CN105675563B - A kind of ***e fast quantitative measurement method for detecting suitable for Site Detection - Google Patents
A kind of ***e fast quantitative measurement method for detecting suitable for Site Detection Download PDFInfo
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- CN105675563B CN105675563B CN201610037309.7A CN201610037309A CN105675563B CN 105675563 B CN105675563 B CN 105675563B CN 201610037309 A CN201610037309 A CN 201610037309A CN 105675563 B CN105675563 B CN 105675563B
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- G—PHYSICS
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- 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
- G01N21/6402—Atomic fluorescence; Laser induced fluorescence
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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"
- G01N21/643—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" non-biological material
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- 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"
- G01N2021/6432—Quenching
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Abstract
The present invention discloses a kind of ***e fast quantitative measurement method for detecting suitable for Site Detection, includes the following steps：(1) paper substrate detection device is prepared：Paper is chosen as detection substrate, hydrophilic detection zone, covalent coupling water-soluble upconversion fluorescence nano material and aptamers ACA 1 are drawn a circle to approve in paper surface with hydrophobic barrier；(2) it chooses gold nano grain and in its surface modification aptamers ACA 2, obtains 2 solution of AuNPs ACA；(3) concentration optimization of 2 solution of AuNPs ACA；(4) standard solution of ***e is prepared；(5) ***e examination criteria curve is drawn；(6) in body fluid to be measured ***e concentration measure.The present invention solves the problems, such as ***e detection field Quantitative detection urgently to be resolved hurrily, realizes quick, quantitative, the highly sensitive detection of ***e concentration in humoral sample, and quantitative detection sensitivity is up to 0.05 μM.
Description
Technical field
The invention belongs to ***e Quantitative detection technical fields, and in particular to one kind is based on up-conversion fluorescence nanometer material
The paper substrate detection method of quick, quantitative, the highly sensitive detection ***e of material.
Background technology
Drug abuse is one of social concern for endangering mankind's most serious at present, and ***e is most widely used in all kinds of drugs
General one kind.Cocaine abuse can generate very big threat to human body so that human heart rate is accelerated, and is short of breath or even shakes
Quiver, spasm, faint from fear phenomena such as, grave danger is formed to human security, social stability and world peace.Therefore, it studies novel, clever
Quick, quantitative ***e detection method has very important practice significance for the prohibition of drug, poisonous substance detection, criminal identification etc. and should
With value.
The traditional ***e detection method reported at present has：Optical Analysis Method, electrochemical methods, efficient capillary electricity
Swimming method, nuclear magnetic resonance spectrometry etc., these traditional detection methods there are detection sensitivity it is inadequate, it is complicated for operation take, rely on essence
Close expensive instrument, the shortcomings of professional is needed to be operated, it cannot usually meet the requirement researched and analysed with clinical detection.With list
Field quick detection drugs method based on clonal antibody is most widely used, but such method has some limitations,
Such as antibody self-defect, Antibody preparation heavy workload, the non-specific binding of separate sources antibody generate false positive signal.Closely
Nian Lai, the drugs field fast detection method based on aptamer are centainly developed, the inspection based on aptamer
Survey method specificity is good, and sensitivity is relative to antibody test higher, but most of mesh is also limited to the laboratory research stage, uncomfortable
Preferably it is used for Site Detection.
In conclusion this field still lacks the satisfactory ***e Quantitative detection side for being suitable for Site Detection
Method, therefore, there is an urgent need to develop to have many advantages, such as ***e Site Detection skill sensitive, quick, accurate, that specificity is good
Art.
Invention content
The technical problems to be solved by the invention in order to overcome the deficiencies of the prior art, provide kind of one kind suitable for Site Detection
Cocaine fast quantitative measurement method for detecting, ***e can be directly obtained and quantify testing result, without secondary detection.
The technical solution that there is provided to solve above-mentioned technical problem of the present invention is：
A kind of ***e fast quantitative measurement method for detecting suitable for Site Detection includes the following steps：
(1) paper substrate detection device is prepared：Paper is chosen as detection substrate, is existed using water-insoluble solvent as hydrophobic barrier
Paper surface draws a circle to approve hydrophilic detection zone, and water-soluble upconversion fluorescence nano material and single-chain nucleic acid aptamers ACA-1 is covalent
The hydrophilic detection zone is coupled at, obtains paper substrate detection device；
(2) gold nano grain (AuNPs) is chosen and in its surface markers single-chain nucleic acid aptamers ACA-2, products therefrom note
Make AuNPs-ACA-2, be scattered in buffer solution, obtain AuNPs-ACA-2 solution；
(3) concentration optimization of AuNPs-ACA-2 solution：Respectively in the hydrophilic inspection of the paper substrate detection device described in step (1)
It surveys and the AuNPs-ACA-2 solution that volume is the ***e solution of V1 fixed concentrations and volume is V2 various concentrations is added in region,
Unreacted AuNPs-ACA-2 is removed after the completion of reaction；980nm laser is used after paper substrate detection device drying after gained is reacted
Light source activation shoots the fluorescent image of hydrophilic detection zone under dark condition and reads green intensity in fluorescent image with software
F；When setting a concentration of the 0 of AuNPs-ACA-2 solution, gained green intensity blank sample intensity is denoted as F0, calculate various concentration
Fluorescent quenching efficiency (F corresponding to AuNPs-ACA-2 solution0-F)/F0, obtain corresponding during fluorescent quenching efficiency maximum
AuNPs-ACA-2 solution concentrations C；
(4) standard solution of ***e is prepared：The ***e solution of various concentration, as ***e standard are prepared with body fluid
Solution；
(5) ***e examination criteria curve is drawn：Respectively in the hydrophilic detection zone of the paper substrate detection device described in step (1)
The ***e standard solution that volume is V1 and the AuNPs-ACA-2 solution that volume is a concentration of C of V2 are added in domain, after the completion of reaction
Remove unreacted AuNPs-ACA-2；It is excited after paper substrate detection device drying after gained is reacted with 980nm laser light sources,
The fluorescent image of hydrophilic detection zone is shot under dark condition and reads green intensity F ' in fluorescent image with software；Setting can
Cacaine standard solution a concentration of 0 when, gained green intensity blank sample intensity is denoted as F0', with fluorescent quenching efficiency (F0’-F’)/
F0' it is ordinate, draw standard curve by abscissa of the logarithm of ***e concentration of standard solution；
(6) in body fluid to be measured ***e concentration measure：Respectively in the hydrophilic inspection of the paper substrate detection device described in step (1)
It surveys and the body fluid to be measured that volume is V1 and the AuNPs-ACA-2 solution that volume is a concentration of C of V2 is added in region, removed after the completion of reaction
Remove unreacted AuNPs-ACA-2；It is excited after paper substrate detection device drying after gained is reacted with 980nm laser light sources, in
The fluorescent image of hydrophilic detection zone is shot under dark condition and reads green intensity Fx in fluorescent image with software；Calculate fluorescence
Quench efficiency (F0’-Fx)/F0', it substitutes into standard curve and obtains the ***e concentration in body fluid to be measured.
By said program, the shape circular of the hydrophilic detection zone, the preferred 2-6mm of internal diameter.
By said program, the water-soluble upconversion fluorescence nano material pattern is spherical shape, and grain size is in 30-100nm, table
Face is modified with amino.Wherein, the matrix of the upconversion fluorescence nano material is upconversion fluorescence nano material NaYF4、NaGdF4
Deng amido modified polyethyleneimine, aminoethyl phosphonic acid etc. of may be used is as modifying agent, to realize water solubility.
By said program, in step (1) during covalent coupling, water-soluble upconversion fluorescence nano material is configured to a concentration of
The solution of 0.2-0.8mg/ml, single-chain nucleic acid aptamers ACA-1 are configured to a concentration of 0.5-4 μM of solution, and the two addition is equal
For 1.3-12 μ L, total volume is the summation of V1 and V2；Or the mixed solution of the two is configured to, addition is adjusted in right amount.
By said program, the gold nano grain pattern is spherical shape, and grain size has citric acid in 10-30nm, surface modification
Root.
By said program, the single-chain nucleic acid ACA-1 and single-chain nucleic acid ACA-2, sequence are respectively：5’-NH2-
TTTTTCAAGAACTGAGGG-3 ' and 5 '-SH-TTTTTACAGCAGGGTGAAGTAACTTCTTG-3 '.
By said program, the paper need to be hydrophily paper, including printing paper, ordinary filter paper, cellulosic filter paper, nitre
Acid cellulose film etc., preferably cellulosic filter paper.
By said program, the water-insoluble solvent in step (1) is selected from hydrophobic inks, paraffin, photoresist, alkyl ketene
Dimer etc..
By said program, pH of buffer range described in step (2) can select NaCl containing 100mM in 7.0-7.5
20mM Tris-HCl buffer solutions；Buffer solution is identical with step (2) in step (3).
By said program, the preparation steps of the ***e solution in step (3) are that solvent in ***e standard items is complete
Buffer solution is added in after volatilization, and (buffer solution can select：The 20mM Tris-HCl, pH of the NaCl containing 100mM is 7.4 buffering
Liquid), ice-bath ultrasonic 30min obtains ***e storing solution, then storing solution is diluted to certain concentration with same buffer.The cocker
Because the certain concentration of solution can be set as 10 μM, the concentration range of AuNPs-ACA-2 solution is usually 0-0.305 μM.
By said program, washed to remove unreacted AuNPs-ACA-2 with buffer solution in step (3).The buffer solution is
The 20mM Tris-HCl buffer solutions of the NaCl containing 100mM, and wherein the volumn concentration of polyethylene glycol (PEG) is 1%-2%,
The volumn concentration of polysorbas20 (Tween 20) is 0.01%-0.05%, the pH ranging from 7.0-7.5 of the buffer solution；It is described
Washing the specific steps are：First paper substrate detection device is inverted, makes hydrophilic detection zone downward, 5-10 μ L washings are added in from top
Buffer solution, and promote solution flowing in lower section pad blotting paper, it washes repeatedly 3-5 times.
By said program, the concentration range of ***e standard solution is 0-50 μM in the step (4).
By said program, the step (3) and volume V1 in step (5), (6) and volume V2 and hydrophilic detection zone are straight
Diameter it is square directly proportional.When hydrophilic detection zone is internal diameter 2-6mm circles, volume V1 and the preferred 1.3-12 μ L of volume V2.
By said program, the reaction temperature in the step (3) and step (5), (6) is 20-37 DEG C, and the reaction time is anti-
Answer 20-60min.
By said program, mobile phone may be used in the step (3) and step (5), (6) and shoot hydrophilic detection zone fluorescence
Image reads green intensity in fluorescent image with mobile phone RGB softwares.
By said program, the body fluid specifically includes blood, saliva, tear, urine, sweat etc..
The principle of the present invention is：Fluorescence resonance energy transfer is mutually tied with the specific recognition function of ***e aptamers
It closes, realizes that ***e quantitatively detects.Cocaine aptamers are divided into two independent DNA chain (ACA-1 and ACA-2) by the present invention,
The two generates the secondary structure of aptamers there is no not interacting during ***e in the presence of ***e.The present invention
Note detection region is covalently fixed on using as the upconversion fluorescence nano material of fluorogenic donor and ACA-1, as fluorescent receptor
Gold nano grain surface modification ACA-2, when in body fluid to be measured contain ***e when, ACA-1 and ACA-2 react shape with ***e
Into aptamers secondary structure (Fig. 1), AuNPs is fixed on paper surface so that between upconversion fluorescence nano material and AuNPS
Distance further, in fluorescence resonance energy transfer distance range (1-10nm), AuNPs can effectively quench up-conversion fluorescence and receive
The fluorescence of rice material, there are correlations with ***e concentration for fluorescent quenching efficiency, and the paper substrate detection device reacted is placed in inspection
Survey in device (Fig. 2) and shoot fluorescence picture with mobile phone, fluorescence picture color with there are correlation, present invention profits between ***e concentration
The quantitative detection of ***e is realized with this principle.
Compared with prior art, the beneficial effects of the invention are as follows：
1st, the present invention directly with the naked eye can carry out rational judgment according to green intensity, while can also be by the use of mobile phone as letter
Number collection device, directly obtains ***e and quantifies testing result, without secondary detection, and Site Detection sensitivity can be with experiment
Room detection method is suitable, far above existing in-situ check and test method；
2nd, the present invention rationally designs the aptamer of specific recognition ***e into two sections of sequences, avoids aptamers sheet
The interference of body secondary structure reduces background signal, improves analysis detection sensitivity；
3rd, up-conversion fluorescence resonance energy is combined by the present invention with aptamers specific recognition capability, by turning in optimization
The relative concentration of fluorescent nano material and aptamers is changed, effectively improves the fluorescent quenching efficiency of system, effectively increases detection spirit
Sensitivity, qualitative detection sensitivity is up to 0.05 μM, and quantitative detection sensitivity is up to 0.5 μM.
4th, the present invention is using the excitation of upconversion fluorescence nano material near infrared light, the advantage of VISIBLE LIGHT EMISSION, directly multiple
It is detected in miscellaneous humoral sample, it is easy to operate without pre-treatment step early period, it is more applicable for Site Detection.
Description of the drawings
Fig. 1 is a kind of detection principle diagram of the ***e fast quantitative measurement method for detecting suitable for Site Detection.
Fig. 2 is a kind of detection device model of the ***e fast quantitative measurement method for detecting suitable for Site Detection.
Fig. 3 is AuNPs-ACA-2 concentration and fluorescent quenching relationship between efficiency figure in embodiment 1.
Fig. 4 is ***e concentration and fluorescent quenching relationship between efficiency figure in saliva in embodiment 1.
Specific embodiment
In order to which those skilled in the art is made to more fully understand technical scheme of the present invention, with reference to specific embodiment pair
The present invention is described in further detail.
1st, in following embodiments, the water-soluble upconversion fluorescence nano material (NaYF4：Yb, Er, wherein Y, Yb, Er
Molar ratio is 80:18:2, it is denoted as UCPs) specific preparation method is：The spherical up-conversion fluorescence that selecting surface modification has oleic acid is received
Rice material NaYF4：Yb, Er (are denoted as OA-UCPs), are dispersed in chloroform, obtain the OA-UCPs chlorine of a concentration of 1 μm of ol/L
Imitative solution；30ml diglycols and 600mg polyethyleneimines are added in 100mL there-necked flasks, system vacuumize after
110 DEG C are heated under magnetic agitation and argon gas protection, reacts 1h, Ran Housheng after 1mL OA-UCPs chloroformic solutions are added dropwise
For temperature to 240 DEG C of the reaction was continued 6h, the system for the treatment of, which is cooled to room temperature in backward flask, adds in isometric ethyl alcohol, and centrifugation is precipitated, will
It is scattered in pure water to get to upconversion fluorescence nano material after precipitation second alcohol and water centrifuge washing 3 times, is denoted as PEI-
UCPs.The upconversion fluorescence nano material pattern is spherical shape, and grain size has amino in 30-100nm, surface modification.
Certainly, as long as water-soluble upconversion fluorescence nano material can be suitably used for the present invention.
2nd, in following embodiments, the preparation of the gold nano grain and its surface markers carry out as follows：
(1) gold nano grain is prepared using trisodium citrate reduction gold chloride method：Gold chloride is first made into mass fraction is
2% aqueous solution, takes 2mL in round-bottomed flask and adds in 50mL high purity waters and be heated to boiling, and adds in vigorous stirring
The trisodium citrate aqueous solution of 5mL38.8mM continues heating and boils 15min, and flaxen aqueous solution of chloraurate becomes claret；
After system cooled to room temperature with after 0.22 μm of membrane filtration to get to the gold nano grain water of a concentration of 3.7 μm of ol/L
Solution is placed in spare in 4 DEG C of refrigerators.The gold nano grain pattern is spherical shape, and grain size has citric acid in 10-30nm, surface modification
Root.
(2) surface markers：1nmol single-chain nucleic acid aptamers ACA-2 are mixed with (2- carboxyethyls) phosphines of 5nmol tri- (TCEP)
1h is reacted at room temperature afterwards to open the disulfide bond of ACA-2；Solution is mixed with 3.7 μm of ol/L gold nano grain solution of 1.5mL after having reacted
Merge in room temperature reaction 16h, it is 0.1mol/L then gradually to adjust solution salt concentration with 2mol/L NaCl solutions, is placed in room
Warm secondary response again is for 24 hours；Products therefrom high purity water centrifuge washing three times, is then dispersed in Tris-HCl buffer solutions after having reacted
It saves backup, as AuNPs-ACA-2 solution.Wherein the composition of buffer solution is buffered for the 20mMTris-HCl of the NaCl containing 100mM
Liquid, pH ranging from 7.0-7.5.
3rd, in the present invention, paper substrate detection device is prepared：Paper is chosen as detection substrate, using water-insoluble solvent as thin
Water barrier draws a circle to approve hydrophilic detection zone in paper surface, by water-soluble upconversion fluorescence nano material and single-chain nucleic acid aptamers
ACA-1 covalent couplings are in the hydrophilic detection zone, you can obtain paper substrate detection device.
(1) paper substrate described in following embodiment is prepared as follows：Detection zone pattern is drawn on computers
And it is printed with normal printer in paper surface；The plastic formwork identical with hydrophilic detection zone pattern form is covered in
Paper substrate surface, with oil pen along detection zone pattern edge draw ink line, drawn be placed on be stored at room temperature make it is hydrophobic
Solvent in ink line volatilizees completely, and resin, organic matter remained in inside paper etc. forms hydrophobic barrier.
(2) in following embodiment by water-soluble upconversion fluorescence nano material and single-chain nucleic acid ACA-1 covalent couplings in paper
Surface is opened to be prepared as follows：Prepare NaIO4The mixed aqueous solution of a concentration of a concentration of 47mM of 26mM, LiCl, by 0.5g (really
Paper substrate is immersed in 75mL mixed aqueous solutions in fact in mass) reacts 1h in 55 DEG C；Then gained paper substrate will be impregnated
Washing, which is placed in baking oven for 2 times, dries, then added in each hydrophilic detection zone 2.6-24 μ L (volume here be V1 and
The summation of V2) PEI-UCPs and ACA-1 mixed solution (in the mixed solution, PEI-UCPs a concentration of 0.5mg/ml, ACA-1
A concentration of 0.5-4 μM, sodium cyanoborohydride NaCNBH3A concentration of 200mM, solvent are buffered for 100mM HEPES, and pH value of solution is
7.2), and in room temperature reaction 30min；The polysorbas20 (Tween 20) that paper substrate volume fraction is 0.02% after having reacted is molten
Liquid washing by soaking 5min, washing are dried afterwards twice, as paper substrate detection device, are saved backup.
Embodiment 1
A kind of ***e fast quantitative measurement method for detecting suitable for Site Detection includes the following steps：
(1) paper substrate detection device is prepared as previously described, and wherein paper selects cellulosic filter paper, circular hydrophilic detection zone
Internal diameter is 3mm, and the mixed solution of 6 μ L PEI-UCPs and ACA-1 is added in each hydrophilic detection zone, wherein in water solubility
Fluorescent nano material converted a concentration of a concentration of 2 μM of 0.5mg/mL and single-chain nucleic acid ACA-1 of PEI-UCPs；
(2) AuNPs-ACA-2 solution is prepared as previously described；
(3) concentration optimization of AuNPs-ACA-2 solution：Respectively in the hydrophilic inspection of the paper substrate detection device described in step (1)
Survey region in add in 10 μM of 3 μ L ***e solution and 3 μ L various concentrations AuNPs-ACA-2 solution (concentration is respectively 0,
0.07,0.105,0.14,0.175,0.21,0.28,0.315 μM), 40min is reacted under the conditions of 25 DEG C, with 6 μ L washing buffers
Liquid washs 5 times and removes unreacted AuNPs-ACA-2；980nm laser is used after paper substrate detection device drying after gained is reacted
Light source activation shoots the fluorescent image of hydrophilic detection zone with mobile phone under dark condition and is read with mobile phone RGB softwares
Green intensity F in fluorescent image；When setting a concentration of the 0 of AuNPs-ACA-2 solution, gained green intensity blank sample intensity is denoted as
F0, calculate the fluorescent quenching efficiency (F corresponding to various concentration AuNPs-ACA-2 solution0-F)/F0, obtain fluorescent quenching efficiency most
Greatly (54.4%) when corresponding AuNPs-ACA-2 solution concentrations C be 0.28 μM；
(4) standard solution of ***e is prepared：With saliva dilution ***e stoste, the ***e for preparing various concentration is molten
Liquid, as ***e standard solution, concentration are respectively 0,0.05,0.1,0.5,1,5,10,50 μM；
(5) ***e examination criteria curve is drawn：Respectively in the hydrophilic detection zone of the paper substrate detection device described in step (1)
The ***e standard solution of 3 μ L and the AuNPs-ACA-2 solution of 3 0.28 μM of μ L concentration are added in domain, is reacted under the conditions of 25 DEG C
40min washs 5 times with 6 μ L washing buffers and removes unreacted AuNPs-ACA-2；Paper substrate detection dress after gained is reacted
It is excited after putting drying with 980nm laser light sources, the fluorescent image for shooting hydrophilic detection zone with mobile phone under dark condition is used in combination
Mobile phone RGB softwares read green intensity F ' in fluorescent image；When setting a concentration of the 0 of ***e standard solution, gained is green
Luminous intensity blank sample intensity is denoted as F0', with fluorescent quenching efficiency (F0’-F’)/F0' it is ordinate, it is dense with ***e standard solution
The logarithm of degree draws standard curve, calibration curve equation Y=0.18X+0.60 for abscissa；
(6) in saliva to be measured ***e concentration measure：Respectively in the hydrophilic inspection of the paper substrate detection device described in step (1)
It surveys in region and adds in the body fluid to be measured of 3 μ L and the AuNPs-ACA-2 solution of 3 0.28 μM of μ L concentration, reacted under the conditions of 25 DEG C
40min washs 5 times with 6 μ L washing buffers and removes unreacted AuNPs-ACA-2；Paper substrate detection dress after gained is reacted
It is excited after putting drying with 980nm laser light sources, the fluorescent image for shooting hydrophilic detection zone with mobile phone under dark condition is used in combination
Mobile phone RGB softwares read green intensity Fx in fluorescent image；Calculate fluorescent quenching efficiency (F0’-Fx)/F0', substitute into standard
The ***e concentration that curve obtains in body fluid to be measured is 0.463 μM.
Cocaine concentration is 0.5 μM in known body fluid to be measured, and accuracy rate of testing result of the invention can reach 92.6%,
The standard deviation of duplicate measurements is 11% three times, and the accuracy and repeatability of testing result are good.
As shown in Figure 3：The concentration of AuNPs-ACA-2 influences fluorescence in a certain range in the detection method that the present invention establishes
Efficiency is quenched, AuNPs-ACA-2 concentration is bigger, and fluorescent quenching efficiency is higher.
As shown in Figure 4：The detection method that the present invention establishes can realize that ***e detects in the range of a certain concentration, cocker
Because concentration is bigger, the green-emitting fluorescent intensity of fluorescent image is weaker, and fluorescent quenching efficiency presents good with ***e concentration logarithm
Linear relationship, show that the detection method can realize qualitative detection and the quantitative detection to saliva ***e, sensitivity point
Not up to 0.05 μM and 0.5 μM.
As shown in Table 1：For the saliva sample of ***e of various concentration, the detection method testing result of the invention established
Relative standard deviation of the accuracy rate between 88.8%-103.2%, repeated detection result is less than 15%, shows that the present invention establishes
Detection method has good Stability and veracity.
Cocaine detects recovery testu in 1 saliva sample of table
| Sample number into spectrum | Add in concentration (μM) | Test concentrations (μM) | The rate of recovery (%) | Relative standard deviation (%) |
| 1 | 0.05 | 0.0516 | 103.2 | 14.6 |
| 2 | 0.5 | 0.463 | 92.6 | 11.0 |
| 3 | 2 | 1.775 | 88.8 | 8.8 |
Embodiment 2
A kind of ***e fast quantitative measurement method for detecting suitable for Site Detection includes the following steps：
(1) paper substrate detection device is prepared as previously described, and wherein paper selects cellulosic filter paper, circular hydrophilic detection zone
Internal diameter is 2mm, and the mixed solution of 2.6 μ L PEI-UCPs and ACA-1 is added in each hydrophilic detection zone, wherein water-soluble
A concentration of 0.5 μM of upconversion fluorescence nano material a concentration of 0.5mg/mL and single-chain nucleic acid ACA-1；
(2) AuNPs-ACA-2 solution is prepared as previously described；
(3) concentration optimization of AuNPs-ACA-2 solution：Respectively in the hydrophilic inspection of the paper substrate detection device described in step (1)
Surveying the AuNPs-ACA-2 solution of the ***e solution that 10 μM of 1.3 μ L are added in region and 1.3 μ L various concentrations, (concentration is respectively
0,0.07,0.105,0.14,0.175,0.21,0.28,0.315 μM), 20min is reacted under the conditions of 20 DEG C, is washed with 3 μ L slow
Fliud flushing washs 3 times and removes unreacted AuNPs-ACA-2；Swashed after paper substrate detection device drying after gained is reacted with 980nm
Radiant excites, and shoots the fluorescent image of hydrophilic detection zone with mobile phone under dark condition and is read with mobile phone RGB softwares
Take green intensity F in fluorescent image；When setting a concentration of the 0 of AuNPs-ACA-2 solution, gained green intensity blank sample intensity note
For F0, calculate the fluorescent quenching efficiency (F corresponding to various concentration AuNPs-ACA-2 solution0-F)/F0, obtain fluorescent quenching efficiency
Corresponding AuNPs-ACA-2 solution concentrations C is 0.14 μM when maximum；
(4) standard solution of ***e is prepared：Cocaine stoste is diluted with human serum, the ***e for preparing various concentration is molten
Liquid, as ***e standard solution, concentration are respectively 0,0.05,0.1,0.5,1,5,10,50 μM；
(5) ***e examination criteria curve is drawn：Respectively in the hydrophilic detection zone of the paper substrate detection device described in step (1)
The ***e standard solution of 1.3 μ L and the AuNPs-ACA-2 solution of 1.3 0.14 μM of μ L concentration are added in domain, under the conditions of 20 DEG C
20min is reacted, 3 times is washed with 3 μ L washing buffers and removes unreacted AuNPs-ACA-2；Paper substrate inspection after gained is reacted
It is excited after surveying device drying with 980nm laser light sources, shoots the fluorescent image of hydrophilic detection zone with mobile phone under dark condition
And read green intensity F ' in fluorescent image with mobile phone RGB softwares；When setting a concentration of the 0 of ***e standard solution, institute
It obtains green intensity blank sample intensity and is denoted as F0', with fluorescent quenching efficiency (F0’-F’)/F0' it is ordinate, it is molten with ***e standard
The logarithm of liquid concentration draws standard curve, calibration curve equation Y=0.11X+0.43 for abscissa；
(6) in test serum ***e concentration measure：Respectively in the hydrophilic inspection of the paper substrate detection device described in step (1)
It surveys in region and adds in the body fluid to be measured of 1.3 μ L and the AuNPs-ACA-2 solution of 1.3 0.14 μM of μ L concentration, it is anti-under the conditions of 20 DEG C
20min is answered, washing 3 times with 3 μ L washing buffers removes unreacted AuNPs-ACA-2；Paper substrate detection after gained is reacted
It is excited after device drying with 980nm laser light sources, the fluorescent image of hydrophilic detection zone is shot simultaneously with mobile phone under dark condition
Green intensity Fx in fluorescent image is read with mobile phone RGB softwares；Calculate fluorescent quenching efficiency (F0’-Fx)/F0', substitute into mark
The ***e concentration that directrix curve obtains in body fluid to be measured is 0.185 μM.
Cocaine concentration is 0.2 μM in known test serum, and accuracy rate of testing result of the invention can reach 92.5%,
The standard deviation of duplicate measurements is 9.8% three times, and the accuracy and repeatability of testing result are good.
Embodiment 3
A kind of ***e fast quantitative measurement method for detecting suitable for Site Detection includes the following steps：
(1) paper substrate detection device is prepared as previously described, and wherein paper selects cellulosic filter paper, circular hydrophilic detection zone
Internal diameter is 6mm, and the mixed solution of 24 μ L PEI-UCPs and ACA-1 is added in each hydrophilic detection zone, wherein in water solubility
A concentration of 4 μM of fluorescent nano material converted a concentration of 0.5mg/mL and single-chain nucleic acid ACA-1；
(2) AuNPs-ACA-2 solution is prepared as previously described；
(3) concentration optimization of AuNPs-ACA-2 solution：Respectively in the hydrophilic inspection of the paper substrate detection device described in step (1)
Survey region in add in 10 μM of 12 μ L ***e solution and 12 μ L various concentrations AuNPs-ACA-2 solution (concentration is respectively 0,
0.07,0.105,0.14,0.175,0.21,0.28,0.315 μM), 40min is reacted under the conditions of 37 DEG C, with 24 μ L washing buffers
Liquid washs 3 times and removes unreacted AuNPs-ACA-2；980nm laser is used after paper substrate detection device drying after gained is reacted
Light source activation shoots the fluorescent image of hydrophilic detection zone with mobile phone under dark condition and is read with mobile phone RGB softwares
Green intensity F in fluorescent image；When setting a concentration of the 0 of AuNPs-ACA-2 solution, gained green intensity blank sample intensity is denoted as
F0, calculate the fluorescent quenching efficiency (F corresponding to various concentration AuNPs-ACA-2 solution0-F)/F0, obtain fluorescent quenching efficiency most
Corresponding AuNPs-ACA-2 solution concentrations C is 0.315 μM when big；
(4) standard solution of ***e is prepared：Cocaine stoste is diluted with tear, the ***e for preparing various concentration is molten
Liquid, as ***e standard solution, concentration are respectively 0,0.05,0.1,0.5,1,5,10,50 μM；
(5) ***e examination criteria curve is drawn：Respectively in the hydrophilic detection zone of the paper substrate detection device described in step (1)
The ***e standard solution of 12 μ L and the AuNPs-ACA-2 solution of 12 0.315 μM of μ L concentration are added in domain, it is anti-under the conditions of 37 DEG C
40min is answered, washing 3 times with 24 μ L washing buffers removes unreacted AuNPs-ACA-2；Paper substrate detection after gained is reacted
It is excited after device drying with 980nm laser light sources, the fluorescent image of hydrophilic detection zone is shot simultaneously with mobile phone under dark condition
Green intensity F ' in fluorescent image is read with mobile phone RGB softwares；When setting a concentration of the 0 of ***e standard solution, gained
Green intensity blank sample intensity is denoted as F0', with fluorescent quenching efficiency (F0’-F’)/F0' it is ordinate, with ***e standard solution
The logarithm of concentration draws standard curve, calibration curve equation Y=0.21X+0.55 for abscissa；
(6) in tear to be measured ***e concentration measure：Respectively in the hydrophilic inspection of the paper substrate detection device described in step (1)
It surveys in region and adds in the body fluid to be measured of 12 μ L and the AuNPs-ACA-2 solution of 12 0.315 μM of μ L concentration, reacted under the conditions of 37 DEG C
40min washs 3 times with 24 μ L washing buffers and removes unreacted AuNPs-ACA-2；Paper substrate detection dress after gained is reacted
It is excited after putting drying with 980nm laser light sources, the fluorescent image for shooting hydrophilic detection zone with mobile phone under dark condition is used in combination
Mobile phone RGB softwares read green intensity Fx in fluorescent image；Calculate fluorescent quenching efficiency (F0’-Fx)/F0', substitute into standard
The ***e concentration that curve obtains in body fluid to be measured is 11.025 μM.
Cocaine concentration is 10 μM in known tear to be measured, and accuracy rate of testing result of the invention can reach
110.25%, the standard deviation of duplicate measurements is 8.3% three times, and the accuracy and repeatability of testing result are good.
Embodiment 4
A kind of ***e fast quantitative measurement method for detecting suitable for Site Detection includes the following steps：
(1) paper substrate detection device is prepared as previously described, and wherein paper selects cellulosic filter paper, circular hydrophilic detection zone
Internal diameter is 4mm, the mixed solution of 10 μ L PEI-UCPs and ACA-1 is added in each hydrophilic detection zone, wherein water-soluble
A concentration of 1 μM of upconversion fluorescence nano material a concentration of 0.5mg/mL and single-chain nucleic acid ACA-1；
(2) AuNPs-ACA-2 solution is prepared as previously described；
(3) concentration optimization of AuNPs-ACA-2 solution：Respectively in the hydrophilic inspection of the paper substrate detection device described in step (1)
Survey region in add in 10 μM of 5 μ L ***e solution and 5 μ L various concentrations AuNPs-ACA-2 solution (concentration is respectively 0,
0.07,0.105,0.14,0.175,0.21,0.28,0.315 μM), 60min is reacted under the conditions of 30 DEG C, with 10 μ L washing buffers
Liquid washs 5 times and removes unreacted AuNPs-ACA-2；980nm laser is used after paper substrate detection device drying after gained is reacted
Light source activation shoots the fluorescent image of hydrophilic detection zone with mobile phone under dark condition and is read with mobile phone RGB softwares
Green intensity F in fluorescent image；When setting a concentration of the 0 of AuNPs-ACA-2 solution, gained green intensity blank sample intensity is denoted as
F0, calculate the fluorescent quenching efficiency (F corresponding to various concentration AuNPs-ACA-2 solution0-F)/F0, obtain fluorescent quenching efficiency most
Corresponding AuNPs-ACA-2 solution concentrations C is 0.28 μM when big；
(4) standard solution of ***e is prepared：Cocaine stoste is diluted with urine, the ***e for preparing various concentration is molten
Liquid, as ***e standard solution, concentration are respectively 0,0.05,0.1,0.5,1,5,10,50 μM；
(5) ***e examination criteria curve is drawn：Respectively in the hydrophilic detection zone of the paper substrate detection device described in step (1)
The ***e standard solution of 5 μ L and the AuNPs-ACA-2 solution of 5 0.28 μM of μ L concentration are added in domain, is reacted under the conditions of 30 DEG C
60min washs 5 times with 10 μ L washing buffers and removes unreacted AuNPs-ACA-2；Paper substrate detection dress after gained is reacted
It is excited after putting drying with 980nm laser light sources, the fluorescent image for shooting hydrophilic detection zone with mobile phone under dark condition is used in combination
Mobile phone RGB softwares read green intensity F ' in fluorescent image；When setting a concentration of the 0 of ***e standard solution, gained is green
Luminous intensity blank sample intensity is denoted as F0', with fluorescent quenching efficiency (F0’-F’)/F0' it is ordinate, it is dense with ***e standard solution
The logarithm of degree draws standard curve, calibration curve equation Y=0.15X+0.57 for abscissa；
(6) in urine to be measured ***e concentration measure：Respectively in the hydrophilic inspection of the paper substrate detection device described in step (1)
It surveys in region and adds in the body fluid to be measured of 5 μ L and the AuNPs-ACA-2 solution of 5 0.28 μM of μ L concentration, reacted under the conditions of 30 DEG C
60min washs 5 times with 10 μ L washing buffers and removes unreacted AuNPs-ACA-2；Paper substrate detection dress after gained is reacted
It is excited after putting drying with 980nm laser light sources, the fluorescent image for shooting hydrophilic detection zone with mobile phone under dark condition is used in combination
Mobile phone RGB softwares read green intensity Fx in fluorescent image；Calculate fluorescent quenching efficiency (F0’-Fx)/F0', substitute into standard
The ***e concentration that curve obtains in body fluid to be measured is 26.783 μM.
Cocaine concentration is 25 μM in known urine to be measured, and accuracy rate of testing result of the invention can reach
107.13%, the standard deviation of duplicate measurements is 6.9% three times, and the accuracy and repeatability of testing result are good.
Embodiment 5
A kind of ***e fast quantitative measurement method for detecting suitable for Site Detection includes the following steps：
(1) paper substrate detection device is prepared as previously described, and wherein paper selects cellulosic filter paper, circular hydrophilic detection zone
Internal diameter is 5mm, the mixed solution of 16 μ L PEI-UCPs and ACA-1 is added in each hydrophilic detection zone, wherein water-soluble
A concentration of 2 μM of upconversion fluorescence nano material a concentration of 0.5mg/mL and single-chain nucleic acid ACA-1；
(2) AuNPs-ACA-2 solution is prepared as previously described；
(3) concentration optimization of AuNPs-ACA-2 solution：Respectively in the hydrophilic inspection of the paper substrate detection device described in step (1)
Survey region in add in 10 μM of 8 μ L ***e solution and 8 μ L various concentrations AuNPs-ACA-2 solution (concentration is respectively 0,
0.07,0.105,0.14,0.175,0.21,0.28,0.315 μM), 40min is reacted under the conditions of 37 DEG C, with 16 μ L washing buffers
Liquid washs 5 times and removes unreacted AuNPs-ACA-2；980nm laser is used after paper substrate detection device drying after gained is reacted
Light source activation shoots the fluorescent image of hydrophilic detection zone with mobile phone under dark condition and is read with mobile phone RGB softwares
Green intensity F in fluorescent image；When setting a concentration of the 0 of AuNPs-ACA-2 solution, gained green intensity blank sample intensity is denoted as
F0, calculate the fluorescent quenching efficiency (F corresponding to various concentration AuNPs-ACA-2 solution0-F)/F0, obtain fluorescent quenching efficiency most
Corresponding AuNPs-ACA-2 solution concentrations C is 0.21 μM when big；
(4) standard solution of ***e is prepared：Cocaine stoste is diluted with sweat, the ***e for preparing various concentration is molten
Liquid, as ***e standard solution, concentration are respectively 0,0.05,0.1,0.5,1,5,10,50 μM；
(5) ***e examination criteria curve is drawn：Respectively in the hydrophilic detection zone of the paper substrate detection device described in step (1)
The ***e standard solution of 8 μ L and the AuNPs-ACA-2 solution of 8 0.21 μM of μ L concentration are added in domain, is reacted under the conditions of 37 DEG C
40min washs 5 times with 16 μ L washing buffers and removes unreacted AuNPs-ACA-2；Paper substrate detection dress after gained is reacted
It is excited after putting drying with 980nm laser light sources, the fluorescent image for shooting hydrophilic detection zone with mobile phone under dark condition is used in combination
Mobile phone RGB softwares read green intensity F ' in fluorescent image；When setting a concentration of the 0 of ***e standard solution, gained is green
Luminous intensity blank sample intensity is denoted as F0', with fluorescent quenching efficiency (F0’-F’)/F0' it is ordinate, it is dense with ***e standard solution
The logarithm of degree draws standard curve, calibration curve equation Y=0.169X+0.63 for abscissa；
(6) in sweat to be measured ***e concentration measure：Respectively in the hydrophilic inspection of the paper substrate detection device described in step (1)
It surveys in region and adds in the body fluid to be measured of 8 μ L and the AuNPs-ACA-2 solution of 8 0.21 μM of μ L concentration, reacted under the conditions of 37 DEG C
40min washs 5 times with 16 μ L washing buffers and removes unreacted AuNPs-ACA-2；Paper substrate detection dress after gained is reacted
It is excited after putting drying with 980nm laser light sources, the fluorescent image for shooting hydrophilic detection zone with mobile phone under dark condition is used in combination
Mobile phone RGB softwares read green intensity Fx in fluorescent image；Calculate fluorescent quenching efficiency (F0’-Fx)/F0', substitute into standard
The ***e concentration that curve obtains in body fluid to be measured is 0.046 μM.
Cocaine concentration is 0.05 μM in known body fluid to be measured, and accuracy rate of testing result of the invention can reach 92%,
The standard deviation of duplicate measurements is 12.8% three times, and the accuracy and repeatability of testing result are good.
The above is only the preferred embodiment of the present invention, it is noted that those of ordinary skill in the art are come
It says, without departing from the concept of the premise of the invention, several modifications and variations can also be made, these belong to the present invention's
Protection domain.
Claims (10)
1. a kind of ***e fast quantitative measurement method for detecting suitable for Site Detection, it is characterised in that include the following steps：
(1) paper substrate detection device is prepared：Paper is chosen as detection substrate, using water-insoluble solvent as hydrophobic barrier in paper
Hydrophilic detection zone is drawn a circle to approve on surface, by water-soluble upconversion fluorescence nano material and single-chain nucleic acid aptamers ACA-1 covalent couplings
In the hydrophilic detection zone, paper substrate detection device is obtained；
(2) gold nano grain is chosen and in its surface modification single-chain nucleic acid aptamers ACA-2, products therefrom is denoted as AuNPs-ACA-
2, it is scattered in buffer solution, obtains AuNPs-ACA-2 solution；
(3) concentration optimization of AuNPs-ACA-2 solution：Respectively in the hydrophilic detection zone of the paper substrate detection device described in step (1)
The AuNPs-ACA-2 solution that volume is the ***e solution of V1 fixed concentrations and volume is V2 various concentrations, reaction are added in domain
Unreacted AuNPs-ACA-2 is removed after the completion；980nm laser light sources are used after paper substrate detection device drying after gained is reacted
Excitation shoots the fluorescent image of hydrophilic detection zone under dark condition and reads green intensity F in fluorescent image with software；If
When determining a concentration of the 0 of AuNPs-ACA-2 solution, gained green intensity blank sample intensity is denoted as F0, calculate various concentration AuNPs-
Fluorescent quenching efficiency (F corresponding to ACA-2 solution0-F)/F0, obtain AuNPs- corresponding during fluorescent quenching efficiency maximum
ACA-2 solution concentrations C；
(4) standard solution of ***e is prepared：The ***e solution of various concentration is prepared with body fluid, as ***e standard is molten
Liquid；
(5) ***e examination criteria curve is drawn：Respectively in the hydrophilic detection zone of the paper substrate detection device described in step (1)
The ***e standard solution that volume is V1 and the AuNPs-ACA-2 solution that volume is a concentration of C of V2 are added in, is removed after the completion of reaction
Unreacted AuNPs-ACA-2；It is excited after paper substrate detection device drying after gained is reacted with 980nm laser light sources, in black
The fluorescent image of hydrophilic detection zone is shot under dark condition and reads green intensity F ' in fluorescent image with software；Set ***e
Standard solution a concentration of 0 when, gained green intensity blank sample intensity is denoted as F0', with fluorescent quenching efficiency (F0’-F’)/F0' be
Ordinate draws standard curve by abscissa of the logarithm of ***e concentration of standard solution；
(6) in body fluid to be measured ***e concentration measure：Respectively in the hydrophilic detection zone of the paper substrate detection device described in step (1)
The body fluid to be measured that volume is V1 and the AuNPs-ACA-2 solution that volume is a concentration of C of V2 are added in domain, is removed not after the completion of reaction
The AuNPs-ACA-2 of reaction；It is excited after paper substrate detection device drying after gained is reacted with 980nm laser light sources, in dark
Under the conditions of shoot hydrophilic detection zone fluorescent image and with software read fluorescent image in green intensity Fx；Calculate fluorescent quenching
Efficiency (F0’-Fx)/F0', it substitutes into standard curve and obtains the ***e concentration in body fluid to be measured.
2. a kind of ***e fast quantitative measurement method for detecting suitable for Site Detection according to claim 1, feature exist
In the hydrophilic detection zone shape for circle, internal diameter 2-6mm.
3. a kind of ***e fast quantitative measurement method for detecting suitable for Site Detection according to claim 1, feature exist
It is spherical shape in the water-soluble upconversion fluorescence nano material pattern, grain size has amino in 30-100nm, surface modification.
4. a kind of ***e fast quantitative measurement method for detecting suitable for Site Detection according to claim 1, feature exist
It is spherical shape in the gold nano grain pattern, grain size has citrate in 10-30nm, surface modification.
5. a kind of ***e fast quantitative measurement method for detecting suitable for Site Detection according to claim 1, feature exist
It is respectively in the single-chain nucleic acid ACA-1 and single-chain nucleic acid ACA-2, sequence：5’-NH2-TTTTTCAAGAACTGAGGG-
3 ' and 5 '-SH-TTTTTACAGCAGGGTGAAGTAACTTCTTG-3 '.
6. a kind of ***e fast quantitative measurement method for detecting suitable for Site Detection according to claim 1, feature exist
Buffer solution described in step (2) is Tris-HCl buffer solutions, and pH ranges are in 7.0-7.5.
7. a kind of ***e fast quantitative measurement method for detecting suitable for Site Detection according to claim 1, feature exist
The concentration range of AuNPs-ACA-2 solution is 0-0.305 μM in step (3).
8. a kind of ***e fast quantitative measurement method for detecting suitable for Site Detection according to claim 1, feature exist
It is washed with buffer solution to remove unreacted AuNPs-ACA-2, the pH ranging from 7.0- of the buffer solution in step (3)
7.5。
9. a kind of ***e fast quantitative measurement method for detecting suitable for Site Detection according to claim 1, feature exist
The concentration of ***e standard solution is respectively 0-50 μM in the step (4).
10. a kind of ***e fast quantitative measurement method for detecting suitable for Site Detection according to claim 1, feature exist
Mobile phone may be used in the step (3) and step (5), (6) and shoot hydrophilic detection zone fluorescent image, use mobile phone
RGB softwares read green intensity in fluorescent image.
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