CN102944549B - Electrogenerated chemiluminescence bacterium sensing method and multi-functional probe - Google Patents
Electrogenerated chemiluminescence bacterium sensing method and multi-functional probe Download PDFInfo
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Abstract
The invention belongs to the technical field of electrogenerated chemiluminescence and particularly relates to an electrogenerated chemiluminescence bacterium sensing method and a multi-functional probe. Modified glucose oxidase is gathered on nanogold particles, the multifunctional probe is built, and bacteria are detected through an electrogenerated chemiluminescence method. The glucose oxidase on the probe has double functions of identification and catalysis. First, the glucose oxidase is glycoprotein, carried oligosaccharide can be combined with lectin specificity, the glucose oxidase can catalyze glucose to oxidize to generate hydrogen peroxide, and the hydrogen peroxide can promote electrogenerated chemiluminescence of luminol. In addition, nanogold particles with large specific surface area have an effect of enriching enzyme and can catalyze luminol to emit light. The method is fast, simple, reliable, cheap and high in sensitivity and is used for detecting bacteria.
Description
Technical field
The invention belongs to electrogenerated chemiluminescence technical field, particularly a kind of electrogenerated chemiluminescence bacterium method for sensing and multiprobe.
Background technology
Bacterium is very extensive in occurring in nature distribution, is the important participant of cycle of matter in the earth, closely bound up with the life of the mankind.But the existence of some pathogenic bacteria drastically influence food security, water security, the mankind are made to produce various disease, the health of harm humans.Within such as 2011, caused by enteropathogenic E.Coli in the food poisoning epidemic situation of Europe outburst, it can make people produce severe diarrhea and septicemia, even dead.In addition, the pseudomonas aeruginosa extensively existed can make the many patient's wound infections in hospital, causes abscess, pulmonary infection, bacteremia and septicemia, very harmful.Therefore, the detection of bacterium, Identification and determination have very important significance for the prevention of disease and diagnosis.Along with the progress of science and technology, various new technology is used to the detection of bacterium, from early stage microscopic examination and counting, euzymelinked immunosorbent assay (ELISA), differential pulse voltammetry till now, mass spectrum, polymerase chain reaction, Surface enhanced raman spectroscopy, impedance, QCM (Quartz Crystal Microbalance), surface plasma body resonant vibration etc., often kind of method has all shown unique advantage, but still there is shortcoming, such as most technical operating procedure is loaded down with trivial details, complicated, the instrument used, medicine costly, survey process consuming time longer etc.Therefore, the detection that quick, easy, reliable, cheap, the highly sensitive method of development is used for bacterium is badly in need of.
Electrogenerated chemiluminescence, is called for short ECL, is a kind of reliably sensitive detection method, is more and more paid close attention in analytical chemistry.It is combined with chemiluminescence detection at electrochemical techniques, and having the features such as sensitivity is good, instrument simple, reaction controllability is good, detectability is low, luminescent substance recyclable regenerative, is good Bacteria Detection technology.If combined having identification with the nano particle with catalysis and inrichment with the glucoproteinase of catalysis, build multiprobe, this detection method will be made easier, cheap, and sensitivity is higher.
Glycoprotein to be connected with protein covalency the molecule formed by short oligonucleotide chain, extensively exist in biosome, many enzymes, hormone, agglutinin, antibody etc. are all glycoprotein, these glycoprotein play an important role in vivo, the process such as participate in immunity, substance transportation, secretion, blood coagulation, cell migration, cell are gone back to the nest.In glycoprotein, oligonucleotide chain is made up of sialic acid, mannose, aminoglucose, galactose, fucose, galactosamine etc., is connected with amino acid in the mode of oxygen-glycosidic bond or nitrogen-glycosidic bond.These oligonucleotide chains carry the metabolism whereabouts information of protein, as recognition unit, can make glycoprotein and some other protein-interactings, play its physiological function.Such as cell surface sialic acid can help lymphocyte normally to go back to the nest spleen, and after having excised sialic acid, its liver of then going back to the nest.In addition, the oligonucleotide chain on glycoprotein can also play raising protein stability, keeps protein biological activity, and gives protein special properties, as antiprotease hydrolysis, heat resistanceheat resistant inactivation, frost resistance, lubricity etc.Glucose oxidase is a kind of glycoprotein, surface is containing abundant glycosyl, specific binding can be produced with some agglutinins, glucose oxidase can produce gluconic acid and hydrogen peroxide by oxidizing glucose simultaneously, and hydrogen peroxide can the luminescence of some chemiluminescent substances of catalysis, as luminol, therefore, the identification of glucose oxidase and catalysis can be utilized to build biology sensor.In addition, over the years, golden nanometer particle is widely used in bio-sensing field.On the one hand, nm of gold has catalytic activity, can promote some generations of reacting; On the other hand, it has larger specific surface area and extraordinary biocompatibility, greatly can promote the charge capacity of enzyme and not affect the activity of enzyme.Meanwhile, golden nanometer particle has excellent electron transmission performance, the electron transmission in energy Promotion system between enzyme active center and electrode surface.Therefore, the glucoproteinase and nm of gold with identification and catalysis are combined, easy, reliable, cheap, the highly sensitive sensor of structure is had great importance.
Summary of the invention
Not enough for prior art, the invention provides a kind of electrogenerated chemiluminescence bacterium method for sensing and multiprobe.
The present invention constructs the multiprobe that a kind of glucose oxidase is combined with nm of gold.Modify can carry out agglutinin---the concanavalin A agglutinin of specific binding with the mannose that extensively exists in the glucoproteinase such as bacterium surface and glucose oxidase or glucose in gold electrode surfaces, bacterium and probe can combine with the concanavalin A agglutinin on gold electrode competitively, detect finally by high-sensitive electrogenerated chemiluminescence method.
A kind of electrogenerated chemiluminescence bacterium method for sensing, its concrete steps are as follows:
A. the preparation of probe:
Get 3 milliliters of nano-Au solutions, concentration is 1.87 × 10
-9mole often liter, then add 0.05 ~ 5 milligram of glucose oxidase, stirred at ambient temperature 24 hours; Then under the rotating speed of 10000 rpms centrifugal 10 ~ 20 minutes and spend deionized water, finally use 0.1 ~ 6 milliliter, pH is 7.2 ~ 7.4, and concentration is the sterile phosphate buffer dispersion of 0.01 mole often liter, the gold nanoparticle probe solution of finishing glucose oxidase;
B. the process of gold electrode and assembling:
The gold electrode aluminium oxide polishing grinding of 0.3 micron, and supersound washing in second alcohol and water respectively; Then be in the sulfuric acid solution of 0.5 mole often liter in concentration, under-0.2 ~ 1.65 volts of current potentials, 0.1 volt of sweep velocity per second, sweep 40 circles to activate gold electrode by cyclic voltammetry; Then deionized water dries up with nitrogen after cleaning;
Be 7.2 ~ 7.4 with pH, concentration is that concanavalin A agglutinin is mixed with the solution that concentration is 0.5 ~ 2 milligram every milliliter by the sterile phosphate buffer of 0.01 mole often liter; Get the concanavalin A agglutinin solution that 50 ~ 100 microlitres prepare, adding each 5 lli is respectively the lime chloride that often rises of 10 ~ 20 mMs and manganese chloride solution, the amount of substance of the two is than being 1:1, gold electrode surfaces after activation process is good is immersed in ConA agglutinin solution, leaves standstill modification 14 hours; Then by gold electrode take out, be 7.2 ~ 7.4 with pH, concentration be 0.01 mole often liter sterile phosphate buffer washing after put into again 50 ~ 100 lli be 0.5 ~ 2 milligram every milliliter bovine serum albumin component five solution close, leave standstill 1 hour; Then being taken out by gold electrode, is 7.2 ~ 7.4 with pH, and concentration is the phosphate buffer washing of 0.01 mole often liter; The bacterium dispersant liquid drop getting 5 ~ 10 microlitres is added in gold electrode surfaces, soaks 1 ~ 2 hour under room temperature; Be 7.2 ~ 7.4 with pH, concentration is after the sterile phosphate buffer washing of 0.01 mole often liter, then is immersed in by gold electrode in gold nanoparticle probe solution that 50 ~ 100 microlitres prepare, at room temperature reacts 0.5 ~ 2 hour;
C. electrochemiluminescdetection detection
Be 6.0 ~ 11.0 with pH, concentration is the electrolyte solution of the glucose of the luminol that often rises containing 100 ~ 500 micromoles of the phosphate buffer buffer preparation of 0.1 mole often liter and 0.05 ~ 1 mole often liter; Add in small beaker 2 milliliters configure electrolyte solution, the gold electrode assembled and platinum are built into three-electrode system to electrode and silver-silver chloride reference electrode, and then the chemiluminogenic method of electricity consumption detects; Scanning voltage scope is 0 ~ 0.6 volt, sweep speed be 100 millivolts per second, Photomultiplier tube voltage is 600 volts;
Or according to the preparation of above-mentioned materials ratio, step and detection method.
In described nano-Au solution, the mean grain size of nano Au particle is 10 ~ 50 nanometers.
Described bacterium dispersion liquid is for being 7.2 ~ 7.4 with pH, and concentration is the concentration of the sterile phosphate buffer dispersion of 0.01 mole often liter is 4.17 × 10
4~ 4.17 × 10
8the Escherichia coli of individual bacterium colony every milliliter or pseudomonas aeruginosa solution.
A kind of multiprobe, it fixes multiple glucose oxidase in nano-scale gold particle sub-surface, can amplification detection signal.
The mean grain size of described nano Au particle is 10 ~ 50 nanometers, and has the ability of catalysis luminol luminescence.
Described glucose oxidase has identification and catalysis dual-use function, and glucose oxidase can produce hydrogen peroxide by catalysis glucose oxidase, and the sugar chain on its surface can be combined with concanavalin A agglutinin specific recognition.
Beneficial effect of the present invention is:
Result shows, is 4.17 × 10 based on the electrochemical sensor of concanavalin A agglutinin to pseudomonas aeruginosa and the colibacillary quantitative measurement range of linearity
2~ 4.17 × 10
6individual bacterium colony, detects and is limited to 263 bacterium colonies.Operating process is easy, and sensitivity is better.
The present invention adopts finishing to have the sub-probe of the gold nano of glucose oxidase to detect, and this probe has multi-functional.Whole assembling process is easy, large to the catalytic action of luminol luminescence, makes the sensitivity of detection better.
Accompanying drawing explanation
Fig. 1 is the electrochemiluminescence signal figure of the pseudomonas aeruginosa of different content, and wherein a, b, c, d, e and f curve represents that clump count is 0,417,4.17 × 10 respectively
3, 4.17 × 10
4, 4.17 × 10
5with 4.17 × 10
6time pseudomonas aeruginosa electrochemiluminescence signal figure;
The electrogenerated chemiluminescence response linear graph that Fig. 2 is the pseudomonas aeruginosa of different content shown in Fig. 1;
Fig. 3 is the application schematic diagram of a kind of multiprobe prepared by the present invention in Electrochemiluminescsensor sensor.
Embodiment
The invention provides a kind of electrogenerated chemiluminescence bacterium method for sensing and multiprobe, below in conjunction with the drawings and specific embodiments, the present invention will be further described.
Embodiment 1
Build the electrogenerated chemiluminescence method for detecting Escherichia coli ATCC11775 based on multiprobe:
A. the preparation of probe:
All glass apparatus for the preparation of golden nanometer particle first soak 24 hours with chloroazotic acid, then clean with deionized water.In 250 milliliters of there-necked flasks, add the gold chloride that 92.5 ml deionized water and 2.5 ml concns are 4 grams every milliliter, being heated with stirring to condenser pipe has drop to be back to flask equably.Add the two citric acid monohydrate trisodiums that 5 ml concns are 10 milligrams every milliliter, heat after 15 minutes and stop, then stirring and be cooled to room temperature, preserve under 4 degree celsius temperature.
Getting 3 ml concns before each experiment is 1.87 × 10
-9the nano-Au solution of mole often liter, adds 3 milligrams of glucose oxidases, stirred at ambient temperature 24 hours.Under the rotating speed of 10000 rpms centrifugal 10 minutes, and spend deionized water, be 7.4 with the pH of 1.5 milliliters again, concentration is the sterile phosphate buffer dispersion of 0.01 mole often liter, the finishing gold nanoparticle probe solution of glucose oxidase.
B. the cultivation of bacterium and process
The Escherichia coli ATCC11775 that this experiment adopts is provided by China national nanometer centers.Microbionation in the glass test tube that cultured solution of broth is housed, shaken cultivation 24 hours in the shaking table of 37 degrees Celsius.Then centrifugal 3 minutes separation of bacterial under the rotating speed of 8000 rpms, be 7.4 with pH, concentration is the sterile phosphate buffer washing of 0.01 mole often liter, and centrifuge washing three times is 7.4 with pH afterwards, and concentration is that the sterile phosphate buffer of 0.01 mole often liter disperses.Determine bacterium solubility by the UV, visible light absorbance surveying bacterium dispersion liquid, then be 7.4 with pH, concentration is the bacterium dispersion liquid that the sterile phosphate buffer of 0.01 mole often liter is diluted to each required concentration.
C. the process of gold electrode and assembling
Gold electrode particle diameter is the aluminium oxide polishing grinding of 0.3 micron, respectively supersound washing in second alcohol and water; Then be in the sulfuric acid solution of 0.5 mole often liter in concentration, under the current potential of-0.2 ~ 1.65 volts, 0.1 volt of sweep velocity per second, sweep 40 circles to activate gold electrode by cyclic voltammetry; Then deionized water dries up with nitrogen after cleaning.
Be 7.4 with pH, concentration is that 2 milligrams of concanavalin A agglutinins are mixed with the solution that concentration is 2 milligrams every milliliter by the sterile phosphate buffer of 0.01 mole often liter.Get 100 microlitre concanavalin A agglutinin solution, adding each 5 lli is respectively the lime chloride that often rises of 10 ~ 20 mMs and manganese chloride solution, gold electrode surfaces after activation is immersed in concanavalin A agglutinin solution than being 1:1, leaving standstill reaction 14 hours by the amount of substance of the two.Then being taken out by gold electrode, is 7.4 with pH, and concentration is that bovine serum albumin component five solution putting into 100 microlitre 2 milligrams every milliliter after the sterile phosphate buffer washing of 0.01 mole often liter is closed, and leaves standstill 1 hour.Then being taken out by gold electrode, is 7.4 with pH, and concentration is the sterile phosphate buffer washing of 0.01 mole often liter.The Escherichia coli dispersant liquid drop to be measured getting 10 microlitre desired concns is added in gold electrode surfaces, soaks 2 hours under room temperature.Be 7.4 with pH, concentration is after the sterile phosphate buffer washing of 0.01 mole often liter, then reacts 1 hour under room temperature in the gold nanoparticle probe solution prepared gold electrode being immersed in 100 microlitres.Be finally 7.4 with pH, concentration is the sterile phosphate buffer washing of 0.01 mole often liter, for use in Electrochemical Detection analysis.
D. electrochemiluminescdetection detection
Be 7.86 with pH, concentration is the electrolyte solution of the glucose of the luminol that often rises containing 100 micromoles of the phosphate buffered saline of 0.1 mole every milliliter and 0.1 mole often liter.In small beaker, add 2 milliliters of electrolyte solutions, the gold electrode assembled and platinum are built into three-electrode system to electrode and silver-silver chloride reference electrode, then the chemiluminogenic method of electricity consumption detects.Scanning voltage scope is 0 ~ 0.6 volt, sweep velocity be 100 millivolts per second, Photomultiplier tube voltage is 600 volts.Result shows, is 4.17 × 10 based on the electrochemical sensor of concanavalin A agglutinin to the responsing linear range of Escherichia coli ATCC11775
2~ 4.17 × 10
6individual bacterium colony.
Embodiment 2
Build the electrogenerated chemiluminescence method for detecting pseudomonas aeruginosa ATCC27853 based on multiprobe:
A. the preparation of probe
All glass apparatus for the preparation of golden nanometer particle first soak 24 hours with chloroazotic acid, then clean with deionized water.In 250 milliliters of there-necked flasks, add the gold chloride that 92.5 ml deionized water and 2.5 ml concns are 4 grams every milliliter, being heated with stirring to condenser pipe has drop to be back to flask equably.Add the two citric acid monohydrate trisodiums that 5 ml concns are 10 milligrams every milliliter, heat after 15 minutes and stop, then stirring and be cooled to room temperature, preserve under 4 degree celsius temperature.
Getting 3 ml concns before each experiment is 1.87 × 10
-9the nano-Au solution of mole often liter, adds 3 milligrams of glucose oxidases, stirred at ambient temperature 24 hours.Under the rotating speed of 10000 rpms centrifugal 10 minutes, and spend deionized water, be 7.4 with the pH of 1.5 milliliters again, concentration is the sterile phosphate buffer dispersion of 0.01 mole often liter, the finishing gold nanoparticle probe solution of glucose oxidase.
B. the cultivation of bacterium and process
The pseudomonas aeruginosa ATCC27853 that this experiment adopts is provided by China national nanometer centers.Microbionation in the glass test tube that cultured solution of broth is housed, shaken cultivation 24 hours in the shaking table of 37 degrees Celsius.Then centrifugal 3 minutes separation of bacterial under the rotating speed of 8000 rpms, be 7.4 with pH, concentration is the sterile phosphate buffer washing of 0.01 mole often liter, and centrifuge washing three times is 7.4 with pH afterwards, and concentration is that the sterile phosphate buffer of 0.01 mole often liter disperses.Determine bacterium solubility by the UV, visible light absorbance surveying bacterium dispersion liquid, then be 7.4 with pH, concentration is the bacterium dispersion liquid that the sterile phosphate buffer of 0.01 mole often liter is diluted to each required concentration.
C. the process of gold electrode and assembling
Gold electrode particle diameter is the aluminium oxide polishing grinding of 0.3 micron, respectively supersound washing in second alcohol and water; Then be in the sulfuric acid solution of 0.5 mole often liter in concentration, under the current potential of-0.2 ~ 1.65 volts, 0.1 volt of sweep velocity per second, sweep 40 circles to activate gold electrode by cyclic voltammetry; Then deionized water dries up with nitrogen after cleaning.
Be 7.4 with pH, concentration is that 2 milligrams of concanavalin A agglutinins are mixed with the solution that concentration is 2 milligrams every milliliter by the sterile phosphate buffer of 0.01 mole often liter.Get 100 microlitre concanavalin A agglutinin solution, adding each 5 lli is respectively the lime chloride that often rises of 10 ~ 20 mMs and manganese chloride solution, gold electrode surfaces after activation is immersed in concanavalin A agglutinin solution than being 1:1, leaving standstill reaction 14 hours by the amount of substance of the two.Then being taken out by gold electrode, is 7.4 with pH, and concentration is that bovine serum albumin component five solution putting into 100 microlitre 2 milligrams every milliliter after the sterile phosphate buffer washing of 0.01 mole often liter is closed, and leaves standstill 1 hour.Then being taken out by gold electrode, is 7.4 with pH, and concentration is the sterile phosphate buffer washing of 0.01 mole often liter.The pseudomonas aeruginosa dispersant liquid drop to be measured getting 10 microlitre desired concns is added in gold electrode surfaces, soaks 2 hours under room temperature.Be 7.4 with pH, concentration is after the sterile phosphate buffer washing of 0.01 mole often liter, then reacts 1 hour under room temperature in the gold nanoparticle probe solution prepared gold electrode being immersed in 100 microlitres.Be finally 7.4 with pH, concentration is the sterile phosphate buffer washing of 0.01 mole often liter, for use in Electrochemical Detection analysis.
D. electrochemiluminescdetection detection
Be 7.86 with pH, concentration is the electrolyte solution of the glucose of the luminol that often rises containing 100 micromoles of the phosphate buffered saline of 0.1 mole every milliliter and 0.1 mole often liter.In small beaker, add 2 milliliters of electrolyte solutions, the gold electrode assembled and platinum are built into three-electrode system to electrode and silver-silver chloride reference electrode, then the chemiluminogenic method of electricity consumption detects.Scanning voltage scope is 0 ~ 0.6 volt, sweep velocity be 100 millivolts per second, Photomultiplier tube voltage is 600 volts.Result shows, is 4.17 × 10 based on the electrochemical sensor of concanavalin A agglutinin to the responsing linear range of pseudomonas aeruginosa ATCC27853
2~ 4.17 × 10
6individual bacterium colony.
Claims (3)
1. an electrogenerated chemiluminescence bacterium method for sensing, is characterized in that, concrete steps are as follows:
A. the preparation of probe:
Getting 3 ml concns is 1.87 × 10
-9the nano-Au solution of mole often liter, then adds 0.05 ~ 5 milligram of glucose oxidase, stirred at ambient temperature 24 hours; Then under the rotating speed of 10000 rpms centrifugal 10 ~ 20 minutes and spend deionized water, finally use 0.1 ~ 6 milliliter, pH is 7.2 ~ 7.4, and concentration is the sterile phosphate buffer dispersion of 0.01 mole often liter, the gold nanoparticle probe solution of finishing glucose oxidase;
B. the process of gold electrode and assembling:
Be 7.2 ~ 7.4 with pH, concentration is that concanavalin A agglutinin is mixed with the solution that concentration is 0.5 ~ 2 milligram every milliliter by the sterile phosphate buffer of 0.01 mole often liter; Get the concanavalin A agglutinin solution that 50 ~ 100 microlitres prepare, adding each 5 lli is respectively the lime chloride that often rises of 10 ~ 20 mMs and manganese chloride solution, the amount of substance of the two is than being 1:1, gold electrode surfaces after activation process is good is immersed in concanavalin A agglutinin solution, leaves standstill modification 14 hours; Then by gold electrode take out, be 7.2 ~ 7.4 with pH, concentration be 0.01 mole often liter sterile phosphate buffer washing after put into again 50 ~ 100 lli be 0.5 ~ 2 milligram every milliliter bovine serum albumin component five solution close, leave standstill 1 hour; Then being taken out by gold electrode, is 7.2 ~ 7.4 with pH, and concentration is the phosphate buffer washing of 0.01 mole often liter; The bacterium dispersant liquid drop getting 5 ~ 10 microlitres is added in gold electrode surfaces, soaks 1 ~ 2 hour under room temperature; Be 7.2 ~ 7.4 with pH, concentration is after the sterile phosphate buffer washing of 0.01 mole often liter, then is immersed in by gold electrode in gold nanoparticle probe solution that 50 ~ 100 microlitres prepare, at room temperature reacts 0.5 ~ 2 hour;
C. electrochemiluminescdetection detection
Be 6.0 ~ 11.0 with pH, concentration is the electrolyte solution of the glucose of the luminol that often rises containing 100 ~ 500 micromoles of the phosphate buffer buffer preparation of 0.1 mole often liter and 0.05 ~ 1 mole often liter; Add in small beaker 2 milliliters configure electrolyte solution, the gold electrode assembled and platinum are built into three-electrode system to electrode and silver-silver chloride reference electrode, and then the chemiluminogenic method of electricity consumption detects; Scanning voltage scope is 0 ~ 0.6 volt, sweep speed be 100 millivolts per second, Photomultiplier tube voltage is 600 volts.
2. method according to claim 1, is characterized in that: in described nano-Au solution, the mean grain size of nano Au particle is 10 ~ 50 nanometers.
3. method according to claim 1, is characterized in that: described bacterium dispersion liquid is for being 7.2 ~ 7.4 with pH, and concentration is the concentration of the sterile phosphate buffer dispersion of 0.01 mole often liter is 4.17 × 10
4~ 4.17 × 10
8the Escherichia coli of individual bacterium colony every milliliter or pseudomonas aeruginosa solution.
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| CN106093021B (en) * | 2016-06-03 | 2018-11-30 | 浙江省农业科学院 | The Escherichia coli of acidity regulation and agglutinin identification visualize bio-sensing method |
| CN106916809A (en) * | 2017-02-20 | 2017-07-04 | 南昌大学 | The new method that gram negative pathogenic bacterium is separate in septicemia |
| CN107064118B (en) * | 2017-04-14 | 2020-11-10 | 济南大学 | Construction method of paper-based high-flux photo-electrochemical biosensor |
| CN109350739B (en) * | 2018-11-12 | 2021-11-05 | 苏州大学 | Novel anti-cancer immunologic adjuvant and preparation method thereof |
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