CN104388528A - Bacteria detection method based on supramolecular recognition and application thereof - Google Patents
Bacteria detection method based on supramolecular recognition and application thereof Download PDFInfo
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- CN104388528A CN104388528A CN201410689764.6A CN201410689764A CN104388528A CN 104388528 A CN104388528 A CN 104388528A CN 201410689764 A CN201410689764 A CN 201410689764A CN 104388528 A CN104388528 A CN 104388528A
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Abstract
The invention relates to a bacteria detection method based on supramolecular recognition. By adopting carborane derivatives or doxorubicin having supramolecular recognition function and stable electrochemical activity as bacterial marker probe molecules, bacterial liquid is dropwise added on an electrode, the marker probe molecules are immediately dropwise added and specifically bound with bacteria, and then differential impulse response signals are detected; or by using a bacterial suspension as an electrolyte and adopting carborane derivatives or doxorubicin having supramolecular recognition function and stable electrochemical activity as bacterial marker probe molecules, the marker probe molecules are added into the bacterial suspension and specifically bound with bacteria and then an alternating current impedance curve is detected. By the bacteria detection method, viable counting, identification of bacterial species as well as the differentiation of drug-resistant strains from sensitive strains in clinical samples can be achieved, the use of expensive immunological reagent or detection kits is avoided and the bacteria detection method has the advantages of strong detection universality, high speed, high sensitivity, good specificity and high detection efficiency.
Description
Technical field
The present invention relates to a kind of method of detecting bacterium based on Supramolecular Recognition and application thereof.
Background technology
Malignant bacteria is serious harm human health " stealthy killer " always.Need the research of the science of carrying out as live bacterial count, taxonomic history, sensitivity etc. in order to reduce it to the harm of the mankind.The most frequently used at present and relative viable count method is accurately agar plate colony counting method, this method is by cultivating 24 hours after Sample Dilution to suitable multiple, bacterium is cultivated and forms discrete bacterium colony, then manually count.Discriminating bacteria kind then will be carried out a series of biochemical test to realize after above-mentioned cultivation.This method not only detection time long, also waste a large amount of human and material resources.The research of the method for quick of bacterium just started to be studied as far back as the sixties in 20th century, and sustainable development always till now.The method for quick of current bacterium can be divided into following a few class: the detection of living cells count, instrument, detection of nucleic acids etc.Though aforesaid method shortens detection time to some extent, required immunoreagent is expensive and toxicity is comparatively large usually, and required instrument is expensive too, and complicated operation, specificity and poor selectivity.
Electrochemistry is the science of related law in mutual conversion between research electric energy and chemical energy and conversion process.Electrochemical test method has: l. is simple, can measure changing the electrical parameter easily measured between the chemical parameters being difficult to measure.2. highly sensitive, even the material change of trace also can be measured by the change of electric current or electricity.3. tell to act charitably in fact, utilize high-precision feature, micro-reacting weight can be detected, and it is carried out quantitatively.Electrochemistry differential pulse voltammetry has highly sensitive: reversible electrode reaction substance sensitive Du Keda lO.7mol/l, irreversible electrode reaction substance sensitive Du Keda lO-Omol/l; Resolving power is strong; Selectivity is strong: before allowing, the amount of discharging substance is large, the high 50000 times of also not interference measurements of the concentration ratio test substance concentration of front discharging substance.Alternating-current impedance is also electrochemical impedance spectroscopy (EIS), it be a kind of with the sine wave potential of little amplitude (or electric current) for disturbing signal, little on the impact of system, and approximate linear between disturbance and the response of system, and this becomes simple with regard to making the digital processing of measuring result.
Molecular recognition is the interested problem of AC always.The various function such as change of colour-change, electrochemical signals is there will be with the formation of supramolecular complex.Be expected to accordingly set up highly selective, sensitive Molecular identification methods fast.
Carborane derivative is the polyhedral boranes cluster compound formed by boron and carbon, under water, oxygen and other various conditions, have very high chemical stability.Carborane is easy to chemically derived, and its boron hydrogen summit is easy to electrophilic substitution reaction occurs, and highly basic can also capture the proton on hydrocarbon summit.The peculiar property of carborane makes the much-talked-about topic that caborane compounds becomes materials chemistry, biological chemistry is paid close attention to jointly of design and synthesizing new.
Summary of the invention
The invention provides the method for Applied Electrochemistry method rapid detection bacterium, can realize live bacterial count, bacterial species is differentiated and Resistant strain in clinical sample and the differentiation of sensitive strain, detection universality is strong, and speed is fast, highly sensitive, specificity is good, and detection efficiency is high; Avoid and use expensive immunoreagent or detection kit; Less demanding to sample, does not need through complex process, all can carry out under the sample conditions of complexity; Equipment used can realize microminiaturization and portability.
The method of described Applied Electrochemistry method rapid detection bacterium is, application Supramolecular Recognition and there is the chemically active carborane derivative of stable electrical or Zorubicin as bacterial identification thing probe molecule, electrode drips bacterium liquid, immediately drip marker probe molecule and bacterium specific combination, then detect differential pulse response signal.
Preferably, the differential pulse spectrum utilizing single standard strain suspensions to obtain carrys out the kind of discriminating bacteria; Or the value of the differential pulse spike potential utilizing single standard strain suspensions to obtain is to differentiate the drug-resistant intensity height of analysis of clinic pathogenic microorganism; Or the value of the differential pulse peak current utilizing single standard strain suspensions to obtain is to calculate the amount of bacterium.Described single standard bacterial strain is preferably streptococcus aureus, intestinal bacteria, Acinetobacter bauamnnii, Klebsiella pneumonia or Proteus mirabilis.
The method of another kind of Applied Electrochemistry method rapid detection bacterium is, with bacteria suspension as electrolytic solution, application Supramolecular Recognition and there is the chemically active carborane derivative of stable electrical or Zorubicin as bacterial identification thing probe molecule, in bacteria suspension, add marker probe molecule and bacterium specific binding, then detect alternating-current impedance curve.
Preferably, the system impedance spectrum utilizing single standard strain suspensions to obtain contrasts with gained resistance value, the kind of discriminating bacteria; Or the electrochemical impedance value utilizing single standard strain suspensions to obtain is to calculate the amount of target bacteria.Described single standard bacterial strain is preferably streptococcus aureus, intestinal bacteria, Acinetobacter bauamnnii, Klebsiella pneumonia or Proteus mirabilis.
The method of another kind of Applied Electrochemistry method rapid detection bacterium is, on electrode modify survey bacterium and cultivate, application Supramolecular Recognition and there is the chemically active carborane derivative of stable electrical or Zorubicin as bacterial identification thing probe molecule. make the bacterium specific binding that detection marker probe molecule and electrode are modified, then detection differential pulse voltammetry signal.
Preferably, the differential pulse spectrum utilizing single standard strain suspensions to obtain carrys out the kind of discriminating bacteria; Or the value of the differential pulse spike potential utilizing single standard strain suspensions to obtain is to differentiate the drug-resistant intensity height of analysis of clinic pathogenic microorganism; Or the value of the differential pulse peak current utilizing single standard strain suspensions to obtain is to calculate the amount of bacterium.Described single standard bacterial strain is preferably streptococcus aureus, intestinal bacteria, Acinetobacter bauamnnii, Klebsiella pneumonia or Proteus mirabilis.
Preferably, when applying above-mentioned electrochemical method rapid detection bacterium, selected range of frequency is 102Hz-108Hz.
The present invention utilizes different probe molecules and bacterium specific binding, causes the difference of electrochemical response signal to come the kind of bacterial detection, quantity or drug-resistant intensity.Concrete operation can be: the electrochemical response signal of detection probes molecule is as blank value, and the electrochemical response signal then after detection probes molecule and bacterium specific binding, compares with blank value.The response spectrogram that bacterium liquid to be measured and aforementioned single standard strain suspensions obtain is contrasted, thus the kind of acquisition bacterial detection, quantity or drug-resistant intensity.
Voltammetric measuring be adopt electrochemical workstation with by a working electrode, the three-electrode system that forms electrode and reference electrode.Working electrode adopts the electrode such as carbon dioxide process carbon electrode or tin indium oxide, and be platinum filament to electrode, reference electrode is filamentary silver, forms common three-electrode system.
In three-electrode system, only need take a small amount of bacterium liquid and identify that molecular composition detects liquid, utilize differential pulse method to obtain the response spectrogram of single bacterium.
On electrode, measure or modify on electrode the method that then bacterium uses probe molecule to be combined with it after measurement scheme can use bacterium to mix with probe molecule.For the former, there is quicker more instant advantage.
Beneficial effect of the present invention: (1) several probe molecule all has specificity and the susceptibility of height, to strains tested streptococcus aureus, intestinal bacteria, Acinetobacter bauamnnii, Klebsiella pneumonia, Proteus mirabilis etc. have good detection of active.(2) unrelated probe molecule and nanocomposite formulations thereof have well detect separating capacity to two kinds of streptococcus aureuses (susceptibility and resistance), can be distinguished by observation peak current and spike potential and impedance etc.
Electrochemical method rapid detection bacterium is adopted also to be in the exploratory stage at present both at home and abroad.
Embodiment
EXAMPLE l
The preparation of 1.1 probe molecule solutions: several probe molecule derivative is mixed with high density mother liquor with PBS respectively, then to be diluted to concentration respectively with PBS be 1*10
-4mol/l, 4*10
-4mol/l, 8*10
-4mol/l, 12*10
-4mol/l.Medicine probe is in-4 DEG C of preservations.Before experiment, probe medicament is taken out and shakes up, carry out electrochemistry experiment.
1.2 experimental strains and cultural method: reference culture: intestinal bacteria (ATCC8739), streptococcus aureus (CGMCCI. 89), Klebsiella pneumonia (ATCC700600), Acinetobacter bauamnnii (ATCC19606), Proteus mirabilis (ATCC12453) etc.Clinical drug-resistant bacterial strain: streptococcus aureus (SA321), Klebsiella pneumonia (KP450), Acinetobacter bauamnnii (AB135), Proteus mirabilis (PM102).LB liquid nutrient medium: 1% (w/v) Tryptones, 0. 5% (w/v) yeast extract, 1% (w/v) sodium-chlor, after high pressure steam sterilization 30min, adjusts pH to 7.2-7.4 with the NaOH of 2mol/ml.LB agar plate is add 0.5% (w/v) agar powder in LB liquid nutrient medium, after high pressure steam sterilization, is cooled to 50 DEG C of a kind of rhyme scheme in Chinese operas serving as the prelude to a complete score for voices, condensation and get final product.Cultural method: fall from a small amount of mattress of picking 4 DEG C of solid slant culture bases preserved with transfering loop, be inoculated in solid LB media plane, after cultivating 24h in 37 DEG C of thermostat containers, with transfering loop picking 3-5 colony lift in sterile test tube, dilute with LB substratum and mix, adjustment bacterial concentration to 1.0 × 10
5-5.0 × 10
5cFU/ml makes MIC mensuration bacteria suspension.
Get ITO electrode, be cut to the strip of 0.5*5.Ocm, respectively with acetone, dehydrated alcohol, redistilled water clean dry up for subsequent use.Doxorubicin solution 15ul is dripped on electrode, makes it sprawl on the area of 0.5*0.5cm, add electrode and reference electrode, obtain DPV curve.This curve test as a comparison in blank test.
The bacterium liquid lOul getting modulated good concentration drips on electrode, and dripping lOul concentration is subsequently 4*10
-4the probe solution of mol/l, obtains relevant electric active molecule probe and the differential pulse voltammetry curve after different bacterium effect.Find out that the peak value of different strain is in different positions by curve, phase extent is enough to the naked eye find out therebetween.Relevant peaks electric current and the spike potential of the electroactive probe molecule obtained under the effect of different strain also have obvious difference.With not having the blank test of bacterium liquid to compare, the position at peak and size have obvious change.
Claims (5)
1. based on the method for detecting bacterium of Supramolecular Recognition, it is characterized in that, apply Supramolecular Recognition and there is the chemically active Zorubicin of stable electrical as bacterial identification thing probe molecule, electrode drips bacterium liquid, immediately drip marker probe molecule and bacterium specific combination, then detect differential pulse response signal.
2. as claimed in claim 1 based on the application of the method for detecting bacterium of Supramolecular Recognition, it is characterized in that, the differential pulse spectrum utilizing single standard strain suspensions to obtain carrys out the kind of discriminating bacteria.
3. as claimed in claim 1 based on the application of the method for detecting bacterium of Supramolecular Recognition, it is characterized in that, the value of the differential pulse spike potential utilizing single standard strain suspensions to obtain is to differentiate the drug-resistant intensity height of analysis of clinic pathogenic microorganism.
4., as claimed in claim 1 based on the application of the method for detecting bacterium of Supramolecular Recognition, it is characterized in that, the value of the differential pulse peak current utilizing single standard strain suspensions to obtain is to calculate the amount of bacterium.
5. the application of the method for detecting bacterium based on Supramolecular Recognition as described in any one of claim 2-4, is characterized in that, described single standard bacterial strain is streptococcus aureus, intestinal bacteria, Acinetobacter bauamnnii, Klebsiella pneumonia or Proteus mirabilis.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106353493A (en) * | 2016-09-21 | 2017-01-25 | 成都测迪森生物科技有限公司 | Ochratoxin test rod |
| CN110146572A (en) * | 2019-06-06 | 2019-08-20 | 西北大学 | A kind of electrochemical AC impedance biosensor and preparation method thereof detecting CD44 albumen |
-
2014
- 2014-11-26 CN CN201410689764.6A patent/CN104388528A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106353493A (en) * | 2016-09-21 | 2017-01-25 | 成都测迪森生物科技有限公司 | Ochratoxin test rod |
| CN110146572A (en) * | 2019-06-06 | 2019-08-20 | 西北大学 | A kind of electrochemical AC impedance biosensor and preparation method thereof detecting CD44 albumen |
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