)-t slope of a curve.Exponential phase of growth E.coli cell metabolism is most fast, and unit time heat production is faster, therefore k value is also bigger at this stage.Therefore, if after bacteriostatic agent is added The k of exponential growth is smaller, then illustrates that fungistatic effect is better.By with blank control, find same size cube aoxidize Under cuprous effect, as the concentration and growth rate constant k value of cuprous oxide are negatively correlated (Fig. 4), i.e. cuprous oxide pair The growth of E.coli cell has the effect of inhibiting.
As seen from Figure 5, by with blank control, find in exponential phase of growth, the generation time of E.coli cell and bottom The concentration of cuprous oxide is positively correlated in object, dense with cuprous oxide under the cube cuprous oxide effect of same size The increase of degree, E.coli cell quantity, which doubles the required time, to be increased, and the growth that cuprous oxide inhibits becomes its growth Slowly.In addition, size is smaller, to the inhibitory effect of E.coli cell growth and breeding under the cuprous oxide effect of same concentration Better, the longest generation time is in 42 nm, 120 μ gmL-1When the generation time be 42.493min.
Fig. 6 is stage exponential phase of growth, is aoxidized in maximum thermal power caused by E.coli cell growth metabolism and substrate The relational graph of cuprous concentration, the maximum thermal power and substrate oxidation that E.coli cell growth and breeding generates as seen from the figure are cuprous Concentration is also negatively correlated, under the cube cuprous oxide effect of same size, with the increase of the concentration of cuprous oxide, The thermal power that E.coli cell generates is smaller, illustrates that its growth metabolism of Bacillus coli cells is more and more slower.In addition, same dense Under the cuprous oxide effect of degree, size is smaller, better to the inhibitory effect of E.coli cell growth and breeding.In 42nm, 120 μ g mL-1When thermal power only have 30.987 μ w.
The relationship of 2.4 cuprous oxide dimensional effects and Escherichia coli inhibiting rate
In order to further analyze cube cuprous oxide bactericidal agent to the growth of E.coli cell, E.coli is being obtained Cell is after the growth rate constant under the effect of various concentration cube cuprous oxide bactericidal agent, according to the following formula:
Wherein, k0And k1Respectively Escherichia coli non-oxidation is cuprous has and has growth rate constant existing for cuprous oxide. The difference of growth rate constant is inhibiting rate compared to normal growth rate when normal growth rate constant and addition bacteriostatic agent.Root Mlc (the C that inhibiting rate is 50% is calculated according to growth inhibition ratio I and bacteriostatic agent concentration c curveI, 50), as semilethal rate Concentration.By comparing (the C under different conditionI, 50) and measure its fungistatic effect, it is found that concentration is smaller and illustrates that bacterium is quicker to it Sense, fungistatic effect are better.
By calculating (the C compared under different conditionI, 50) cube cuprous oxide fungistatic effect is measured, find cube The smaller bacterium of cuprous oxide concentration is more sensitive to it, and fungistatic effect is better.It is sub- by comparing E.coli cell and cube oxidation Copper (k-c), (tG- c) and (Pmax- c) relationship discovery, the continuous increase of the nano cuprous oxide of same size with concentration, suppression The effect of Escherichia coli Growth breeding processed is better, when concentration is 120 μ gmL-1When almost inhibit the lifes of Escherichia coli It is long；When the various sizes of cuprous oxide bacteriostatic agent of same concentration acts on Escherichia coli, nano cuprous oxide size is smaller, suppression Bacterium activity is higher, and cuprous oxide is best antibacterial having a size of 42nm.This illustrates the dimensional effect of cube cuprous oxide to antibacterial effect The negatively correlated trend of fruit provides theoretical basis to prepare the antibacterial research of smaller szie cuprous oxide, and develops a kind of monitoring The new method of nano material bacteriostatic activity.
Specific embodiments of the present invention are described above.It is to be appreciated that the invention is not limited to above-mentioned Particular implementation, those skilled in the art can make a variety of changes or modify within the scope of the claims, this not shadow Ring substantive content of the invention.In the absence of conflict, the feature in embodiments herein and embodiment can any phase Mutually combination.

Claims (5)

1. a kind of new method for monitoring nano material bacteriostatic activity, it is characterised in that: aoxidized using micro- calorimeter real-time monitoring sub- Fungistatic effect of the copper to Escherichia coli.
2. a kind of new method for monitoring nano material bacteriostatic activity as described in claim 1, it is characterised in that: utilize micro heat The heat of instrument real-time perfoming reaction process, dynamics data acquisition, the production of E.coli cell growth metabolism when being successively recorded in 35 DEG C Thermal power (P-t) curve, time 34546s.
3. a kind of new method for monitoring nano material bacteriostatic activity as described in claim 1, it is characterised in that: including walking as follows It is rapid:
S1, in the sample cell of the micro- calorimeter of RD 496-CK 2000, respectively not by 1.34mL culture medium, 160 μ L difference sizes With the Cu of concentration2O is placed in the 2mL glass small casing on micro- calorimeter sample cell upper layer, and the fluid nutrient medium of 0.1mL bacterium solution is set It in the big glass bushing of the 15mL of lower layer, then is put into togerther in stainless steel tube, covers stainless steel lid, by stainless steel sample pond It is carefully placed into micro- calorimeter；
S2, in reference cell be added 1.6mL LB culture medium as control, corresponding response parameter is set, constant temperature to 35 DEG C, to After baseline stability, being broken with cable release makes LB culture medium in the tubule of upper layer and cuprous oxide solution and the big pipe of lower layer in glass tube In E.coli cell contact and mix, the heat of micro- automatic real-time perfoming reaction process of calorimeter, dynamics data acquisition are continuous to remember Record heat production power (P-t) curve of the E.coli cell growth metabolism at 35 DEG C, time 34546s；
The S3, (C by calculating compared under different conditionI, 50) measure cube cuprous oxide fungistatic effect；
S4, by comparing E.coli cell and cube cuprous oxide (k-c), (tG- c) and (Pmax- c) relationship, obtain cube Influence of the dimensional effect of body cuprous oxide to fungistatic effect.
4. a kind of new method for monitoring nano material bacteriostatic activity as claimed in claim 3, it is characterised in that: every time before measurement Bacterium solution OD value is measured, bacteria colony count is determined by standard curve fit equation, so that bacterium solution bacterial population used is about every time 108CFU/mL。
5. a kind of new method for monitoring nano material bacteriostatic activity as described in claim 1, it is characterised in that: cuprous oxide is most Good antibacterial having a size of 42nm, the optimum allowable concentrations of cuprous oxide are 120 μ gmL-1。
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