CN106092982A - Cancerous cell and the preparation of the cell surface polysaccharide detection double mode paper chip of fluorescence/colorimetric - Google Patents
Cancerous cell and the preparation of the cell surface polysaccharide detection double mode paper chip of fluorescence/colorimetric Download PDFInfo
- Publication number
- CN106092982A CN106092982A CN201610384836.5A CN201610384836A CN106092982A CN 106092982 A CN106092982 A CN 106092982A CN 201610384836 A CN201610384836 A CN 201610384836A CN 106092982 A CN106092982 A CN 106092982A
- Authority
- CN
- China
- Prior art keywords
- paper chip
- fluorescence
- cancerous cell
- chain
- colorimetric
- 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.)
- Granted
Links
Classifications
-
- 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
- G01N21/6402—Atomic fluorescence; Laser induced fluorescence
-
- 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
-
- 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
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
Landscapes
- Health & Medical Sciences (AREA)
- Physics & Mathematics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Engineering & Computer Science (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Optics & Photonics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Plasma & Fusion (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
The method that the invention discloses the preparation of a kind of cancerous cell and the cell surface polysaccharide detection double mode paper chip of fluorescence/colorimetric.Wax printing technique is utilized to prepare paper chip, silver-colored and the aptamers at fluorescence coating working region growth gold nanorods and porous, and then carry out the capture of cancerous cell, racemosus chain hybridization technique is utilized to realize the amplification of signaling molecule, thus realize the accurate measurement of cancerous cell, the paper chip prepared is folded, drips chromophoric solution at conversion zone, thus realize the visual preliminary assay of cancerous cell.It is subsequently added polysaccharide inhibitor, thus realizes the dynamic characterization of cell surface polysaccharide.
Description
Technical field
The present invention relates to low cost, be easy to carry about with one, visual cancerous cell and cell surface polysaccharide analysis and detection technology neck
Territory, is prepared by a kind of double mode sensor of fluorescence/colorimetric that can detect cancerous cell and cell surface polysaccharide in particular, this
Invention further relates to have employed racemosus hybridization chain signal amplification technique.
Background technology
In recent years, cancer has become the number one killer of the mankind, greatly threatens human health, the transfer of cancerous cell and
Diffusion is the major reason that cancer is fatal, and detection the most early and in time treatment are the basic methods overcoming cancer.Polysaccharide is thin
The important feature of cellular surface, polysaccharide is distributed in surface of cell membrane and inside by highdensity with albumen or lipid covalent effect.
The dynamically change of cell surface glycoprotein and the transfer of cell, break up closely related, the therefore detection tool to cell surface polysaccharide
Significant.At present, a lot of methods (such as high performance liquid chromatography, mass spectrum, nuclear magnetic resonance, NMR etc.) are applied to the analysis of polysaccharide, but
The destructiveness being these methods is very strong, and the dynamic characterization making liver cell surface polysaccharide express is infeasible.Additionally, these methods are taken
Time, need complicated sample pre-treatments and expensive instrument.And the micro-fluidic visual colorimetric detection of paper substrate analytical equipment is with glimmering
Achieve the combining of light detection low cost, be easy to carry about with one, visual initial survey and the perfect adaptation that accurately measures.
In order to improve cancerous cell and the sensitivity of polysaccharide detection, we have employed racemosus cross chain reaction, miscellaneous with traditional
Interlinkage reaction difference, racemosus cross chain reaction can produce the product of the length of many support arms, and it can adsorb more signaling molecule
Thus realize effective signal and amplify.Simultaneously in order to improve cancerous cell and the sensitivity of polysaccharide colorimetric detection, use noble metal multiple
Conjunction nano material is as analogue enztme, and because its specific surface area is big, good biocompatibility, catalytic performance advantages of higher, in science and technology
Field causes huge concern.Growth has the paper chip of this noble metal composite nano materials, not only increases paper chip
Specific surface area, and effectively reduce the background fluorescence of paper.More it is made to urge due to the collaborative and electronic effect between different component
Change performance to be greatly improved, thus be widely used in various types of biosensor.Use quantum dot as biological
Probe, due to physics and the chemical property of its uniqueness, such as good biocompatibility, easily prepares, wide excitation spectrum, anti-light bleaching,
Narrow, symmetrical, adjustable emission spectrum so that quantum dot is used for label biological materials, has than using organic fluorescent dye
Preferably fluorescent characteristic.Supplement with quantum dot or part replaces organic fluorescence marker material, hypersensitive, high stable can be started
And the bioanalysis detection technique of long luminescence lifetime.In view of above advantage, quantum dot will become the most promising fluorescence
Label, thus quantum dot substitute fluorescent dye substantially increase as fluorescent marker liver cell surface polysaccharide detection can
Row.
Summary of the invention
It is an object of the invention to provide a kind of length and have gold nanorods and the paper chip of porous silver and platinoid nano chain and oxidation
The racemosus hybridization chain signal amplification technique of modified by graphene quantum dot realizes the preparation method of the double mode sensor of fluorescence/colorimetric,
For cancerous cell and quick, the Sensitive Detection of cancer cell surfaces polysaccharide.
In order to solve above-mentioned technical problem, the present invention is to have gold nanorods and the paper chip of porous silver and platinum by a kind of length
The racemosus hybridization chain signal amplification technique that copper nano chain and graphene oxide quantum dot are modified realizes, it is characterized in that including with
Lower step:
(1) design paper chip fluorescence coating and than the hydrophobic wax print area of chromatograph and hydrophilic working region (accompanying drawing 1)；
(2) at the working region of α fluorescence coating growth gold nanorods and porous silver layer；
(3) aptamers chain is fixed on the working region of the paper chip of step (2) gained, uses bovine serum albumin to close subsequently
Avtive spot, last aptamers chain capture cancerous cell；
(4) racemosus hybridization chain platinoid (PtCu) nano chain and graphene quantum dot (GQDs) modified is fixed on step (3) institute
The working region of the paper chip obtained；
(5) as shown in Figure 2, the paper chip of step (4) gained is folded, β than the working region of chromatograph drip chromogenic substrate,
PH is buffer solution and the hydrogen peroxide of 4 ~ 6, carries out colorimetric preliminary assay；
(6) accurately fluoremetry: put in fluorescence equipment by the paper chip of step (4) gained, at excitation wavelength 360 nm with send out
Fluoremetry is carried out under ejected wave length 460 nm；
(7) 10 ~ 30 L polysaccharide inhibitor tunicamycins are dripped in the paper chip working region of step (4) gained；
(8) by the paper chip of step (7) gained for the mensuration of cell surface glycosyl, step (5) and step (6) are repeated.
Fluorescence/colorimetric double mode paper chip pattern Adobe illustrator CS4 software design designed by the present invention
The hydrophobic wax print pattern of paper chip, the paper of paper chip used is chromatographic paper.
The working region of fluorescence coating of the present invention grows the step of gold nanorods and porous silver layer:
(1) synthesis gold seeds: the secondary water measuring 160 mL is placed in there-necked flask, is heated to 90 C, is subsequently added 1.6
ML gold chloride, heating in water bath, to 96 C, reacts 1 min, adds 5.6 mL 1% sodium citrates immediately, and stirs 15 min extremely
Solution becomes claret；The gold seeds taking 20 L is added drop-wise to the working region of fluorescence coating, at room temperature place 45 min；
(2) synthesis gold nanorods and porous silver layer: take 1% gold chloride of 20.0 L and 200 mM oxammonium hydrochloride .s of fresh preparation
It is added drop-wise to the working region of gold seeds, growth at RT 10 min；Subsequently by 20.0 L 24 mM nitre of fresh preparation
Acid silver and 200 mM ascorbic acid are added drop-wise to above-mentioned length to be had on the paper of gold nanorods, growth at RT 10 min；Finally, water is used
Thoroughly clean, be dried 30 min under room temperature, prepare gold nanorods and porous silver layer in paper chip.
The working region of the paper chip that aptamers chain is fixed on step (2) gained of the present invention, uses cattle subsequently
Serum albumin closes avtive spot, and the step of last aptamers chain capture cancerous cell is:
20 L aptamers are fixed on gold nanorods and on paper chip working region that porous modified by silver is crossed, are subsequently added 20
L bovine serum albumin, to close avtive spot, is eventually adding the cancerous cell of 20 L variable concentrations.
Racemosus hybridization chain PtCu nano chain and GQDs modified of the present invention is fixed on the refill of step (3) gained
The working region of sheet, step is:
(1) synthesis PtCu nano chain: 1 mL tbp, 18 mg six water Nickel dichloride .s and bis-water of 22 mL are joined
In 100 mL there-necked flasks, ultrasonic 15 min after shaking up, under nitrogen protection, strong agitation limit, limit drips the 5 fresh preparations of mL
2 mg mL-1Sodium borohydride, immediately adds 10 mL 6 mM potassium platinic chloride and the mixed liquors of copper chloride, by above-mentioned molten
Ultrasonic 30 min of liquid, afterwards heating in water bath 2 h in 60 C water-baths, finally with ethanol and washing, be dried to obtain PtCu
Nano chain；
(2) synthesis GQDs: weighing 2 g citric acids and be put in 5 mL beakers, be heated to 180 C, solution colour becomes orange, adjusts
Joint pH, to neutral, i.e. can get GQDs；
(3) the racemosus hybridization chain that PtCu nano chain and GQDs modify: I-Con A Concanavalin (I-Con A) and H1-GQDs amber
The method of amber acid imide coupling obtains；I-Con A, H1-GQDs and H2 PTC-200 is heated to 95 C and keeps 10
Min, and in 30 s, it is cooled to 4 C, subsequently, 100 L I-Con A are added to 1 mL H1-GQDs and H2 (1:1)
In, 37 C react overnight；Finally, in above-mentioned solution, add 1 mL PtCu nano chain, react 15 min, centrifugal, will
To product be dispersed in buffer solution；
(4) the racemosus hybridization chain that fixing PtCu nano chain and GQDs modify: 20 L PtCu nano chain and GQDs are modified
Racemosus hybridization chain is fixed on the paper chip working region of modified.
Chromogenic substrate of the present invention is 3,3,5,5-tetramethyl benzidine, o-phenylenediamine, 2,2-azine groups-bis--(3-
Ethyl benzo thiazoline quinoline-6-sulfonic acid) di-ammonium salts.
PH of the present invention be the buffer solution of 4 ~ 6 be NaAc_HAc buffer solution, phosphate buffered solution,
Tris hydrochloric acid buffer solution.
Cell surface glycosyl colorimetric preliminary assay step of the present invention, drip chromogenic substrate respectively, pH be 4 ~ 6 buffering molten
Liquid and hydrogen peroxide, carry out the mensuration of sample, draws the standard curve of gray scale and cancerous cell concentration, it is achieved the visualization of cancerous cell
Preliminary examination, fixes cell concentration constant subsequently, changes the concentration of tunicamycin, draws the pass of gray scale and glycosyl inhibition agent tunicamycin concentration
System, it is achieved the visualization preliminary examination of cancer cell surfaces polysaccharide.
Cell surface glycosyl fluoremetry step of the present invention, puts into paper chip in fluorescence ware, carries out the survey of sample
Fixed, draw the standard curve of fluorescence intensity and cancerous cell concentration, it is achieved the accurate detection of cancerous cell, fix cell concentration subsequently not
Become, change the concentration of tunicamycin, draw the relation of fluorescence intensity and glycosyl inhibition agent tunicamycin concentration, it is achieved cancer cell surfaces is many
The accurate detection of sugar.
Beneficial effects of the present invention
(1) gold nanorods and the use of porous silver layer, increases specific surface area and effectively reduces the background fluorescence of paper, improve inspection
The sensitivity surveyed.
(2) employing of racemosus hybridization chain, effectively achieves the amplification of fluorescence and colorimetric signal, makes signal obtain significantly
Raising.
(3) replacing fluorescent dye with quantum dot, ratio uses organic fluorescence molecule to have more preferable fluorescent characteristic, and starts super
Sensitive, high stable and the bioanalysis detection technique of long luminescence lifetime.
(4) by the combination of colorimetric/fluorescent dual module formula biosensor, it is achieved that cancerous cell and cell surface polysaccharide can
Depending on change colorimetric initial survey and carry out accurate fluorescence measurement.
Accompanying drawing explanation
Fig. 1: paper chip α fluorescence coating and β are than the hydrophobic wax print pattern of chromatograph；
The size of Fig. 2: paper chip and each layer function；The folding mode of paper chip.
Detailed description of the invention
Embodiment 1
The application in MCF-7 cell of fluorescence/colorimetric double mode paper chip:
(1) design paper chip fluorescence coating and ratio the hydrophobic wax print area of chromatograph and hydrophilic working region on computers, will figure
Case is printed upon A4 size chromatographic paper；
(2) in the working region of step (1) gained paper chip fluorescence coating growth gold nanorods and porous silver layer: measure 160 mL's
Secondary water is placed in there-necked flask, is heated to 90 C, is subsequently added 1.6 mL gold chlorides, heating in water bath to 96 C, reacts 1
Min, adds 5.6 mL 1% sodium citrates immediately, and stirs 15 min and become claret to solution；The gold seeds taking 20 L drips
It is added to the working region of fluorescence coating, at room temperature place 45 min；
200 mM oxammonium hydrochloride .s of 1% gold chloride and fresh preparation that take 20.0 L are added drop-wise on the paper of gold seeds,
Growth at RT 10 min；Subsequently 20.0 L 24 mM silver nitrate and the 200 mM ascorbic acid of fresh preparation are added drop-wise to
State in the long sheet hole having gold nanorods, at room temperature grow 10 min；Finally, thoroughly clean with water, and be dried at room temperature for
30 min；
(3) aptamers is fixed on the working region of the paper chip of step (2) gained, 20 L aptamers are fixed on gold nano
On the paper chip working region of rod and porous silver layer modified, it is subsequently added 20 L bovine serum albumin to close active sites
Point, is eventually adding the MCF-7 cell of the 20 a series of variable concentrations of L；
(4) the racemosus hybridization chain that PtCu nano chain and GQDs modify is fixed on the working region of the paper chip of step (3) gained, closes
Become PtCu nano chain: 1 mL tbp, 18 mg six water Nickel dichloride .s and bis-water of 22 mL are joined 100 mL tri-mouthfuls
In flask, ultrasonic 15 min after shaking up, under nitrogen protection, strong agitation limit, limit drips 2 mg mL of the 5 fresh preparations of mL-1
Sodium borohydride, immediately adds 10 mL 6 mM potassium platinic chloride and the mixed liquors of copper chloride, by ultrasonic for above-mentioned solution 30
Min, afterwards heating in water bath 2 h in 60 C water-baths, finally with ethanol and washing, be dried to obtain PtCu nano chain；
Synthesis GQDs: weighing 2 g citric acids and be put in 5 mL beakers, be heated to 180 C, solution colour becomes orange, regulates pH
To neutrality, i.e. can get GQDs；
The racemosus hybridization chain that PtCu nano chain and GQDs modify: the method that I-Con A and H1-GQDs couples with butanimide
Obtain；I-Con A, H1-GQDs and H2 PTC-200 is heated to 95 C and keeps 10 min, and cool down in 30 s
To 4 C, subsequently, 100 L I-Con A are added in 1 mL H1-GQDs and H2 (1:1), and 37 C react overnight；Finally,
In above-mentioned solution, add 1 mL PtCu nano chain, react 15 min, centrifugal, the product obtained is dispersed in phosphate and delays
In dissolved liquid；
The racemosus hybridization chain that fixing PtCu nano chain and GQDs modify: the racemosus that 20 L PtCu nano chain and GQDs are modified
Hybridization chain is fixed on the paper chip working region of modified；
(5) paper chip is put in fluorescence ware, carry out the mensuration of sample, draw fluorescence intensity bent with the standard of cancerous cell concentration
Line, it is achieved the accurate detection of MCF-7 cell, folds paper chip, drips 30 L 3,3,5,5-tetramethyl benzidine, pH respectively
It is 4 Acetic acid-sodium acetate buffer and hydrogen peroxide, carries out the mensuration of sample, draw the standard curve of gray scale and cancerous cell concentration,
Realize the visualization preliminary examination of MCF-7 cell.
Embodiment 2
The application in MCF-7 cell surface polysaccharide detects of fluorescence/colorimetric double mode paper chip:
(1) design paper chip fluorescence coating and ratio the hydrophobic wax print area of chromatograph and hydrophilic working region on computers, will figure
Case is printed upon A4 size chromatographic paper；
(2) in the working region of step (1) gained paper chip fluorescence coating growth gold nanorods and porous silver layer: measure 160 mL's
Secondary water is placed in there-necked flask, is heated to 90 C, is subsequently added 1.6 mL gold chlorides, heating in water bath to 96 C, reacts 1
Min, adds 5.6 mL 1% sodium citrates immediately, and stirs 15 min and become claret to solution；The gold seeds taking 20 L drips
It is added to the working region of fluorescence coating, at room temperature place 45 min；
200 mM oxammonium hydrochloride .s of 1% gold chloride and fresh preparation that take 20.0 L are added drop-wise on the paper of gold seeds,
Growth at RT 10 min；Subsequently 20.0 L 24 mM silver nitrate and the 200 mM ascorbic acid of fresh preparation are added drop-wise to
State in the long sheet hole having gold nanorods, at room temperature grow 10 min；Finally, thoroughly clean with water, and be dried at room temperature for
30 min；
(3) aptamers is fixed on the working region of the paper chip of step (2) gained, 20 L aptamers are fixed on through Jenner
On the paper chip working region of rice rod and porous silver layer modified, it is subsequently added 20 L bovine serum albumin to close active sites
Point, is eventually adding 20 L fixed concentrations (106Cells/mL) MCF-7 cell；
(4) the racemosus hybridization chain that PtCu nano chain and GQDs modify is fixed on the working region of the paper chip of step (3) gained, closes
Become PtCu nano chain: 1 mL tbp, 18 mg six water Nickel dichloride .s and bis-water of 22 mL are joined 100 mL tri-mouthfuls
In flask, ultrasonic 15 min after shaking up, under nitrogen protection, strong agitation limit, limit drips 2 mg mL of the 5 fresh preparations of mL-1
Sodium borohydride, immediately adds 10 mL 6 mM potassium platinic chloride and the mixed liquors of copper chloride, by ultrasonic for above-mentioned solution 30
Min, afterwards heating in water bath 2 h in 60 C water-baths, finally with ethanol and washing, be dried to obtain PtCu nano chain；
Synthesis GQDs: weighing 2 g citric acids and be put in 5 mL beakers, be heated to 180 C, solution colour becomes orange, regulates pH
To neutrality, i.e. can get GQDs；
The racemosus hybridization chain that PtCu nano chain and GQDs modify: the method that I-Con A and H1-GQDs couples with butanimide
Obtain；I-Con A, H1-GQDs and H2 PTC-200 is heated to 95 C and keeps 10 min, and cool down in 30 s
To 4 C, subsequently, 100 L I-Con A are added in 1 mL H1-GQDs and H2 (1:1), and 37 C react overnight；Finally,
In above-mentioned solution, add 1 mL PtCu nano chain, react 15 min, centrifugal, the product obtained is dispersed in phosphate and delays
In dissolved liquid；
The racemosus hybridization chain that fixing PtCu nano chain and GQDs modify: the racemosus that 20 L PtCu nano chain and GQDs are modified
Hybridization chain is fixed on the paper chip working region of modified；
(5) 20 L sugar chain inhibitor tunicamycins of variable concentrations are dripped；
(6) paper chip is put in fluorescence ware, carry out the mensuration of sample, draw fluorescence intensity and sugar chain inhibitor tunicamycin concentration
Relation, it is achieved the accurate detection of MCF-7 cell surface polysaccharide, paper chip is folded, drips 30 L 3,3,5,5-four respectively
Methyl biphenyl amine, pH are 4 Acetic acid-sodium acetate buffer and hydrogen peroxide, carry out the mensuration of sample, draw gray scale and glycosyl inhibition
The relation of agent tunicamycin concentration, it is achieved the visualization preliminary examination of MCF-7 cell surface polysaccharide.
SEQUENCE LISTING
<110>University Of Ji'nan
<120>cancerous cell and the preparation of the cell surface polysaccharide detection double mode paper chip of fluorescence/colorimetric
<130> 2016
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 33
<212> DNA
<213>artificial sequence
<400> 1
aatctatttc tagagcacaa tcacaggagc cag 33
<210> 2
<211> 59
<212> DNA
<213>artificial sequence
<400> 2
ttttttttct ggctcctgtg attgtgctct agtttacatc gctagagcac aatcacagg 59
<210> 3
<211> 58
<212> DNA
<213>artificial sequence
<400> 3
ttttctagag cacaatcaca ggagccagtt acctgtgatt gtgctctagc gatgtttt 58
Claims (9)
1. cancerous cell and the preparation of the cell surface polysaccharide detection double mode paper chip of fluorescence/colorimetric, is characterized in that including following step
Rapid:
1.1 design paper chip fluorescence coatings and than the hydrophobic wax print area of chromatograph and hydrophilic working region；
1.2 at the working region of α fluorescence coating growth gold nanorods and porous silver layer；
The working region of 1.3 paper chip that aptamers chain is fixed on step 1.2 gained, uses bovine serum albumin to close subsequently
Avtive spot, last aptamers chain capture cancerous cell；
The racemosus hybridization chain that platinoid (PtCu) nano chain and graphene quantum dot (GQDs) are modified by 1.4 is fixed on step 1.3 institute
The working region of the paper chip obtained；
1.5 as shown in Figure 2, the paper chip of step 1.4 gained is folded, drips colour developing at β respectively than the working region of chromatograph
Substrate, pH are buffer solution and the hydrogen peroxide of 4 ~ 6, carry out colorimetric preliminary assay；
1.6 accurate fluoremetries: again the paper chip of step 1.4 gained is put in fluorescence equipment, at excitation wavelength 360 nm and
Launch and carry out fluoremetry under wavelength 460 nm；
1.7 in the paper chip working region of step 1.4 gained dropping 10 ~ 30 L polysaccharide inhibitor tunicamycins；
The paper chip of step 1.7 gained is used for the mensuration of cell surface glycosyl by 1.8, repeats step 1.5 and step 1.6.
2. the preparation of the double mode paper chip of fluorescence/colorimetric is detected according to cancerous cell described in claims 1 and cell surface polysaccharide,
It is characterized in that, design the hydrophobic wax print pattern of paper chip and hydrophilic work with Adobe illustrator CS4 software
Zone map, the paper of paper chip used is chromatographic paper.
3. the preparation of the double mode paper chip of fluorescence/colorimetric is detected according to cancerous cell described in claims 1 and cell surface polysaccharide,
It is characterized in that, at the working region of α fluorescence coating growth gold nanorods and porous silver layer, specifically comprise the following steps that
First gold seeds is synthesized: the secondary water measuring 160 mL is placed in there-necked flask, is heated to 90 C, is subsequently added 1.6
ML gold chloride, heating in water bath, to 96 C, reacts 1 min, adds 5.6 mL 1% sodium citrates immediately, and stirs 15 min extremely
Solution becomes claret；The gold seeds taking 20 L is added drop-wise to the working region of fluorescence coating, at room temperature place 45 min；
Synthesis gold nanorods and porous silver layer: 200 mM oxammonium hydrochloride .s of 1% gold chloride and fresh preparation that take 20.0 L drip
It is added on the paper of gold seeds, at room temperature grows 10 min；Subsequently by 20.0 L 24 mM silver nitrate of fresh preparation
It is added drop-wise in the above-mentioned long sheet hole having gold nanorods with 200 mM ascorbic acid, at room temperature grows 10 min；Finally, water is used
Thoroughly clean, and be dried at room temperature for 30 min.
4. the preparation of the double mode paper chip of fluorescence/colorimetric is detected according to cancerous cell described in claims 1 and cell surface polysaccharide,
It is characterized in that, the working region of the described paper chip that aptamers is fixed on step 1.2 gained, use Sanguis Bovis seu Bubali pure subsequently
Protein blocking avtive spot, last aptamers chain capture cancerous cell, concrete preparation process is as follows:
20 L aptamers are fixed on gold nanorods and on paper chip working region that porous modified by silver is crossed, are subsequently added 20 L
Bovine serum albumin, to close avtive spot, is eventually adding the cancerous cell of 20 L variable concentrations.
5. the preparation of the double mode paper chip of fluorescence/colorimetric is detected according to cancerous cell described in claims 1 and cell surface polysaccharide,
It is characterized in that, described racemosus hybridization chain PtCu nano chain and GQDs modified is fixed on the paper chip of step 1.3 gained
Working region, concrete preparation technology is as follows:
First synthesis PtCu nano chain: 1 mL tbp, 18 mg six water Nickel dichloride .s and bis-water of 22 mL are joined
In 100 mL there-necked flasks, ultrasonic 15 min after shaking up, under nitrogen protection, strong agitation limit, limit drips the 5 fresh preparations of mL
2 mg mL-1Sodium borohydride, immediately adds 10 mL 6 mM potassium platinic chloride and the mixed liquors of copper chloride, by above-mentioned molten
Ultrasonic 30 min of liquid, afterwards heating in water bath 2 h in 60 C water-baths, finally with ethanol and washing, be dried to obtain PtCu
Nano chain；
Synthesis GQDs: weighing 2 g citric acids and be put in 5 mL beakers, be heated to 180 C, solution colour becomes orange, regulates pH
To neutrality, i.e. can get GQDs；
The racemosus hybridization chain that PtCu nano chain and GQDs modify: I-Con A Concanavalin (Con A) and H1-GQDs succinyl
The method of imines coupling obtains；I-Con A, H1-GQDs and H2 PTC-200 is heated to 95 C and keeps 10 min,
And in 30 s, it is cooled to 4 C, and subsequently, 100 L I-Con A are added in 1 mL H1-GQDs and H2 (1:1), and 37
C reacts overnight；Finally, in above-mentioned solution, add 1 mL PtCu nano chain, react 15 min, centrifugal, the product that will obtain
It is dispersed in buffer solution；
The racemosus hybridization chain that fixing PtCu nano chain and GQDs modify: the racemosus that 20 L PtCu nano chain and GQDs are modified
Hybridization chain is fixed on the paper chip working region of modified.
6. the preparation of the double mode paper chip of fluorescence/colorimetric is detected according to cancerous cell described in claims 1 and cell surface polysaccharide,
It is characterized in that, described chromogenic substrate is 3,3,5,5-tetramethyl benzidine, o-phenylenediamine, 2,2-azine groups-bis--(3-second
Base benzo thiazoline quinoline-6-sulfonic acid) di-ammonium salts.
7. the preparation of the double mode paper chip of fluorescence/colorimetric is detected according to cancerous cell described in claims 1 and cell surface polysaccharide,
It is characterized in that, pH be the buffer solution of 4 ~ 6 be NaAc_HAc buffer solution, phosphate buffered solution, Tris hydrochloride buffer
Solution.
8. the preparation of the double mode paper chip of fluorescence/colorimetric is detected according to cancerous cell described in claims 1 and cell surface polysaccharide,
It is characterized in that, the colorimetric preliminary assay step of sample, drip chromogenic substrate respectively, pH be 4 ~ 6 buffer solution and hydrogen peroxide,
Carry out the mensuration of sample, draw the standard curve of gray scale and cancerous cell concentration, it is achieved the visualization preliminary examination of cancerous cell, fix subsequently
Cell concentration is constant, changes the concentration of tunicamycin, draws the relation of gray scale and sugar chain inhibitor tunicamycin concentration, it is achieved cancerous cell
The visualization preliminary examination of surface polysaccharide.
9. the preparation of the double mode paper chip of fluorescence/colorimetric is detected according to cancerous cell described in claims 1 and cell surface polysaccharide,
It is characterized in that paper chip is put in fluorescence ware by the fluoremetry step of sample, carry out the mensuration of sample, draw fluorescence strong
Degree and the standard curve of cancerous cell concentration, it is achieved the accurate detection of cancerous cell, fix cell concentration subsequently constant, change tunicamycin
Concentration, draw the relation of fluorescence intensity and sugar chain inhibitor tunicamycin concentration, it is achieved the accurate detection of cancer cell surfaces polysaccharide.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610384836.5A CN106092982B (en) | 2016-06-02 | 2016-06-02 | The preparation of cancer cell and cell surface polysaccharide detection fluorescence/colorimetric double mode paper chip |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610384836.5A CN106092982B (en) | 2016-06-02 | 2016-06-02 | The preparation of cancer cell and cell surface polysaccharide detection fluorescence/colorimetric double mode paper chip |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106092982A true CN106092982A (en) | 2016-11-09 |
| CN106092982B CN106092982B (en) | 2018-08-14 |
Family
ID=57447084
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610384836.5A Expired - Fee Related CN106092982B (en) | 2016-06-02 | 2016-06-02 | The preparation of cancer cell and cell surface polysaccharide detection fluorescence/colorimetric double mode paper chip |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106092982B (en) |
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107576637A (en) * | 2017-08-16 | 2018-01-12 | 广西师范大学 | It is a kind of to regulate and control graphene quantum dot catalytically active surface plasmon absorption spectroscopic assay Pb with aptamers2+Method |
| CN108226074A (en) * | 2017-12-26 | 2018-06-29 | 河南师范大学 | It is applied based on twin-channel nanometer of analogue enztme of colorimetric fluorescence and its in analysis detects |
| CN108254368A (en) * | 2017-12-27 | 2018-07-06 | 中国农业科学院农产品加工研究所 | A kind of method of the gold nanorods detection heavy metal ion based on DNA regulation and control |
| CN108802356A (en) * | 2018-07-03 | 2018-11-13 | 济南大学 | A kind of preparation method of double mode three-dimensional paper chip device for cancer cell detection |
| CN109307773A (en) * | 2018-10-31 | 2019-02-05 | 福州大学 | A kind of protein glycosylation detection kit, detection method and application |
| CN109425598A (en) * | 2017-09-05 | 2019-03-05 | 中国人民解放军军事医学科学院微生物流行病研究所 | A kind of capillary micro-fluid self-driven micro-fluidic chip and the preparation method and application thereof |
| CN109633115A (en) * | 2019-01-08 | 2019-04-16 | 杭州霆科生物科技有限公司 | A kind of water quality comprehensive toxicity detection paper chip |
| CN109628557A (en) * | 2019-01-07 | 2019-04-16 | 济南大学 | Dual signal enhances application of the paper base biosensor in miRNA detection |
| CN109781811A (en) * | 2019-02-28 | 2019-05-21 | 中国科学院电子学研究所 | A kind of paper chip aptamer sensor and preparation method thereof |
| CN110954517A (en) * | 2019-12-11 | 2020-04-03 | 西北大学 | One-step fluorescence derivatization method for reducing sugar and application thereof |
| US11680908B2 (en) | 2017-10-06 | 2023-06-20 | Corning Incorporated | Assembly having nanoporous surface layer with hydrophobic layer |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102914536A (en) * | 2012-10-19 | 2013-02-06 | 大连大学 | Patterned multilayer array paper chip, preparation method and application thereof |
| CN102980998A (en) * | 2012-11-21 | 2013-03-20 | 济南大学 | High-flux microfluidics paper chip for instantly and quickly detecting multiple human tumor markers |
| CN103091302A (en) * | 2012-12-27 | 2013-05-08 | 济南大学 | Preparation of ECL (electro chemical luminescence) DNA (Deoxyribose Nucleic Acid) sensor based on 3D paper chip, and application of sensor to simultaneous detection on Hg<2+> and Ag<+> |
| CN104374769A (en) * | 2014-11-17 | 2015-02-25 | 济南大学 | Method for detecting glucose by paper-based colorimetric assay |
| CN104483310A (en) * | 2014-12-03 | 2015-04-01 | 济南大学 | Construction method of visual self-energized glucose biosensor |
| CN104569371A (en) * | 2015-01-29 | 2015-04-29 | 华南师范大学 | PDA modified paper-based micro-fluidic chip and application of PDA modified paper-based micro-fluidic chip to DNA colorimetric detection |
| CN104807987A (en) * | 2014-01-27 | 2015-07-29 | 广州阳普医疗科技股份有限公司 | Paper chip, making method thereof, and bio-molecule detection method |
| CN104865241A (en) * | 2015-05-15 | 2015-08-26 | 济南大学 | Method for preparing electrochemical luminescence cell sensor paper chips based on alloy nanoparticles modification |
-
2016
- 2016-06-02 CN CN201610384836.5A patent/CN106092982B/en not_active Expired - Fee Related
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102914536A (en) * | 2012-10-19 | 2013-02-06 | 大连大学 | Patterned multilayer array paper chip, preparation method and application thereof |
| CN102980998A (en) * | 2012-11-21 | 2013-03-20 | 济南大学 | High-flux microfluidics paper chip for instantly and quickly detecting multiple human tumor markers |
| CN103091302A (en) * | 2012-12-27 | 2013-05-08 | 济南大学 | Preparation of ECL (electro chemical luminescence) DNA (Deoxyribose Nucleic Acid) sensor based on 3D paper chip, and application of sensor to simultaneous detection on Hg<2+> and Ag<+> |
| CN104807987A (en) * | 2014-01-27 | 2015-07-29 | 广州阳普医疗科技股份有限公司 | Paper chip, making method thereof, and bio-molecule detection method |
| CN104374769A (en) * | 2014-11-17 | 2015-02-25 | 济南大学 | Method for detecting glucose by paper-based colorimetric assay |
| CN104483310A (en) * | 2014-12-03 | 2015-04-01 | 济南大学 | Construction method of visual self-energized glucose biosensor |
| CN104569371A (en) * | 2015-01-29 | 2015-04-29 | 华南师范大学 | PDA modified paper-based micro-fluidic chip and application of PDA modified paper-based micro-fluidic chip to DNA colorimetric detection |
| CN104865241A (en) * | 2015-05-15 | 2015-08-26 | 济南大学 | Method for preparing electrochemical luminescence cell sensor paper chips based on alloy nanoparticles modification |
Non-Patent Citations (4)
| Title |
|---|
| LINLIN LIANG 等: ""Aptamer-based fluorescent and visual biosensor for multiplexed monitoring of cancer cells in microfluidic paper-based analytical devices"", 《SENSORS AND ACTUATORS B: CHEMICAL》 * |
| LINLIN LIANG 等: ""Fluorescence "turn-on" determination of H2O2 using multilayer porous SiO2/NGQDs and PdAu mimetics enzymatic/oxidative cleavage of single-stranded DNA"", 《BIOSENSORS ANDBIOELECTRONICS》 * |
| LINLIN LIANG 等: ""Microfluidic paper-based multiplex colorimetric immunodevice based on the catalytic effect of Pd/Fe3O4@C peroxidase mimetics on multiple chromogenic reactions"", 《ANALYTICA CHIMICA ACTA》 * |
| 王方方 等: ""纸芯片制作及其在化学发光法检测葡萄糖和尿酸中的应用"", 《分析科学学报》 * |
Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107576637A (en) * | 2017-08-16 | 2018-01-12 | 广西师范大学 | It is a kind of to regulate and control graphene quantum dot catalytically active surface plasmon absorption spectroscopic assay Pb with aptamers2+Method |
| CN107576637B (en) * | 2017-08-16 | 2019-09-27 | 广西师范大学 | It is a kind of to regulate and control quantum dot catalytic absorption spectroscopic assay Pb with aptamers2+Method |
| CN109425598A (en) * | 2017-09-05 | 2019-03-05 | 中国人民解放军军事医学科学院微生物流行病研究所 | A kind of capillary micro-fluid self-driven micro-fluidic chip and the preparation method and application thereof |
| US11680908B2 (en) | 2017-10-06 | 2023-06-20 | Corning Incorporated | Assembly having nanoporous surface layer with hydrophobic layer |
| CN108226074A (en) * | 2017-12-26 | 2018-06-29 | 河南师范大学 | It is applied based on twin-channel nanometer of analogue enztme of colorimetric fluorescence and its in analysis detects |
| CN108254368A (en) * | 2017-12-27 | 2018-07-06 | 中国农业科学院农产品加工研究所 | A kind of method of the gold nanorods detection heavy metal ion based on DNA regulation and control |
| CN108802356A (en) * | 2018-07-03 | 2018-11-13 | 济南大学 | A kind of preparation method of double mode three-dimensional paper chip device for cancer cell detection |
| CN109307773A (en) * | 2018-10-31 | 2019-02-05 | 福州大学 | A kind of protein glycosylation detection kit, detection method and application |
| CN109628557A (en) * | 2019-01-07 | 2019-04-16 | 济南大学 | Dual signal enhances application of the paper base biosensor in miRNA detection |
| CN109633115A (en) * | 2019-01-08 | 2019-04-16 | 杭州霆科生物科技有限公司 | A kind of water quality comprehensive toxicity detection paper chip |
| CN109781811A (en) * | 2019-02-28 | 2019-05-21 | 中国科学院电子学研究所 | A kind of paper chip aptamer sensor and preparation method thereof |
| CN110954517A (en) * | 2019-12-11 | 2020-04-03 | 西北大学 | One-step fluorescence derivatization method for reducing sugar and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106092982B (en) | 2018-08-14 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106092982A (en) | Cancerous cell and the preparation of the cell surface polysaccharide detection double mode paper chip of fluorescence/colorimetric | |
| Chen et al. | A facile fluorescence lateral flow biosensor for glutathione detection based on quantum dots-MnO2 nanocomposites | |
| Hasanzadeh et al. | Immunosensing of breast cancer prognostic marker in adenocarcinoma cell lysates and unprocessed human plasma samples using gold nanostructure coated on organic substrate | |
| CN105524612B (en) | A kind of isophorone class fluorescence probe and its preparation and application | |
| CN107064264B (en) | A kind of construction method of the optical electro-chemistry sensor for no enzyme detection dopamine | |
| CN106018373B (en) | The preparation of 3-dimensional metal enhancing fluorescence/colorimetric bimodulus paper chip and ATP detections | |
| Wei et al. | A disposable amperometric immunosensor for chlorpyrifos-methyl based on immunogen/platinum doped silica sol–gel film modified screen-printed carbon electrode | |
| Wang et al. | A novel hydrazide Schiff base self-assembled nanoprobe for selective detection of human serum albumin and its applications in renal disease surveillance | |
| CN111687408B (en) | Fluorescent copper nanocluster, preparation method and application thereof | |
| CN108982630A (en) | A kind of preparation method and application of the electrochemical immunosensor of interlayer type detection prostate-specific antigen | |
| CN104502593B (en) | The preparation method of the unmarked immunosensor of a kind of galvanochemistry stomach neoplasms tumor markers | |
| Sha et al. | Electroluminescent aptasensor based on RuSiO2 nanoparticles for detection cytochrome c using ferrocene as quenching probe | |
| CN107389636A (en) | A kind of preparation and application of the water soluble fluorescence sensor that endogenous glutathione can be detected in cancer cell | |
| CN114295694B (en) | Electrochemiluminescence aptamer sensor for detecting breast cancer HER-2 and detection method thereof | |
| Pourreza et al. | A novel metal enhanced fluorescence bio probe for insulin sensing based on poly vinyl alcohol-borax hydrogel functionalized by Ag dots | |
| Shojaeifard et al. | Collaboration of cyclometalated platinum complexes and metallic nanoclusters for rapid discrimination and detection of biogenic amines through a fluorometric paper-based sensor array | |
| Wu et al. | Advances in gold nanoparticles for mycotoxin analysis | |
| CN107936035A (en) | A kind of cysteine-modifying graphene quantum dot GQCY and preparation method are with preparing the application on dopamine luciferase assay reagent | |
| Feng et al. | A novel strategy for multiplexed immunoassay of tumor markers based on electrochemiluminescence coupled with cyclic voltammetry using graphene-polymer nanotags | |
| CN110658168A (en) | Method for detecting testosterone by using gold nanocluster-gold nanorod novel immunosensor | |
| Huang et al. | Electrochemiluminescent sensor based on Ru (bpy) 32+-doped silica nanoprobe by incorporating a new co-reactant NBD-amine for selective detection of hydrogen sulfide | |
| CN106092999B (en) | Based on H in fluorescence and colorimetric method detection cancer cell2O2The preparation of paper chip | |
| Suresh et al. | Amperometric immunosensor for ricin by using on graphite and carbon nanotube paste electrodes | |
| CN106526182B (en) | A kind of structure of paper substrate aptamer fluorescent optical sensor for fibrin ferment detection | |
| Chaturvedi et al. | Emerging technologies for non-invasive quantification of physiological oxygen transport in plants |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20180814 Termination date: 20200602 |