CN205720081U - A kind of 16SrRNA electrochemica biological chip - Google Patents
A kind of 16SrRNA electrochemica biological chip Download PDFInfo
- Publication number
- CN205720081U CN205720081U CN201620156838.4U CN201620156838U CN205720081U CN 205720081 U CN205720081 U CN 205720081U CN 201620156838 U CN201620156838 U CN 201620156838U CN 205720081 U CN205720081 U CN 205720081U
- Authority
- CN
- China
- Prior art keywords
- probe
- chip
- 16srrna
- self
- detection
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Abstract
The open a kind of 16SrRNA electrochemica biological chip of the utility model, relates to biomedicine, Electrochemical Detection and unimolecule self assembly field.This chip includes building unimolecule Iy self-assembled layer on the chip surface, and chip surface has chip micropore；Being respectively fixed with capture probe and detection probe in described chip micropore, capture on described capture probe has pathogen 16S rRNA target molecule；Described detection probe forms 16S rRNA molecular probe identification layer.It has the beneficial effects that: use nm of gold to detect probe with the nano combined substance markers of SWCN, utilizes biotin and Avidin height single-minded and biological stability, is used for linking probe and nano-complex particle；Bacterium has advantage simple, rapid, that testing cost is low, highly sensitive, for instruct clinical rational drug use provide by force, strong support.
Description
Technical field
The utility model relates to biomedicine, Electrochemical Detection and self-assembling technique field, specifically a kind of 16SrRNA electrochemica biological chip.
Background technology
Urinary tract infections (Urinary Tract Infections, UTI) is to levy with bladder irritation, i.e. frequent micturition, urgent urination, odynuria, even general infection is the infectious diseases of Clinical symptoms.The predisposing factor of UTI and the cause of disease are very clear and definite, i.e. caused by pathogenic microorganism (mainly bacterium) invasion urethra, the overwhelming majority is gram negative bacilli, such as EHEC, proteus mirabilis, Klebsiella Pneumoniae, pseudomonas aeruginosa, Acinetobacter baumannii, enterobacter cloacae.But UTI to be a class incidence of disease high and clinician is easier to the disease ignored, atypical symptoms, easily recur, malpractice easily causes the appearance of antibody-resistant bacterium, forms intractable urinary tract infections.The diseases such as the renal failure that caused by UTI, hypertension significantly reduce the quality of life of people, bring serious harm to the life and health of people, and therefore diagnosis and treatment UTI is even more important timely and accurately.
Although the diagnostic method of current UTI is more, but there is no acknowledged easy, specifically high effective ways.In many discipline intersection detection architecture of newly-developed, electrochemistry is a kind of method therein.Both studied generation at two-phase interface with a subject of charge transfer phenomenon, set up the relation of a certain chemical reaction and current-responsive based on electrochemical principle, and by the measurement such as physical quantitys such as current potential, electric current, conductance or electricity, tried to achieve the content of material or consider.Self-assembling technique, as a kind of new technique, designs the structure of molecule on a molecular scale, has highly sensitive, selectively good advantage, has obtained huge application in electrochemical analysis field.
At present, the diagnostic method of tradition UTI detects time length, cost height, complex operation, and cross pollution easily occurs, and false positive rate is high；Visible genetic chip technology unrealized quick detection truly.
In sum, needing the new method of a kind of quick detection to Urinary Tract Pathogens: A at present badly, simple to realize to Urinary Tract Pathogens: A many flux, low cost, operation, detection is quick, and can complete detection in common lab, preferably meets the needs of situation of all-level hospitals.
Utility model content
A purpose of the present utility model is to provide a kind of 16SrRNA electrochemica biological chip.
For achieving the above object, a kind of 16SrRNA electrochemica biological chip described in the utility model, including chip, and building unimolecule Iy self-assembled layer on the chip surface, chip surface has chip micropore；Being respectively fixed with capture probe and detection probe in described chip micropore, capture on described capture probe has pathogen 16S rRNA target molecule；Described detection probe forms 16S rRNA molecular probe identification layer, and implementation step is as follows:
A. build unimolecule Iy self-assembled layer at chip surface, carry out activating unimolecule self-chambering layer；
B. capture probe is fixing in chip micropore；
C. capture probe capture pathogen 16S rRNA target molecule；
D., after adding detection probe, 16S rRNA molecular probe identification layer is built；
E. by electrochemica biological chip, molecule hybridization information is exported with the form of electronic signal；
F. horseradish peroxidase is added to change as indication signal using electron transmission, it is achieved the quick detection to variety classes pathogen.
Described chip is interface, a kind of basis, the implementation method at this interface is: the first is exactly to add minimal amount of electronegative material to chip micropore in processing procedure, form a cover layer in advance in micropore, in our previous test, it turned out this molecular layer can reduce the nonspecific reaction between electronegative biomolecule；The second chemical reagent is exactly bovine serum albumin, and it is added in chip micropore, and bovine serum albumin can be coated self-assembled monolayer layer, reduces background signal by reducing the nonspecific reaction of electrode surface, strengthens detection signal strength signal intensity.The third chemical reagent is casein, mixes together with enzyme solutions, the caseic bovine serum albumin that is functionally similar to, but can stop enzyme and self assembled monolayer link, intensified response specific.
Described unimolecule Iy self-assembled layer is 11-Mercaptoundecanoic acid-BPA unimolecule Iy self-assembled layer.
Described capture probe fixing means in chip micropore: 1. organic metal mercaptan connects upper organic double compound or forms monolayer Iy self-assembled layer on end modified after alcohol radical, this layer has the function of the deposition resisting protein or nucleic acid, can expose the end group of functionalization simultaneously；Unimolecule Iy self-assembled layer can be activated in the common effect of Biological cross-linker and Avidin；2. unimolecule Iy self-assembled layer surface after activation, adds capture probe.
Described Biological cross-linker is the one in EDC, NHS.
nullDescribed capture probe and detection probe are for for 6 kinds of urinary tract pathogen (EHECs、Proteus mirabilis、Klebsiella Pneumoniae、Pseudomonas aeruginosa、Acinetobacter baumannii、Enterobacter cloacae) the specific nucleic acid molecules probe system of 16S rRNA，Utilize sequence analysis system ARB (Software environment for sequence data)、NCBI (National Center of Biotechnology Information) and RDP-II: (The Ribosomal Database Project) information system carries out the Preliminary screening of 6 kinds of urinary tract pathogen 16S rRNA specific dna probes；The regional choice of probe system design is as follows:
According to document (SoumiteshChakravorty, DanicaHelb, et al.JMicrobiol CLUSTAL Methods.2007,69 (2): 330-339): 16S rRNA contains nine hypervariable regions of V1-V9, this nine districts guard and specifically have both, and can be used in the structure of molecular probe system.As a example by EHEC, in 1540 bases of EHEC 16S rRNA, selecting to be suitable for detecting conserved sequence district (242-433) the design detection probe between V2 and V3 of EHEC, V3 hypervariable sequence district (433-497) designs capture probe.
For probe data storehouse source in NCBI and RDP-II, input 6 kinds of urinary tract pathogen 16S rRNA respectively and inquire about, obtain the 16S rRNA sequence of this 6 kinds of urinary tract pathogens, set the 16S rRNA base sequence scope of 6 kinds of urinary tract pathogens, carry out FAST and compare screening.Utilize CLUSTAL X software to carry out multisequencing contraposition arrangement, find out specifically high combined sequence.
(such as Fig. 1) as a example by EHEC, in the probe sets designing and filtering out, utilized the detection to EHEC 16S rRNA for the specific higher probe filtering out by molecular biology method, obtain EHEC capture probe and detection probe.
It is also based on above-mentioned steps for proteus mirabilis, Klebsiella Pneumoniae, pseudomonas aeruginosa, Acinetobacter baumannii, detection probe and the design of capture probe structure of enterobacter cloacae, construct common Urinary Tract Pathogens: A 16S rRNA nucleic acid molecular probe storehouse.
Described capture probe is biotinylated capture probe.
The biotinylation DNA detection probe that described detection probe is synthesis, the biotinylation DNA detection probe of described synthesis is provided with decorative layer.
The decorative layer arranging on described detection probe is: SWCN-nano-Au composite；The preparation method of described SWCN-nano-Au composite is: first prepares containing c-terminus single wall layer CNT, then prepares nano gold sol by sodium hypophosphite liquid-phase reduction method；Then the gold grain surface in situ growing single-wall layer carbon nanotube layer in the hydroxylated nano gold sol prepared；Finally utilize solvent extraction to remove organic formwork agent, then after supercritical drying, prepare single wall layer nanotube-nanotube Au composite.
Modification principle on the biotinylation DNA detection probe of synthesis for the described single wall layer carbon nanotube-nano Au composite is: biotin-avidin system is the system of Cascaded amplification detection signal conventional at present, each subunit of Avidin contains a site being combined with biotin, and form high stability compound with biotin or derivatives thereof, its affinity costant is 10-15M-1, the only general covalent bond order of magnitude lower of ratio, but the high 10-100 ten thousand times than antigen-antibody reaction, in conjunction with soon, in irreversible reaction, and at pH, temperature, all can stable existence in organic solvent or the bigger excursion of denaturant；Due to interaction highly single-minded and strong between biotin-labeled pentylamine system, utilize specifically not only single wall layer carbon nanotube-nano Au composite can be connected together with detection probe between biotin (Biotin)-Avidin (Avidin), and there is the effect of Cascaded amplification signal.
It is marked with horseradish peroxidase bioactivator on described detection probe.After detection probe and pathogen nucleic acid hybridization to be checked, under the catalysis and signal amplification of horseradish peroxidase, change as indication signal using each reaction micropore electron transmission of electrochemica biological chip surface, it is achieved the quick of variety classes pathogen is detected
The application process of described detection urinary tract pathogen electrochemica biological chip, as shown in Figure 5:
A. processing the urine specimen that clinic is collected, in cracking urine, pathogen exposes 16SrRNA sequence；
B. being modified with capture probe in chip micropore, biotinylated detection probe will identify target-gene sequence；
C. target gene is captured by capture probe, is simultaneously introduced the Avidin-horseradish peroxidase with electrochemical signals amplification；
D. formed the horseradish peroxidase-Avidin-Biotin-molecular probe stereochemical structure of class " sandwich " by the interaction of chemical bond；
E. by electrochemistry transmission and the signal amplification of enzyme, molecule hybridization information is exported by electrochemical chip with readable electrochemical signals.
Play an important role in the sensitivity when electrochemical electrode array detection is applied for the bio-sensing interface and the degree of accuracy.The mercaptan self assembled monolayer building on gold film is the orderly molecular layer system of a kind of height tissue, protein or nucleic acid deposition can be resisted and stop the non-specific hybridization being easy to produce between biomolecule, end group containing functionalization, is widely used in electrode face finish.
Bacteria Detection principle: common Urinary Tract Pathogens: A, its specific 16SrRNA and corresponding probe hybridization can produce respective specific current signal value, distinguish infectious bacteria species by the difference of current signal value.
A kind of 16SrRNA electrochemica biological chip described in the utility model, it has the beneficial effects that: use nm of gold to detect probe with the nano combined substance markers of SWCN, utilize biotin and Avidin height single-minded and biological stability, be used for linking probe and nano-complex particle；Wherein SWCN is as electronics reserve center, and golden nanometer particle is as electronics high-speed transfer passage, and both combinations can improve that the detection of molecular probe is specific and the overall performance of electrochemica biological sensing chip array；This chip is based on electrochemical detection system, obtains the quantitative relationship between original current value and analytical concentration, then carries out a series of electrochemical analysis process；It is fast that Bacteria Detection analyzes speed, by 16S rDNA gene conserved region is detected, in early days, quickly judge bacterium presence or absence and classification thereof can have advantage simple, rapid, that testing cost is low, highly sensitive, for instruct clinical rational drug use provide by force, strong support.
Brief description
Fig. 1 is EHEC 16S rRNA probe combinations；
Fig. 2 is unimolecule Iy self-assembled layer structural representation；
Fig. 3 is the structure of electrochemica biological chip surface DNA molecular identification layer；
Fig. 4 is nano-complex particle detection probes；
In Fig. 5: A. electrochemical electrode array cyclic voltammogram in TMB-H2O2 solution after different modifying, sweep speed 50mV/s；B. electrochemical electrode array chronoamperogram in TMB-H2O2 solution after different modifying；
In Fig. 6: the differential pulse figure (μm ol/L) under A. target DNA molecule variable concentrations；B. target DNA molecular concentration and peak point current linear relationship；
Fig. 7 is electrochemica biological chip detection flow process.
Detailed description of the invention
Embodiment 1
1. the Collecting and dealing of urine specimen:
1. specimen collection object:
Hospital outpatient and in hospital patients of urinary tract infection: random sampling 400 case, age 7-60 year.
2. processing method:
Collecting above-mentioned hospital outpatient and the urine of clinical patients of urinary tract infection of being in hospital, the sample that separates collected is installed in-70 DEG C of preservations in the glycerine brucella culture medium bottle containing 15%.The sample overnight preserving is inoculated into LB culture medium up in the case of not having miscellaneous bacteria to disturb, till growing into logarithmic phase.Before formal use, the urine cultivated on LB is stored in the environment of-70 DEG C with the form of freezing tubule.
3. the quick detection of pathogen:
A. cultivate urine specimen centrifuge in tubule at LB and centrifuge 5min with 1000 × 10g, then suspension is abandoned, obtain bacterial lysate containing 1mol/l NaOH, 0.1%TritonX-100,2mmol/l every liter EDTA and the TRIS-HCL of the 20mmol/l containing 1mg/ml lysozyme, incubated at room temperature 5 minutes in the solution of Ph8.0；
B.50 μ l containing 2.5% bovine serum albumin and the phosphate buffer of 1mol/l of detection probe of 0.25 μm of ol/l, pH7.4, join under 65 DEG C of environment temperatures in bacterial lysate and hatch 10min, and target probe hybridizes completely；
C.4 the mixed liquor of the cracking bacterium solution of μ l and detection probe is then added to hatch 15min under 65 DEG C of wet conditions on the working electrode in chip, by the time after being dried by wash clean, the horseradish peroxidase (HRP) of 4 μ l is added on working electrode hatch 15min, after chip is rinsed and dried, the mould of plastics of one well in advance is covered at chip surface, the 50 μ l catalytic solutions preparing are joined each nanopores simultaneously, until reactant liquor all covers all of electrode, then with the electrochemical reaction signal on multichannel potentiostat 16 micropores of detection, identify different bacterial species by different output electrochemical signal values.
Embodiment 2
Such as Fig. 6-7；nullAfter electrochemical electrode array surface successfully builds 11-Mercaptoundecanoic acid-BPA unimolecule Iy self-assembled layer，Respectively to 2、8、14、No. 16 reactive tanks add the biotinylation DNA detection probe of 2 0.1 μm of ol/L of μ L，Electrochemical electrode array is put into hatching in box of full nitrogen take out after 10min，Rinse 5s with PBS gently，Respectively again toward 2、8、14、No. 16 reactive tanks add 0.01 μm of ol/L of 1 μ L、1μL 0.10μmol/L、1μL 0.05μmol/L、The e. coli dna target molecule probe sequence of the HRP-Avidin functionalization of 1 0.03 μm of ol/L of μ L，Then will hatch box put in 65 DEG C of water baths fully hatch hybridization 40min after take out，Rinse 5s with PBS，Rapidly 2、8、14、No. 16 reactive tanks are separately added into 20 μ L TMB-H2O2Solution, puts into reader and detects.
Modify HRP-Avidin on the basis of electrochemical electrode array surface successfully builds mercaptan self assembled monolayer.It is respectively adopted cyclic voltammetry and the chronoamperometry biological functional to electrochemical electrode array to be studied, wherein No. 6 reactive tank electrodes are bare electrode, its corresponding response curve is (6), after No. 4 electrode surfaces successfully build 11-Mercaptoundecanoic acid self assembled monolayer, response curve is (4), electric current reduces compared with curve (6), No. 2 electrode surfaces are built with 11-Mercaptoundecanoic acid-BPA molecule assembled layers, because there being the existence of mcroorganism molecule BPA, curve (2) electric current significantly reduces.No. 8 electrode surfaces successfully modify HRP-Avidin, and curve (8) kinetic current significantly increases, because HRP is catalysis H2O2Produce O2 oxidation TMB, occur strong redox reaction to produce obvious curent change.Chrono-amperometric detection curve corresponding with cyclic voltammetry, wherein (10) are that bare electrode is at TMB-H2O2Chronoa mperometric plot, (12) for the response curve after structure 11-Mercaptoundecanoic acid unimolecule Iy self-assembled layer, (14) being to be built with 11-Mercaptoundecanoic acid BPA molecule assembled layers curve, (16) for modifying the response curve of HRP-Avidin at molecular Iy self-assembled layer.Research shows that the electrochemical electrode array performance after modifying through biological functional is good, 10th, the 12nd, the 14th, the chrono-amperometric steady-state value (unit: 1 × 10-5A) of No. 16 electrodes is respectively the 0.0051st, the 0.3020th, the 0.9640th, 1.0900, can detect the 1.26 × 10-6A curent change because building with modify electrochemical electrode array surface generation after different large biological molecule.
Table 1 is common urinary tract infections Pseudomonas and the probe gene order of 16S rDNA phase complementation thereof.
Common urinary tract infections Pseudomonas and the probe gene order of 16SrDNA phase complementation thereof
Claims (4)
1. a 16SrRNA electrochemica biological chip, including chip, it is characterised in that: on the chip surface
Building unimolecule Iy self-assembled layer, chip surface has chip micropore；Described chip micropore is respectively fixed with and catches
Obtaining probe and detection probe, capture on described capture probe has pathogen 16S rRNA target molecule；Described detection
Probe forms 16S rRNA molecular probe identification layer.
2. a kind of 16SrRNA electrochemica biological chip as claimed in claim 1, it is characterised in that: described list
Molecular Iy self-assembled layer is 11-Mercaptoundecanoic acid-BPA unimolecule Iy self-assembled layer.
3. a kind of 16SrRNA electrochemica biological chip as claimed in claim 1, it is characterised in that: described inspection
The biotinylation DNA detection probe that probing pin is synthesis, on the biotinylation DNA detection probe of described synthesis
It is provided with decorative layer.
4. a kind of 16SrRNA electrochemica biological chip as claimed in claim 3, it is characterised in that: described inspection
It is marked with horseradish peroxidase bioactivator on probing pin.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201620156838.4U CN205720081U (en) | 2016-03-02 | 2016-03-02 | A kind of 16SrRNA electrochemica biological chip |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201620156838.4U CN205720081U (en) | 2016-03-02 | 2016-03-02 | A kind of 16SrRNA electrochemica biological chip |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN205720081U true CN205720081U (en) | 2016-11-23 |
Family
ID=57334959
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201620156838.4U Expired - Fee Related CN205720081U (en) | 2016-03-02 | 2016-03-02 | A kind of 16SrRNA electrochemica biological chip |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN205720081U (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105861646A (en) * | 2016-03-02 | 2016-08-17 | 成都市妇女儿童中心医院 | Electrochemical biological chip for detecting urinary tract pathogen 16SrRNA and technical application thereof |
| CN112410446A (en) * | 2020-11-18 | 2021-02-26 | 成都市妇女儿童中心医院 | Detection probe and kit for detecting urinary tract pathogenic bacteria |
-
2016
- 2016-03-02 CN CN201620156838.4U patent/CN205720081U/en not_active Expired - Fee Related
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105861646A (en) * | 2016-03-02 | 2016-08-17 | 成都市妇女儿童中心医院 | Electrochemical biological chip for detecting urinary tract pathogen 16SrRNA and technical application thereof |
| CN112410446A (en) * | 2020-11-18 | 2021-02-26 | 成都市妇女儿童中心医院 | Detection probe and kit for detecting urinary tract pathogenic bacteria |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Yang et al. | Detection of Escherichia coli with a label-free impedimetric biosensor based on lectin functionalized mixed self-assembled monolayer | |
| Bai et al. | Fullerene-doped polyaniline as new redox nanoprobe and catalyst in electrochemical aptasensor for ultrasensitive detection of Mycobacterium tuberculosis MPT64 antigen in human serum | |
| Shahdeo et al. | Graphene based sensors | |
| Jalalvand | Fabrication of a novel and ultrasensitive label-free electrochemical aptasensor for detection of biomarker prostate specific antigen | |
| Zheng et al. | Advanced sensitivity amplification strategies for voltammetric immunosensors of tumor marker: State of the art | |
| Wang et al. | Application of impedance spectroscopy for monitoring colloid Au-enhanced antibody immobilization and antibody–antigen reactions | |
| Zhu et al. | Amperometric immunosensor for simultaneous detection of three analytes in one interface using dual functionalized graphene sheets integrated with redox-probes as tracer matrixes | |
| Huang et al. | A disposable electrochemical immunosensor for carcinoembryonic antigen based on nano-Au/multi-walled carbon nanotubes–chitosans nanocomposite film modified glassy carbon electrode | |
| CN101194162B (en) | Carbon nanotube transistor biosensors with aptamers as molecular recognition elements and method for sensing a target material using the same | |
| White et al. | Rapid, selective, label-free aptameric capture and detection of ricin in potable liquids using a printed floating gate transistor | |
| Lin et al. | Carbon nanomaterial-based biosensors: A review of design and applications | |
| Zhao et al. | Electrochemical immunosensor for the carcinoembryonic antigen based on a nanocomposite consisting of reduced graphene oxide, gold nanoparticles and poly (indole-6-carboxylic acid) | |
| Ming et al. | based microfluidic aptasensors | |
| Gou et al. | Voltammetric immunoassay for Mycobacterium tuberculosis secretory protein MPT64 based on a synergistic amplification strategy using rolling circle amplification and a gold electrode modified with graphene oxide, Fe 3 O 4 and Pt nanoparticles | |
| Zhou et al. | A magnetic bead-based bienzymatic electrochemical immunosensor for determination of H9N2 avian influenza virus | |
| Han et al. | Electrochemical immunoassay for thyroxine detection using cascade catalysis as signal amplified enhancer and multi-functionalized magnetic graphene sphere as signal tag | |
| Hou et al. | Modification-free amperometric biosensor for the detection of wild-type p53 protein based on the in situ formation of silver nanoparticle networks for signal amplification | |
| Liu et al. | Boronic acid-based electrochemical sensors for detection of biomolecules | |
| CN110376380B (en) | Electrochemical enzyme-linked immunosensor and preparation and application thereof to antigen detection | |
| Ouyang et al. | Fabrication of surface protein-imprinted biofuel cell for sensitive self-powered glycoprotein detection | |
| Li et al. | A self-powered rotating paper-based analytical device for sensing of thrombin | |
| Fan et al. | Smartphone-based electrochemical system with multi-walled carbon nanotubes/thionine/gold nanoparticles modified screen-printed immunosensor for cancer antigen 125 detection | |
| Liao et al. | Hybridization chain reaction triggered poly adenine to absorb silver nanoparticles for label-free electrochemical detection of Alzheimer's disease biomarkers amyloid β-peptide oligomers | |
| Wei et al. | Highly sensitive detection of multiple proteins from single cells by MoS2-FET biosensors | |
| Geleta et al. | Electrochemical biosensors for detecting microbial toxins by graphene-based nanocomposites |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20161123 Termination date: 20180302 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |