CN109142293A - A kind of fluorescent optical sensor and its preparation and application based on carbonitride desorption - Google Patents
A kind of fluorescent optical sensor and its preparation and application based on carbonitride desorption Download PDFInfo
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- CN109142293A CN109142293A CN201810840502.3A CN201810840502A CN109142293A CN 109142293 A CN109142293 A CN 109142293A CN 201810840502 A CN201810840502 A CN 201810840502A CN 109142293 A CN109142293 A CN 109142293A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
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Abstract
The invention discloses a kind of fluorescent optical sensor based on carbonitride desorption and its preparations and application.Sensor the preparation method comprises the following steps: with g-C3N4Nanometer sheet is the reinforcing agent of chemical illuminating reagent, using the complementary strand of molecule DNA to be measured as biological identification element；By g-C3N4Nanometer sheet, chemical illuminating reagent, the complementary strand of molecule DNA to be measured and the mixing of Tris-HCI buffer, are made the fluorescent optical sensor based on carbonitride desorption.The sensor have simplicity, fast, it is accurate the features such as, when for detecting HIV nucleic acid small molecule, the range of linearity be 50-2000 nM, detection limit (3 S/N) be 20 nmol/L.
Description
Technical field
The invention belongs to technical field of fluorescence detection, and in particular to a kind of fluorescence sense based on carbonitride desorption
Device and its preparation and application.
Background technique
AIDS is a kind of great infectious disease of harmfulness, is caused by aids infection viral (HIV).AIDS virus exists
After invading human body, groups of people can be presented always whithin a period of time without apparent symptom, that is, incubation period is in each human body
The length of upper latent time is all different.Therefore HIV infection detection is extremely important to the prevention and treatment of HIV.Under normal circumstances, viral
After infecting human body, it can be found by a series of detections, and what can be detected earliest is viral nucleic acid.Detection of nucleic acids can be direct
It checks HIV nucleic acid, HIV infection can be detected before discovery serology variation, and, energy sensitiveer than P24 antigen detection method
Virus infection is found earlier.HIV nucleic acid quantification detection quantitative approach generally has: the two kinds of sides RT-PCR and signal amplification amplification
Method, it usually needs use expensive instrument, and operate relatively complicated.
Summary of the invention
In view of the above-mentioned deficiencies in the prior art, it is an object of the present invention to provide a kind of fluorescence based on carbonitride desorption
Sensor and its preparation and application.The sensor have simplicity, fast, it is accurate the features such as, for measuring HIV nucleic acid small molecule
When, the range of linearity is 50-2000 nM, and detection limit (3 S/N) is 20 nmol/L.
To achieve the purpose of the present invention, it adopts the following technical scheme that
A kind of preparation method of the fluorescent optical sensor based on carbonitride desorption, with g-C3N4Nanometer sheet is chemiluminescence examination
The reinforcing agent of agent, using the complementary strand of molecule DNA to be measured as biological identification element；By g-C3N4Nanometer sheet, chemiluminescence examination
Agent, the complementary strand of molecule DNA to be measured and the mixing of Tris-HCI buffer, are made based on the glimmering of carbonitride desorption
Optical sensor.
g-C3N4The concentration of nanometer sheet in the sensor is 132-330 mg/mL.
The concentration of the complementary strand of molecule DNA to be measured in the sensor is 1-20nM.
A kind of preparation method as described above fluorescent optical sensor obtained based on carbonitride desorption.
A kind of application of the fluorescent optical sensor as described above based on carbonitride desorption is used for small point of HIV nucleic acid
The detection of son.
It further, is with described based on carbonitride desorption when detection for HIV nucleic acid small molecule
Fluorescent optical sensor is matrix, system to be measured is added in the optical electro-chemistry sensor based on nitridation carbon signal amplification,
Joint ultraviolet lamp carries out illumination and the imaging of chemiluminescence fluoroscopic imaging systems, described based on nitrogen in having system existing for HIV
The fluorescent optical sensor for changing carbon desorption can generate luminous intensity enhancing, by the concentration for drawing known HIV nucleic acid small molecule
With the standard curve of luminous intensity, the luminous intensity detected by the fluorescent optical sensor based on carbonitride desorption
It can determine the concentration of HIV nucleic acid small molecule in system to be measured.
Further, when detection for HIV nucleic acid small molecule, the chemical illuminating reagent is MCLA.
Further, when detection for HIV nucleic acid small molecule, the range of linearity to HIV nucleic acid small molecule is 50-
2000 nM, 3 S/N of detection limit are 20 nmol/L.
Further, when detection for HIV nucleic acid small molecule, light is carried out using the ultraviolet lamp of 350-400nm wavelength
According to 60s.
When being used to measure HIV nucleic acid small molecule for sensor of the invention, the specific steps are as follows:
(1) foundation of standard curve:
1. respectively by chemical illuminating reagent MCLA, g-C3N4, the complementary strand of HIV-DNA and the HIV-DNA of isometric various concentration
Standard solution mixing, is added Tris-HCI buffer, a series of test agents is made；
2. test agent is added in ELISA Plate, under 365 nm ultraviolet lamps after illumination 60s, it is placed in chemiluminescence fluorescence imaging system
In the darkroom of system, each time for exposure is 90s；
3. reading and converting by software, luminous intensity corresponding to the HIV-DNA standard solution of various concentration is obtained；
4. by step 3. numeric renderings obtained at standard curve, equation of linear regression are as follows: the e of y=1.6228 + 2.084e7lg
[x / (10-8Mol/L)], related coefficient 0.985；Wherein y is luminous intensity, and x is the concentration of HIV-DNA standard solution；
(2) measurement of actual samples:
By chemical illuminating reagent MCLA, g-C3N4, HIV-DNA complementary strand and to test sample mix, be added Tris-HCI buffer,
It is made to test sample；Will to test sample, through the in step (1), 2., 3. step continues to measure, obtain the luminous intensity to test sample, generation
Enter in equation of linear regression to get to the HIV-DNA concentration in test sample.
Preferably, step 1. in test agent overall accumulated amount be 100 μ L；The dosage of MCLA is 15 μ L, in test agent
In concentration be 0.15 mg/ml；g-C3N4Concentration in test agent is 198 mg/ml；The complementary strand of HIV-DNA is to be measured
Concentration in reagent is 1 nM；The volume of HIV-DNA standard solution is 10 μ L；The concentration range of HIV-DNA standard solution is 0-
2000 nM。
MCLA:[2- methyl -6(-4- methoxyphenyl) -3,7- glyoxalidine simultaneously [1,2-a] piperazine -3-1- hydrochloric acid
Salt], chemical illuminating reagent.
Step 1. described in HIV-DNA complementary strand, sequence are as follows: ACTGCTAGAGATTTTCCACAT.
Step 1. described in Tris-HCI buffer pH value be 7.4.
The step 3. reading and conversion are as follows: the chemiluminescence intensity in each hole of ELISA Plate is read with Image J software,
The software optical density value indicates luminous intensity；The area for first selecting light emitting region passes through the optical density in measurement fixed-area
Value, then to quantify the luminous intensity in every hole.
The mechanism of fluorescence detection of the present invention is:
g-C3N4Nanometer sheet can enhance the photochemical luminescence of MCLA, and provide one very well for fixed Hiv-DNA complementary strand
Platform, DNA molecular in sugar chain (phosphoric acid backbone) have negative electrical charge phosphate group, g-C3N4Nanometer sheet is positively charged, g-
C3N4It can be interacted by π-π and electrostatic force is in conjunction with the ssDNA probe, be adsorbed on g-C3N4The ss DNA on surface is accounted for
According to g-C3N4Active site, to reduce photochemical luminescence signal.There are target Hiv-DNA,
SsDNA probe identifies and combining target Hiv-DNA is to form double helix chain DNA (dsDNA).The dsDNA and g-C of formation3N4Between
Interaction it is very weak, cause conjugate be detached from g-C3N4The surface of nanometer sheet, so as to cause the recovery of photochemiluminescence signal.
The present invention has the advantage that compared with the prior art
Sensor of the invention has simplicity, fast, it is accurate the features such as, when detection for HIV nucleic acid small molecule, the range of linearity
For 50-2000 nM, detection limit (3 S/N) is 20 nmol/L, high sensitivity.
Detailed description of the invention
Fig. 1 is the principle of the present invention schematic diagram；Probe-DNA refers to Hiv-DNA complementary strand；
Fig. 2 is the increase figure of photochemical luminescence intensity under different light application times；(a) 0, (b) 20, (c) 40, (d) 60,
(e) 80, (f) 120, (g) 140 and (h) 160 s；Tris-HCl buffer (pH=7.4), MCLA:0.15 mg/ml, 15 μ
l；g-C3N4: 264 mg/ml；Time for exposure=90 s；Reagent total amount: 100 μ l；
Fig. 3 is g-C3N4Influence diagram of the concentration to PCL；(a) 0, (b) 66, (c) 132, (d) 198, (e) 264, (f)
330, (g) 396 and (h), 462 mg/mL；Tris-HCl buffer (pH=7.4)；MCLA:0.15 mg/ml, 15 μ l；
Time for exposure=90s；Reagent total amount: 100 μ l；
Fig. 4 is influence diagram of the Hiv-DNA complementary strand concentration and probe concentration to PCL；DNA probe: 0,1,2,5,7,10,20,30*10-7
mol/L；g-C3N4:198 mg/ml；Tris-HCl buffer (pH=7.4)；MCLA:0.15 mg/ml, 15 μ l；Time for exposure=
90s；Reagent total amount: 100 μ l；
Fig. 5 is the photochemiluminescence recovery situation figure of MCLA after adding target Hiv-DNA；Target dna: 0,2,5,20,50,
100,200*10-8mol/L；DNA probe: 7e-7；MCLA 0.15 mg/ml；C3N4: 198 mg/ml；Tris-HCl buffer
(pH=7.4)；37 DEG C of 1 h of incubation；
Fig. 6 is the linear graph of luminous intensity and target DNA concentration；
Fig. 7 is mixture Capillary Electrophoresis map；A. probe and g-C3N4；B. probe/g-C3N4/ target dna (lazy weight)；
C. detector/g-C3N4/ target dna (sufficient amount).
Specific embodiment
Further to disclose rather than the present invention is limited, the present invention is described in further detail below in conjunction with example.
A kind of preparation method of the fluorescent optical sensor based on carbonitride desorption, with g-C3N4Nanometer sheet is chemistry hair
The reinforcing agent of light reagent, using the complementary strand of molecule DNA to be measured as biological identification element；By g-C3N4Nanometer sheet, chemiluminescence
Reagent, the complementary strand of molecule DNA to be measured and the mixing of Tris-HCI buffer, are made based on carbonitride desorption
Fluorescent optical sensor.
g-C3N4The concentration of nanometer sheet in the sensor is 132-330 mg/mL.
The concentration of the complementary strand of molecule DNA to be measured in the sensor is 1-20nM.
A kind of preparation method as described above fluorescent optical sensor obtained based on carbonitride desorption.
A kind of application of the fluorescent optical sensor as described above based on carbonitride desorption is used for small point of HIV nucleic acid
The detection of son.
It further, is with described based on carbonitride desorption when detection for HIV nucleic acid small molecule
Fluorescent optical sensor is matrix, system to be measured is added in the optical electro-chemistry sensor based on nitridation carbon signal amplification,
Joint ultraviolet lamp carries out illumination and the imaging of chemiluminescence fluoroscopic imaging systems, described based on nitrogen in having system existing for HIV
The fluorescent optical sensor for changing carbon desorption can generate luminous intensity enhancing, by the concentration for drawing known HIV nucleic acid small molecule
With the standard curve of luminous intensity, the luminous intensity detected by the fluorescent optical sensor based on carbonitride desorption
It can determine the concentration of HIV nucleic acid small molecule in system to be measured.
Further, when detection for HIV nucleic acid small molecule, the chemical illuminating reagent is MCLA.
Further, when detection for HIV nucleic acid small molecule, the range of linearity to HIV nucleic acid small molecule is 50-
2000 nM, 3 S/N of detection limit are 20 nmol/L.
The present invention is described in detail by taking the detection of HIV nucleic acid small molecule as an example.Other test substances are referring to HIV nucleic acid
The detection of small molecule carries out.
In order to obtain optimal detection performance, light application time, g-C are had studied3N4Concentration and HIV concentration and probe concentration are to detection
The influence of HIV DNA response.
1, the influence that light application time detects Hiv-DNA
Fig. 2 has studied the influence that light application time detects Hiv-DNA.It can be seen that PCL reaction is extended to light application time from 20 s
160 s and increase, it is most strong to find that illumination 60s shines in figure.It chooses light application time 60s and does subsequent experimental.
2、 g-C3N4The influence that concentration detects HIV- DNA
Fig. 3 has studied g-C3N4The influence that concentration detects HIV-DNA increases g-C3N4After concentration, due to g-C3N4It is catalyzed MCLA
Chemiluminescence obviously increases；Work as g-C3N4Concentration when being more than 198 mg/ml, catalytic effect weakens, so selection g-C3N4Concentration
Subsequent experimental is done for 198 mg/ml.
3, the influence that concentration and probe concentration detects Hiv- DNA
In the case where other conditions are constant, from fig. 4, it can be seen that with the increase of concentration and probe concentration, chemiluminescence imaging intensity
Increase.When concentration and probe concentration reaches 1 nM, luminous value inhibits most obvious.Therefore, 1 nM of optimum concentration of probe is selected to carry out down
The experiment of one step.
4, in the presence of target Hiv-DNA, with g-C3N4The formation and release of middle dsDNA, CL intensity are remarkably reinforced.Knot
Fruit shows dsDNA and g-C3N4Interaction be weaker than ssDNA and g-C3N4Interaction.Therefore, target analytes are deposited
It can restore g-C3N4To the humidification of MCLA photochemical luminescence.
With the increase of target Hiv-DNA concentration, photochemiluminescence intensity is gradually increased, photochemiluminescence intensity relative to
The curve of aimed concn has good linear relationship for target within the scope of 50-2000 nM.Equation of linear regression is expressed as y
=1.622 e8 +2.084e7lg [x/ (10-8Mol/L)], related coefficient 0.985, according to the inspection of the 3 δ Hiv-DNA calculated
Surveying limit (LOD) is 20 nM.
5, the combination of capillary electrophoresis separation analysis verifying probe and target Hiv-DNA
Capillary electrophoresis be used to prove g-C3N4In the presence of probe and target Hiv-DNA specific recognition.Complementary strand is visited
Needle indicates fluorescent marker.It can be seen from figure 7 that the peak occurred in curve a, indicates probe；Two peaks of appearance in curve b, 1
New peak at min indicates probe and g-C3N4Combination product, the dissociative DNA for still indicating not participate in reaction at 4 min visits
Needle.Said sample and target Hiv DNA are incubated for 20 hours.Analyze Incubation mixtures.Such as Fig. 7 curve c, only one strong peak goes out
Existing, this shows in g-C3N4In the presence of, the specific recognition of probe and target Hiv DNA are extremely successful.
The present invention is based on g-C3N4Unique photocatalysis performance, have developed a kind of novel photochemical luminescence (PCL) imaging
Method.Using the graphite-phase carbonitride (g-C of high dispersive in water3N4) it is catalyst, ultraviolet excitation induction generates active oxygen and increases
The chemiluminescence of Strong oxdiative MCLA, the detection using HIV-DNA complementary strand as biological identification element, for HIV DNA sequence dna.Experiment
The result shows that MCLA shows very strong photoinduction chemiluminescence response in neutral buffered medium；As the suitable g-C of use3N4
When, photochemical luminescence response enhancing.g-C3N4Possess big specific surface area and pi-conjugated structure, it can be with Hiv-DNA complementary strand
It is combined closely by π-π sintering action, encloses g-C3N4Active site, cause MCLA photochemical luminescence be suppressed；
When Hiv-DNA chain to be measured is added, due to the ds DNA (double helix chain DNA) and g-C of formation3N4Between declines,
DNA double chain is tended to far from g-C3N4The surface of nanometer sheet, therefore luminous signal restores.UV light irradiation time, reagent concentration etc. because
Element has an impact to photochemiluminescence intensity, by optimum experimental, obtains optimal conditions are as follows: it shines within illumination 60 seconds, g-C3N4Concentration
198 mg/ml, 1 nM of concentration and probe concentration.The intensity that the photochemical luminescence restores is linear related to the concentration of Hiv-DNA sequence,
The range of linearity is 50-2000 nM, and detection limit (3 S/N) is 20 nmol/L, the experimental results showed that g-C3N4Successful utilization is photic
In luminescence imaging sensor, have it is prominent easy, fast, it is accurate the features such as.
Embodiment 1
A kind of preparation method of the fluorescent optical sensor based on carbonitride desorption measurement HIV nucleic acid small molecule, including it is following
Step:
(1) foundation of standard curve:
1. respectively by chemical illuminating reagent MCLA, g-C3N4, the complementary strand of HIV-DNA and the HIV-DNA of isometric various concentration
Standard solution mixing, is added Tris-HCI buffer, a series of test agents is made；
2. test agent is added in ELISA Plate, under 365 nm ultraviolet lamps after illumination 60s, it is placed in chemiluminescence fluorescence imaging system
In the darkroom of system, each time for exposure is 90s；
3. reading and converting by software, luminous intensity corresponding to the HIV-DNA standard solution of various concentration is obtained；
4. by step 3. numeric renderings obtained at standard curve, equation of linear regression are as follows: the e of y=1.6228 + 2.084e7lg
[x / (10-8Mol/L)], related coefficient 0.985；Wherein y is luminous intensity, and x is the concentration of HIV-DNA standard solution；
(2) measurement of actual samples:
By chemical illuminating reagent MCLA, g-C3N4, HIV-DNA complementary strand and to test sample mix, be added Tris-HCI buffer,
It is made to test sample；Will to test sample, through the in step (1), 2., 3. step continues to measure, obtain the luminous intensity to test sample, generation
Enter in equation of linear regression to get to the HIV-DNA concentration in test sample.
Step 1. in test agent overall accumulated amount be 100 μ L；The dosage of MCLA is 15 μ L, the concentration in test agent
For 0.15 mg/ml；g-C3N4Concentration in test agent is 198 mg/ml；The complementary strand of HIV-DNA is in test agent
Concentration is 1 μM；The volume of HIV-DNA standard solution is 10 μ L；The concentration range of HIV-DNA standard solution is 50-2000 nM.
The step 3. reading and conversion are as follows: the chemiluminescence intensity in each hole of ELISA Plate is read with Image J software,
The software optical density value indicates luminous intensity；The area for first selecting light emitting region passes through the optical density in measurement fixed-area
Value, then to quantify the luminous intensity in every hole.
In step (2) to the overall accumulated amount of test sample be 100 μ L；The dosage of MCLA is 15 μ L, is being to the concentration in test sample
0.15 mg/ml；g-C3N4It is being 198 mg/ml to the concentration in test sample；The complementary strand of HIV-DNA is being to the concentration in test sample
1 nM；Volume to test sample is 10 μ L.
In view of security consideration, the present invention only tests simulation sample, and the results are shown in Table 1.
Table 1: the rate of recovery experimental result of HIV-DNA is measured
The foregoing is merely presently preferred embodiments of the present invention, all equivalent changes done according to scope of the present invention patent with repair
Decorations, are all covered by the present invention.
Sequence table
<110>University of Fuzhou
<120>a kind of fluorescent optical sensor and its preparation and application based on carbonitride desorption
<130> 1
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
<212> DNA
<213> Abedus herberti
<400> 1
actgctagag attttccaca t 21
Claims (9)
1. a kind of preparation method of the fluorescent optical sensor based on carbonitride desorption, it is characterised in that: with g-C3N4Nanometer sheet
For the reinforcing agent of chemical illuminating reagent, using the complementary strand of molecule DNA to be measured as biological identification element；By g-C3N4Nanometer sheet,
Chemical illuminating reagent, the complementary strand of molecule DNA to be measured and the mixing of Tris-HCI buffer, are made and are desorbed based on carbonitride
The fluorescent optical sensor of attached effect.
2. the preparation method of the fluorescent optical sensor according to claim 1 based on carbonitride desorption, feature exist
In: g-C3N4The concentration of nanometer sheet in the sensor is 132-330 mg/mL.
3. the preparation method of the fluorescent optical sensor according to claim 1 based on carbonitride desorption, feature exist
In: the concentration of the complementary strand of molecule DNA to be measured in the sensor is 1-20nM.
4. a kind of preparation method as described in any one of claims 1-3 fluorescence obtained based on carbonitride desorption passes
Sensor.
5. a kind of application of the fluorescent optical sensor as claimed in claim 4 based on carbonitride desorption, it is characterised in that:
Detection for HIV nucleic acid small molecule.
6. the application of the fluorescent optical sensor according to claim 5 based on carbonitride desorption, it is characterised in that: use
When the detection of HIV nucleic acid small molecule, be using the fluorescent optical sensor based on carbonitride desorption as matrix, will be to
Survey system is added in the optical electro-chemistry sensor based on nitridation carbon signal amplification, and joint ultraviolet lamp carries out illumination and change
Luminous fluorescent imaging system images are learned, in having system existing for HIV, the fluorescence based on carbonitride desorption is passed
Sensor can generate luminous intensity enhancing, by drawing the concentration of known HIV nucleic acid small molecule and the standard curve of luminous intensity, by
The luminous intensity that the fluorescent optical sensor based on carbonitride desorption detects can determine HIV core in system to be measured
The concentration of sour small molecule.
7. the application of the fluorescent optical sensor according to claim 6 based on carbonitride desorption, it is characterised in that: use
When the detection of HIV nucleic acid small molecule, the chemical illuminating reagent is MCLA.
8. the application of the fluorescent optical sensor according to claim 6 based on carbonitride desorption, it is characterised in that: use
When the detection of HIV nucleic acid small molecule, the range of linearity to HIV nucleic acid small molecule is 50-2000 nM, and 3 S/N of detection limit is 20
nmol/L。
9. the application of the fluorescent optical sensor according to claim 6 based on carbonitride desorption, it is characterised in that: use
When the detection of HIV nucleic acid small molecule, illumination 60s is carried out using the ultraviolet lamp of 350-400nm wavelength.
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| HUANSHUN YIN,: "Photoelectrochemical immunosensor for microRNA detection", 《SENSORS AND ACTUATORS B》 * |
| KUN HU: "Graphitic carbon nitride nanosheet-basedmulticolour fluorescent nanoprobe for multiplexed analysis of DNA", 《MICROCHIM ACTA》 * |
| 中山大学设备与实验室处: "《实验教学研究与实践》", 31 December 2005, 中山大学出版社 * |
| 田野: "《运动生理学高级教程》", 31 August 2003, 高等教育出版社 * |
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