CN108872173A - A kind of fluorescence enhancement type aptamer sensor and its preparation method and application - Google Patents
A kind of fluorescence enhancement type aptamer sensor and its preparation method and application Download PDFInfo
- Publication number
- CN108872173A CN108872173A CN201810692454.8A CN201810692454A CN108872173A CN 108872173 A CN108872173 A CN 108872173A CN 201810692454 A CN201810692454 A CN 201810692454A CN 108872173 A CN108872173 A CN 108872173A
- Authority
- CN
- China
- Prior art keywords
- aptamer
- enhancement type
- fluorescence
- fluorescence enhancement
- atp
- 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
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
-
- 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"
- G01N2021/6432—Quenching
-
- 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"
- G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
Abstract
The invention belongs to biosensor technology fields, and in particular to a kind of fluorescence enhancement type aptamer sensor and its preparation method and application.Metal Nano structure is introduced in fluorescent molecule detection system, the aptamer for modifying a certain number of base A passes through sulfydryl in conjunction with metal nanoparticle, the launching efficiency that plasma resonance improves fluorophor occurs using metal nanoparticle, so that fluorescent radiation amplitude increases substantially, achieve the purpose that amplify fluorescence signal, a kind of fluorescence enhancement type aptamer sensor that can be used for detecting low concentration intentional object is provided in combination with the specific selection of aptamer, both the sensitivity of analysis detection had been ensure that, accuracy, specificity, the diversity of detection object is realized again, there is low cost simultaneously, free of contamination feature, keep the detection range of fluorescence enhancement type aptamer sensor wider.
Description
Technical field
The invention belongs to biosensor technology fields, and in particular to a kind of fluorescence enhancement type aptamer sensor and its preparation
Methods and applications.
Background technique
In the fields such as clinical diagnosis, food safety and environmental protection increasingly to the highly sensitive testing requirements of biomolecule
Height constructs more sensitive, accurate sensing detection method and has become the emphasis studied in life analysis science research field and heat
Point.Fluorescence aptamer sensor is the fluorescent optical sensor for constructing aptamer as recognition group, which has both fluorescence point
The advantages of analysis and aptamer, can carry out quantitative analysis to a variety of biological micromolecules, and cheap, easy to operate, before
Scape is wide.But general very low (such as insulin, dopamine, aflatoxins, concentration is about for the concentration of biomolecule in actual environment
For ng/ml), and ambient noise is serious that (hemoglobin has fluorescent emission in 473nm or so in 600nm or so, water in such as blood
Peak).Therefore influenced to weaken ambient noise, resolving power and sensitivity of the fluorescence aptamer sensor to measured object are improved, needs to increase
The fluorescence conversion efficiency of fluorophor in strong sensor.
Metal surface plasma body enhancement effect is to improve a kind of extremely important of fluorophor fluorescence conversion efficiency and have
The approach of effect, metal surface enhancing fluorescent effect, which can be divided into surface-enhanced fluorescence effect and local surface, enhances fluorescent effect two
Kind.Wherein metal is existed with two-dimensional structure in surface-enhanced fluorescence effect, and preparation process is complex, and what is be related to sets
Standby cost is high, is not easy to promote and apply.And in local surface enhancing fluorescent effect, metal nanoparticle is directly used in fluorescence increasing
By force, the process for preparing metallic film or array is eliminated, preparation method is simple, and cost is relatively low, has broader practice prospect.
Local surface plasma resonance enhancing fluorescence is a kind of short range effect across space, i.e., only works as fluorescent material and metal nano
There are when certain distance, be possible to generate enhancing fluorescence for particle surface.And when metal nanoparticle and fluorescent material contact,
The fluorescence species of excitation state can transfer energy to nanoparticle in non-radiative form and return to ground state, show as sending out fluorescence
The quenching effect penetrated.Therefore when carrying out fluorescence enhancement using metal nanoparticle, need between metal nanoparticle and fluorophor
Introduce separation layer.Common inorganic barrier material is mainly silicon materials, such as SiO2, nano-stephanoporate silicon dioxide, indefinite form silicon
Deng, but itself do not have selectivity and biocompatibility it is lower；In addition, polyamidoamine dendrimer (PAMAM) and
Surfactant (CTAB etc.) also can be used as insolated layer materials, but PAMAM preparation process is complicated, it is difficult to realize accurate control；Table
Face activating agent is related to organic substance harmful to health again mostly, is unfavorable for practical application.More and more researchers will in recent years
For dsDNA as separation layer, biocompatibility is high, and the quantity by adjusting base-pair in dsDNA, which changes, is isolated thickness, but because
It is general DNA polymer only to the DNA base being complementary to selective, therefore using dsDNA as the metal of separation layer
Enhancing fluorescent effect can only be used to detect DNA sequence dna mostly, and application range is narrow.
Summary of the invention
Present invention incorporates the excellent characteristics of the high specific of aptamer selection and metal nanoparticle, provide one kind
Fluorescence enhancement type aptamer sensor, overcome it is existing by signal amplify improve sensitivity detection technique high operation requirements, at
The disadvantages of this height, harsh testing conditions.Metal Nano structure is introduced in fluorescent molecule detection system, modifies a certain number of alkali
In conjunction with metal nanoparticle, it is glimmering using metal nanoparticle plasma resonance raising to occur for the aptamer of base A by sulfydryl
The launching efficiency of light group achievees the purpose that amplify fluorescence signal, in combination with core so that fluorescent radiation amplitude increases substantially
The specific selection of sour aptamer provides a kind of fluorescence enhancement type aptamer sensor that can be used for detecting low concentration determinand, both guarantees
The sensitivity of analysis detection, accuracy, specificity, and the diversity of detection object is realized, while there is low cost, nothing
The characteristics of pollution, keeps the detection range of fluorescence enhancement type aptamer sensor wider.
To achieve the above object, the present invention provides following technical scheme：
The present invention provides a kind of fluorescence enhancement type aptamer sensor, and the principle foundation of the fluorescence enhancement type aptamer sensor is gold
Belong to enhancing fluorescent effect (Metal-enhanced fluorescence, MEF).By the aptamer and metal nano of object
Particle combines, and the aptamer complementary strand of the object of fluorophor label is added then to get fluorescence enhancement type aptamer sensing
Device.The aptamer complementary strand of the object of fluorophor label follows base pair complementarity principle, passes through hydrogen bond and object
Aptamer combine.
Further, the aptamer of the object is the aptamer of 5 ' terminal modified base A, 5 ' terminal modified base A
Quantity be 27~45 and its corresponding length is 9nm~15nm.
Further, the quantity of 5 ' terminal modified base A is 27 and its corresponding length is 9nm.
The present invention also provides a kind of application of fluorescence enhancement type aptamer sensor in quantitative detection target concentration, institutes
Stating object is ATP.
Separation layer between metal nanoparticle and fluorophor, metal nanoparticle are served as using certain amount base A
Very strong covalent bond is formed by sulfydryl with the aptamer of 5 ' terminal modified certain amount base A, building is based on metallic nanoparticle
The fluorescence enhancement type aptamer sensor of sub- local surface plasma enhancing fluorescent effect, both has DNA molecular biocompatibility
High advantage, and various biomolecules detection is realized using aptamer.
Further, the Concentration Testing range of linearity of the ATP is 0.5nmol/L~1nmol/L.
Further, the detection method of the target concentration is that object is placed in fluorescence enhancement type aptamer sensor
It in solution, stands, centrifugation, supernatant and sediment is subjected to fluorescence detection respectively.
The preparation method of above-mentioned fluorescence enhancement type aptamer sensor, includes the following steps：
Step 1：It is incubated after metal nanoparticle solution is mixed with the aptamer of the object after activation；
Step 2：1 × PBS buffer solution is added；
Step 3：NaCl solution is added, so that the concentration of NaCl is gradually increased to 0.2mol/L in total solution, stands at least for 24 hours；
Step 4：Centrifugation removes supernatant, is centrifuged after resulting sediment is added in 1 × PBS, is finally dissolved in 1 × PBS again；
Step 5：The aptamer complementary strand of the object marked by fluorophor is added, mixes, mild dynamic respons 2 hours,
Obtain fluorescence enhancement type aptamer sensor.
Further, the volume ratio of the aptamer of the metal nanoparticle solution and object is (5~12):1.
Further, the volume ratio of the aptamer of the metal nanoparticle solution and object is 8:1.
Compared with prior art, the beneficial effects of the present invention are：
1, fluorescence enhancement type aptamer sensor of the present invention has hypersensitivity, and common fluorescent optical sensor is all to utilize fluorescent base
The fluorescence intensity change of group measures the concentration of object, and transducer sensitivity is limited to the fluorescence conversion efficiency of fluorophor,
Thus it is difficult to improve.And the present invention is based on metalfluorescent enhancement effects, improve the fluorescence conversion efficiency of fluorophor, are exaggerated glimmering
Light signal strength improves the detection sensitivity of fluorescence aptamer sensor, detects the minimum dense of ATP in the implementation of specific case
Degree is 0.5nmol/L.
2, fluorescence enhancement is realized by adjusting the quantity of base A, Enhancement of Fluorescence is by metal Nano structure local surface
The influence of plasma resonance wavelength (λ) and the distance between fluorophor and metal Nano structure (d).Utilize metal nanoparticle
When carrying out fluorescence enhancement, to avoid fluorophor from transferring energy to metal nanoparticle in non-radiative form, fluorescence is generated
Quenching needs to introduce separation layer between metal nanoparticle and fluorophor.Common insolated layer materials mainly have：
PAMAM、SiO2, dsDNA etc..Wherein PAMAM preparation process is complicated；SiO2Without selectivity and biocompatibility it is lower；
DsDNA biocompatibility is high, but selective just for DNA sequence dna, can not carry out to the biomolecule other than DNA polymer
Concentration Testing.The present invention serves as separation layer using a certain number of base A, and the quantity by adjusting base A controls metal and receives
The local surface plasma of rice corpuscles enhances fluorescent effect, improves the sensitivity of sensor；Both there is DNA molecular bio-compatible
Property high advantage, and realize using aptamer inexpensive, highly selective, the highly sensitive detection of various biomolecules, reduce
The step of other addition selective molecular recognition group, method is more simple, while it is expected that the hybrid design can overcome few core
Bending or distortion of the thuja acid in surfaces of metal nanoparticles.
3, detection object range is wide, and the fluorescence enhancement type aptamer sensor of this method preparation can be by changing aptamer
Type and be applied to different target object, separation layer thickness will not therefore and change, transducer sensitivity only with aptamer
To the selectivity of object in relation to, it can be achieved that biomolecule detection object diversification.
Detailed description of the invention
Fig. 1 is a kind of working principle diagram of fluorescence enhancement type aptamer sensor of the present invention.
Fig. 2 be embodiment 1 in 5 ' terminal modified different number base A ATP aptamer (D1~D6) and FAM-DNA it is glimmering
Light spectrogram, FAM-DNA are the ATP aptamer complementary strand of FAM label.
Fig. 3 is the fluorescence for modifying the ATP aptamer (D1~D6) of different number base A in embodiment 1 under 521nm wavelength
Intensity, abscissa are length (i.e. fluorophor and Jenner corresponding to the base A quantity of different ATP aptamers (D1~D6)
The length of the distance on rice corpuscles surface), ordinate is fluorescence intensity, and three bases are 1 nanometer, and D1~D6 aptamer front end is repaired
The base A of decorations is corresponding to be followed successively by 6nm, 8nm, 9nm, 10nm, 12nm and 15nm.
Fig. 4 be in embodiment 2 under 521nm wavelength gold nanoparticle and ATP aptamer different volumes than when fluorescence it is strong
Degree.
Fig. 5 is the fluorescence spectra of the supernatant (0.5nmol/L~1nmol/L) of various concentration ATP in embodiment 3.
Fig. 6 is the linear relationship chart of various concentration ATP and supernatant fluorescence intensity in embodiment 3.ATP concentration and fluorescence are strong
The linear relationship for spending variation delta I is Y=819.54576+72.66102X, and wherein Y is fluorescence intensity, and X is ATP concentration, related
Coefficient is：R2=0.97914.
Fig. 7 is the fluorescence spectrum that various concentration ATP (0.5nmol/L~1nmol/L) sediment afterwards is added in embodiment 3
Figure.
Fig. 8 is various concentration ATP and sediment fluorescence intensity linear relationship chart, ATP concentration and fluorescence intensity in embodiment 3
The linear relationship of Δ I is Y=1565.898-812.708X, and related coefficient is：R2=0.99712.
Specific embodiment
With reference to the accompanying drawing, embodiments of the present invention are described further.Following embodiment is to preferably say
Bright technical solution of the present invention, rather than limited the scope of the invention with this.
Test material used in embodiment and instrument are as follows：
Tris (2-carboxyethyl) phosphine hydrochloride (TCEP), Tween-20 (Tween-20), ATP core
Sour aptamer 1~6 (D1~D6), the ATP aptamer complementary strand of FAM label, is purchased from Shanghai Sheng Gong Bioisystech Co., Ltd
(China)；
II type centrifuge of GL-16 is purchased from Anting Scientific Instrument Factory, Shanghai；
HZQ-F200 type shaken cultivation case is purchased from Beijing Dong Lianhaer instrument manufacturing Co., Ltd；
IKAVortex-3 type vortex mixer is purchased from Shanghai Chu Bai laboratory equipment Co., Ltd；
07HWS-2 digital display constant temperature blender with magnetic force is purchased from Hangzhou motor for instrument Co., Ltd；
Electronic balance is purchased from Mettler-Toledo Instrument (Shanghai) Co., Ltd.；
F-7000 type Fluorescence Spectrometer is purchased from HITACHI company of Japan.
Wherein, 1, the nucleotides sequence of ATP aptamer 1~6 (D1~D6) is classified as：
D1:5'-SH-AAAAAAAAAAAAAAAAAAACCTGGGGGAGTATTGCGGAGGAAGGT-3'(5 ' terminal modified base A
Length be 6nm), as shown in SEQ ID NO.1；
D2:5'-SH-AAAAAAAAAAAAAAAAAAAAAAAAACCTGGGGGAGTATTGCGGAGGAAGG T-3'(5' is terminal modified
Base A length be 8nm), as shown in SEQ ID NO.2；
D3:5'-SH-AAAAAAAAAAAAAAAAAAAAAAAAAAAACCTGGGGGAGTATTGCGGAGGA AGGT-3'(5' is repaired at end
The length of the base A of decorations is 9nm), as shown in SEQ ID NO.3；
D4:5'-SH-AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAACCTGGGGGAGTATTGCGGAGGAAGGT-3'(5'
The length of terminal modified base A is 10nm), as shown in SEQ ID NO.4；
D5:5'-SH-AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAACCTGGGGGAGTATTGCGGAGGAAGGT-
The length of 3'(5' terminal modified base A is 12nm), as shown in SEQ ID NO.5；
D6:5'-SH-AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAACCTGGGGGAGTATTGCGG
The length of AGGAAGGT-3'(5' terminal modified base A is 15nm), as shown in SEQ IDNO.6；The guarantor of above-mentioned aptamer
The condition of depositing is -20 DEG C, and the pot-life is 1 year.
2, the nucleotides sequence of the ATP aptamer complementary strand of FAM label is classified as：5'-ACCTTCCTCCGCAATACTCCCCC
AGGT-FAM-3', as shown in SEQ ID NO.7, the preservation condition of the aptamer complementary strand is -20 DEG C, and the pot-life is 1
Year.
3,TCEP:2.5mmol/L, pH=5.0；Make solvent dissolution with Tris- acetic acid.
4, PBS buffer solution：(pH=7.4) 1 × PBS：5mL, 10 × PBS is taken to be diluted to 50mL.
5, the preparation (2mol/L) of NaCl solution：351mg sodium chloride is taken to prepare 3mL solution.
6, experimental water is the ultrapure water that resistance is 18.2M Ω.
A kind of working principle of fluorescence enhancement type aptamer sensor of the invention is：Metal nanoparticle solution is added first
Enter into centrifuge tube, is then respectively adding the core of the object of 5 ' terminal modified different number base A of same volume same concentration
Sour aptamer.It is mildly shaken under room temperature, the aptamer complementary strand that the object of fluorophor modification is added prepares detection probe, i.e.,
For fluorescence enhancement type aptamer sensor；It is then centrifuged for, taking precipitate, fluorescence detection is carried out after dissolution, by by fluorescence enhancement type
The fluorescence of the aptamer complementary strand of the object of the fluorophor modification of the fluorescence intensity and respective concentration of aptamer sensor is strong
Degree compares, and determines the best generation distance of metalfluorescent enhancement effect.The detection probe for then taking enhancing highest multiple, adds
The object for entering various concentration, since aptamer and object have stronger affinity, so that a part of fluorophor mark
The aptamer complementary strand of the object of note is dissociated out, and the concentration of the object of addition is bigger, the fluorescence intensity of supernatant
Bigger, the fluorescence intensity in corresponding sediment dies down with the increase of the concentration of object, sees Fig. 1.
The detection of optimum distance between 1 fluorophor of embodiment and gold nanoparticle
The present embodiment is according to the different systems by fluorescence enhancement type aptamer sensor of distance between fluorophor and gold nanoparticle
Standby experiment is divided into 6 groups.
ATP aptamer used is ATP aptamer 1, i.e. D1 in group 1；
ATP aptamer used is ATP aptamer 1, i.e. D2 in group 2；
ATP aptamer used is ATP aptamer 1, i.e. D3 in group 3；
ATP aptamer used is ATP aptamer 1, i.e. D4 in group 4；
ATP aptamer used is ATP aptamer 1, i.e. D5 in group 5；
ATP aptamer used is ATP aptamer 1, i.e. D6 in group 6.
The preparation of every group of fluorescence enhancement type aptamer sensor, includes the following steps：
Step 1：ATP aptamer is centrifuged 1min at 12000rpm, is then diluted to 5 μm of ol/L with TCEP, it is black in room temperature
2h is activated under dark no light condition；The ATP aptamer of 800 μ L solution of gold nanoparticles (AuNPs) and 100 μ L is mixed and incubated
18h。
Step 2：1 × PBS of 100 μ L is added into the solution that step 1 obtains, and reacts 6 hours, the effect of 1 × PBS
To adjust pH value, increase the intensity of solion.
Step 3：The NaCl solution of 2mol/L is added into the solution that step 2 obtains, every 3 hours are repeated once, and repeat 2
It is secondary, so that the concentration of NaCl is gradually increased to 0.2mol/L in total solution, stand for 24 hours.
Step 4：Step 3 solution obtained is centrifuged 15 minutes under the conditions of 14 DEG C, 15,000rpm, is centrifuged altogether three times；
Supernatant is removed, resulting sediment is dissolved into 900 μ L, in 1 × PBS buffer solution, centrifugation is then dissolved in 300 μ L, 1 ×
In PBS buffer solution.
Step 5：500 μ L are added into step 4 acquired solution, the ATP aptamer of 1 μm of ol/L marked by FAM is complementary
Chain, it is dynamic respons 2 hours mild at 37 DEG C；Then it at 14 DEG C, is centrifuged 10 minutes under the conditions of 15,000rpm, removes supernatant, most
Sediment is dissolved in 10mL afterwards, in 1 × PBS buffer solution, obtains fluorescence enhancement type aptamer sensor.
Step 6：Take step 5 400 μ L of fluorescence enhancement type aptamer sensor solution obtained be added in cuvette carry out it is glimmering
Light detection.
The fluorescence enhancement type aptamer sensor that group 1~6 obtains is respectively fluorescence enhancement type D1 aptamer sensor, fluorescence enhancement
Type D2 aptamer sensor, fluorescence enhancement type D3 aptamer sensor, fluorescence enhancement type D4 aptamer sensor, fluorescence enhancement type D5 aptamer
Sensor and fluorescence enhancement type D6 aptamer sensor.
Fluorescence detection is as the result is shown：When fluorophor and gold nanoparticle surface distance are 6nm and 8nm (used
When ATP aptamer is D1 and D2), fluorescence intensity weakens, and resonance energy transfer, fluorescent quenching, when fluorophor and gold occurs
When nanoparticle surface distance is 9nm, 10nm, 12nm and 15nm (when ATP aptamer used is D3, D4, D5 and D6)
Metalfluorescent enhancement effect occurs, and when fluorophor and gold nanoparticle surface distance are 9nm (ATP aptamer is D3)
When, fluorescence intensity enhancing is 2.7 times of the fluorescence intensity of the ATP aptamer complementary strand of FAM label, most beneficial for amplification fluorescence
Signal is shown in Fig. 2 and Fig. 3.
The optimum volume ratio of 2 solution of gold nanoparticles of embodiment and ATP aptamer is tested
According to the volume ratio of solution of gold nanoparticles (AuNPs) and ATP aptamer in the present embodiment, by fluorescence enhancement type aptamer
Sensor preparation experiment is divided into 4 groups, and every group all uses D1~D6 as the aptamer of object respectively.
In group 1, the volume ratio of solution of gold nanoparticles (AuNPs) and ATP aptamer is 5:1；
In group 2, the volume ratio of solution of gold nanoparticles (AuNPs) and ATP aptamer is 8:1；
In group 3, the volume ratio of solution of gold nanoparticles (AuNPs) and ATP aptamer is 10:1,
In group 4, the volume ratio of solution of gold nanoparticles (AuNPs) and ATP aptamer is 12:1.
Every group of fluorescence enhancement type aptamer sensor preparation includes the following steps：
Step 1：ATP aptamer is centrifuged 1min at 12000rpm, is then diluted to 5 μm of ol/L with TCEP, it is black in room temperature
2h is activated under dark no light condition；Solution of gold nanoparticles (AuNPs) and ATP aptamer are mixed and incubate 18h.
Step 2：1 × PBS is added into the solution that step 1 obtains, and reacts 6 hours, the effect of 1 × PBS is to adjust
PH value increases the intensity of solion.
Step 3：The NaCl solution of 2mol/L is added into the solution that step 2 obtains, every 3 hours are repeated once, and repeat 2
It is secondary, so that the concentration of NaCl is gradually increased to 0.2mol/L in total solution, stand for 24 hours.
Step 4：Step 3 solution obtained is centrifuged 15 minutes under the conditions of 14 DEG C, 15,000rpm, is centrifuged altogether three times；
Supernatant is removed, resulting sediment is dissolved into 900 μ L, in 1 × PBS buffer solution, centrifugation is then dissolved in 300 μ L, 1 ×
In PBS buffer solution.
Step 5：The ATP aptamer complementary strand (FAM- of 1 μm of ol/L marked by FAM is added into step 4 acquired solution
DNA), dynamic respons 2 hours mild at 37 DEG C；Then it at 14 DEG C, is centrifuged 10 minutes under the conditions of 15,000rpm, removes supernatant,
Sediment is finally dissolved in 10mL, in 1 × PBS buffer solution, obtains fluorescence enhancement type aptamer sensor.
Step 6：Step 5 400 μ L of fluorescence enhancement type aptamer sensor solution obtained is taken to be added in cuvette,
Fluorescence detection is carried out under 521nm wavelength.
The raw materials used volume of 1 fluorescence enhancement type aptamer sensor of table
Due to can not theoretically predict to have the metal nanoparticle of certain size can generate to neighbouring fluorophor
The effect that quenching effect still enhances, and gold nanoparticle is easy to reunite, it is therefore desirable to Jenner's grain of rice of addition
The volume ratio of sub- solution and ATP aptamer optimizes screening.
Fluorescence detection interpretation of result：
When AuNps and ATP aptamer volume ratio are 8:When 1, fluorescence intensity reinforcing effect is best, is shown in Table 2 and Fig. 4.
2 AuNps of table and 521nm fluorescence intensity level when ATP aptamer different volumes ratio
Embodiment 3
1) fluorescence enhancement type aptamer sensor is prepared, is included the following steps：
Step 1：ATP aptamer D3 is centrifuged 1min at 12000rpm, then 5 μm of ol/L are diluted to TCEP, in room temperature
2h is activated under dark no light condition；The ATP aptamer D3 of 800 μ L solution of gold nanoparticles (AuNPs) and 100 μ L are mixed simultaneously
Incubate 18h.
Step 2：1 × PBS of 100 μ L is added into the solution that step 1 obtains, and reacts 6 hours, the effect of 1 × PBS
To adjust pH value, increase the intensity of solion.
Step 3：The NaCl solution of 2mol/L is added into the solution that step 2 obtains, every 3 hours are repeated once, and repeat 2
It is secondary, so that the concentration of NaCl can be gradually increased to 0.2mol/L in total solution, stand for 24 hours.
Step 4：Step 3 solution obtained is centrifuged 15 minutes under the conditions of 14 DEG C, 15,000rpm, is centrifuged altogether three times；
Supernatant is removed, resulting sediment is dissolved into 900 μ L, in 1 × PBS buffer solution, centrifugation is then dissolved in 300 μ L, 1 ×
In PBS buffer solution.
Step 5：500 μ L are added into step 4 acquired solution, the ATP aptamer D3's of 1 μm of ol/L marked by FAM
Complementary strand, it is dynamic respons 2 hours mild under the conditions of 37 DEG C；Then it at 14 DEG C, is centrifuged 10 minutes, goes under the conditions of 15,000rpm
Except supernatant, finally sediment is dissolved in 1 × PBS buffer solution of 10mL, obtains fluorescence enhancement type aptamer sensor.
Step 6：It takes step 5 fluorescence enhancement type aptamer sensor solution obtained to be equally assigned into 400 every part of μ L, takes 10
Part is spare.
2) ATP of various concentration is prepared
6.05mg adenosine -5 '-triphosphoric acid disodium salt (ATP) accurately is weighed, is placed in 10mL volumetric flask, adds ultrapure water dissolution fixed
Hold, mix, obtain ATP standard mother liquor, concentration is 1 μm of ol/L；
90 μ L of ATP standard mother liquor is taken, is placed in 3mL centrifuge tube, ultrapure water is added to be settled to 3mL, obtains ATP standard intermediate fluid A, it is dense
Degree is 30nmol/L；
30 μ L of ATP standard mother liquor is taken, is placed in 3mL centrifuge tube, ultrapure water is added to be settled to 3mL, obtains ATP standard intermediate fluid B, it is dense
Degree is 10nmol/L；
Taking concentration respectively is 30nmol/L ATP standard intermediate fluid A20 μ L, 10 μ L, and adding ultrapure water constant volume is 1mL, obtains concentration difference
For the ATP standard working solution of 6nmol/L, 3nmol/L；
Taking concentration respectively is 100 μ L of 10nmol/L ATP standard intermediate fluid B, 80 μ L, 60 μ L, 50 μ L, adds the ultrapure water constant volume to be
1mL obtains the ATP standard working solution that concentration is respectively 1nmol/L, 0.8nmol/L, 0.6nmol/L, 0.5nmol/L.
3) ATP is detected
ATP standard working solution 100 μ L (0.5nmol/L, 0.6nmol/L, 0.8nmol/L, 1nmol/ of various concentration are taken respectively
L3nmol/L and 6nmol/L) and the ATP standard intermediate fluid (10nmol/L and 30nmol/L) of various concentration be placed in above-mentioned steps 6
In the fluorescence enhancement type aptamer sensor solution distributed, after standing reaction 25min, 12,000rpm centrifugations 10 minutes, by supernatant
Liquid carries out fluorescence detection, and carries out fluorescence detection after sediment is dissolved in 1 × PBS buffer solution of 400 μ L.
Supernatant and sediment after dense ATP (0.5nmol/L~30nmol/L) different to addition carry out fluorescence respectively
Detection, the results show that the concentration range of linearity of prepared fluorescence enhancement type aptamer sensor detection ATP be 0.5nmol/L~
1nmol/L, detection is minimum to be limited to 0.5nmol/L.The fluorescence intensity of supernatant is directly proportional to the ATP concentration of addition, sees Fig. 5, sinks
The fluorescence intensity of starch and the concentration of ATP are inversely proportional, and see Fig. 8.
After object ATP is added, the complementary strand of the ATP aptamer D3 of FAM label can be dissociated out, deposit after centrifugation
It is in supernatant, as the ATP concentration of addition is bigger, the fluorescence intensity of supernatant is gradually increased, the fluorescence intensity of sediment
It is inversely proportional with the ATP concentration of addition, sees Fig. 6~8.The results show that ATP in 0.5nmol/L~1nmol/L concentration range with it is glimmering
Luminous intensity has good linear relationship：The linear relationship of ATP concentration and the fluorescence intensity of supernatant is Y=819.54576+
72.66102X, wherein Y is fluorescence intensity, and X is ATP concentration, and related coefficient is：R2=0.97914, see Fig. 6；ATP concentration and heavy
The linear relationship of the fluorescence intensity of starch is Y=1565.898-812.708X, and related coefficient is：R2=0.99712, see Fig. 8；
The fluorescence intensity of sediment changes variation tendency with ATP concentration and becomes apparent, and sensitivity is higher, therefore can in biomolecule detection
Centrifugation taking precipitate detection after object is added in selection.When fluorescence enhancement type aptamer sensor is applied to ATP detection, ATP concentration
The detection range of linearity is 0.5nmol/L~1nmol/L, detects minimum be limited to：0.5nmol/L.
The embodiment of the above, only presently preferred embodiments of the present invention, is only used to explain the present invention, not limit
The scope of the present invention processed to those of ordinary skill in the art certainly can be according to skill disclosed in this specification
Art content, makes other embodiments easily by way of replacing or changing, therefore all in the principle of the present invention and technique item
The changes and improvements etc. that part is done, should be included in scope of the present invention patent.
SEQUENCE LISTING
<110>Light Engineering Institutes Of Zhengzhou
<120>A kind of fluorescence enhancement type aptamer sensor and its preparation method and application
<130>Nothing
<160> 7
<170> PatentIn version 3.5
<210> 1
<211> 45
<212> DNA
<213>Artificial sequence
<400> 1
aaaaaaaaaa aaaaaaaaac ctgggggagt attgcggagg aaggt 45
<210> 2
<211> 51
<212> DNA
<213>Artificial sequence
<400> 2
aaaaaaaaaa aaaaaaaaaa aaaaacctgg gggagtattg cggaggaagg t 51
<210> 3
<211> 54
<212> DNA
<213>Artificial sequence
<400> 3
aaaaaaaaaa aaaaaaaaaa aaaaaaaacc tgggggagta ttgcggagga aggt 54
<210> 4
<211> 57
<212> DNA
<213>Artificial sequence
<400> 4
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa acctggggga gtattgcgga ggaaggt 57
<210> 5
<211> 63
<212> DNA
<213>Artificial sequence
<400> 5
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaacct gggggagtat tgcggaggaa 60
ggt 63
<210> 6
<211> 72
<212> DNA
<213>Artificial sequence
<400> 6
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaacctg ggggagtatt 60
gcggaggaag gt 72
<210> 7
<211> 27
<212> DNA
<213>Artificial sequence
<400> 7
accttcctcc gcaatactcc cccaggt 27
Claims (9)
1. a kind of fluorescence enhancement type aptamer sensor, which is characterized in that by the aptamer of object and metal nanoparticle knot
It closes, the aptamer complementary strand of the object of fluorophor label is added then to get fluorescence enhancement type aptamer sensor.
2. fluorescence enhancement type aptamer sensor according to claim 1, which is characterized in that the aptamer of the object
For the aptamer of 5 ' terminal modified base A, the quantity of 5 ' terminal modified base A be 27~45 and its corresponding length be 9nm~
15nm。
3. fluorescence enhancement type aptamer sensor according to claim 2, which is characterized in that the quantity of 5 ' terminal modified base A
For 27 and its corresponding length is 9nm.
4. based on a kind of fluorescence enhancement type aptamer sensor answering in quantitative detection target concentration described in claim 1
With, which is characterized in that the object is ATP.
5. a kind of fluorescence enhancement type aptamer sensor answering in quantitative detection target concentration according to claim 4
With, which is characterized in that the Concentration Testing range of linearity of the ATP is 0.5nmol/L~1nmol/L.
6. a kind of fluorescence enhancement type aptamer sensor answering in quantitative detection target concentration according to claim 4
It is with, which is characterized in that the detection method of the target concentration：Object is placed in fluorescence enhancement type aptamer sensor solution
In, it stands, centrifugation, supernatant and sediment is subjected to fluorescence detection respectively.
7. the preparation method of the described in any item fluorescence enhancement type aptamer sensors of claim 1~6, which is characterized in that including
Following steps：
Step 1：It is incubated after metal nanoparticle solution is mixed with the aptamer of the object after activation；
Step 2：1 × PBS buffer solution is added；
Step 3：NaCl solution is added, so that the concentration of NaCl is gradually increased to 0.2mol/L in total solution, stands at least for 24 hours；
Step 4：Centrifugation removes supernatant, is centrifuged again after resulting sediment is added in 1 × PBS, finally it is dissolved in 1 ×
In PBS；
Step 5：The aptamer complementary strand of the object marked by fluorophor is added, mixes, mild dynamic respons 2 hours,
Obtain fluorescence enhancement type aptamer sensor.
8. preparation method according to claim 7, which is characterized in that the core of the metal nanoparticle solution and object
The volume ratio of sour aptamer is（5~12）:1.
9. preparation method according to claim 7, which is characterized in that the core of the metal nanoparticle solution and object
The volume ratio of sour aptamer is 8:1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810692454.8A CN108872173B (en) | 2018-06-29 | 2018-06-29 | Fluorescence-enhanced aptamer sensor and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810692454.8A CN108872173B (en) | 2018-06-29 | 2018-06-29 | Fluorescence-enhanced aptamer sensor and preparation method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108872173A true CN108872173A (en) | 2018-11-23 |
| CN108872173B CN108872173B (en) | 2020-12-15 |
Family
ID=64296806
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810692454.8A Active CN108872173B (en) | 2018-06-29 | 2018-06-29 | Fluorescence-enhanced aptamer sensor and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108872173B (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111004622A (en) * | 2019-11-28 | 2020-04-14 | 郑州轻工业大学 | Preparation method and application of high-sensitivity fluorescent probe for detecting dopamine |
| CN112557646A (en) * | 2021-01-21 | 2021-03-26 | 天津工业大学 | Fluorescent adenosine triphosphate sensor based on circulation of aptamer and enzymatic target |
| CN112630439A (en) * | 2020-12-11 | 2021-04-09 | 郑州轻工业大学 | Splitting aptamer sensor based on nanogold and preparation method and application thereof |
| CN112834475A (en) * | 2020-06-09 | 2021-05-25 | 郑州轻工业大学 | Aptamer, method for regulating signal intensity of aptamer and application |
| DE102020115332A1 (en) | 2020-06-09 | 2021-12-09 | Forschungszentrum Jülich GmbH | BIOMARKER SENSOR-BASED DETECTION OF DISEASE-SPECIFIC BIOMARKERS |
Citations (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004059279A3 (en) * | 2002-11-26 | 2005-10-06 | Univ Maryland Biotechnology | High-sensitivity assays for pathogen detection using metal-enhanced fluorescence |
| CN101424642A (en) * | 2008-11-14 | 2009-05-06 | 中国科学院上海应用物理研究所 | Target molecule detecting method based on nanometer aurum and nucleic acid structure |
| EP2365096A1 (en) * | 2000-03-22 | 2011-09-14 | Life Technologies Corporation | Methods of using semiconductor nanocrystals in bead-based nucleic acid assays |
| CN102676508A (en) * | 2012-04-20 | 2012-09-19 | 华森新科（苏州）纳米技术有限公司 | Small molecule probe based on nano-gold and aptamer and preparation method of small molecule probe |
| CN104280542A (en) * | 2014-10-21 | 2015-01-14 | 南京基蛋生物科技有限公司 | Dual-enhanced chemiluminescent immunoassay method based on metal enhanced luminescence and nano particle labelled amplification |
| CN104697968A (en) * | 2013-12-06 | 2015-06-10 | 中国科学院深圳先进技术研究院 | Construction method for near infrared fluorescence energy transfer based biosensor |
| WO2016015022A1 (en) * | 2014-07-25 | 2016-01-28 | Shchepinov, Mikhail | Single molecule proteomics |
| CN105352921A (en) * | 2015-10-13 | 2016-02-24 | 北京科技大学 | Preparation and application of mercury ion sensor based on photonic crystal enhanced fluorescence |
| US20160169877A1 (en) * | 2014-12-11 | 2016-06-16 | The Regents Of The University Of California | Patterning silica islands onto thermoplastic shrink film |
| CN106248960A (en) * | 2016-07-28 | 2016-12-21 | 郑州轻工业学院 | A kind of high-throughput nucleic acid aptamer sensor detecting insulin and preparation method thereof |
| WO2017046320A1 (en) * | 2015-09-16 | 2017-03-23 | Fianostics Gmbh | Substrate for fluorescence amplification |
| CN106596971A (en) * | 2016-12-16 | 2017-04-26 | 江南大学 | Precious metal plasma effect enhanced up-conversion fluorescence structure-based method for hypersensitive detection of vascular endothelial growth factor |
-
2018
- 2018-06-29 CN CN201810692454.8A patent/CN108872173B/en active Active
Patent Citations (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2365096A1 (en) * | 2000-03-22 | 2011-09-14 | Life Technologies Corporation | Methods of using semiconductor nanocrystals in bead-based nucleic acid assays |
| WO2004059279A3 (en) * | 2002-11-26 | 2005-10-06 | Univ Maryland Biotechnology | High-sensitivity assays for pathogen detection using metal-enhanced fluorescence |
| CN101424642A (en) * | 2008-11-14 | 2009-05-06 | 中国科学院上海应用物理研究所 | Target molecule detecting method based on nanometer aurum and nucleic acid structure |
| CN102676508A (en) * | 2012-04-20 | 2012-09-19 | 华森新科（苏州）纳米技术有限公司 | Small molecule probe based on nano-gold and aptamer and preparation method of small molecule probe |
| CN104697968A (en) * | 2013-12-06 | 2015-06-10 | 中国科学院深圳先进技术研究院 | Construction method for near infrared fluorescence energy transfer based biosensor |
| WO2016015022A1 (en) * | 2014-07-25 | 2016-01-28 | Shchepinov, Mikhail | Single molecule proteomics |
| CN104280542A (en) * | 2014-10-21 | 2015-01-14 | 南京基蛋生物科技有限公司 | Dual-enhanced chemiluminescent immunoassay method based on metal enhanced luminescence and nano particle labelled amplification |
| US20160169877A1 (en) * | 2014-12-11 | 2016-06-16 | The Regents Of The University Of California | Patterning silica islands onto thermoplastic shrink film |
| WO2017046320A1 (en) * | 2015-09-16 | 2017-03-23 | Fianostics Gmbh | Substrate for fluorescence amplification |
| CN105352921A (en) * | 2015-10-13 | 2016-02-24 | 北京科技大学 | Preparation and application of mercury ion sensor based on photonic crystal enhanced fluorescence |
| CN106248960A (en) * | 2016-07-28 | 2016-12-21 | 郑州轻工业学院 | A kind of high-throughput nucleic acid aptamer sensor detecting insulin and preparation method thereof |
| CN106596971A (en) * | 2016-12-16 | 2017-04-26 | 江南大学 | Precious metal plasma effect enhanced up-conversion fluorescence structure-based method for hypersensitive detection of vascular endothelial growth factor |
Non-Patent Citations (5)
| Title |
|---|
| JING WANG .ET AL: "AGold Nanoparticle-Based Aptamer Target Binding Readout", 《ADVANCED MATERIALS》 * |
| KHULAN SERGELEN.ET AL: ""Plasmon Field-Enhanced Fluorescence Energy Transfer for Hairpin"", 《ACS SENSOR》 * |
| YONG-QIANG LI.ET AL: ""Distance-Dependent Metal-Enhanced Quantum Dots Fluorescence"", 《ANALYTICAL CHEMISTRY》 * |
| 姜利英: ""基于荧光共振能量转移的核酸适体传感器研究"", 《光子学报》 * |
| 陈丽珍: "构建基于金基底的表面增强光谱传感器用于生物分子及有机小分子的检测研究", 《中国优秀硕士学位论文全文数据库 工程科技Ⅰ辑》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111004622A (en) * | 2019-11-28 | 2020-04-14 | 郑州轻工业大学 | Preparation method and application of high-sensitivity fluorescent probe for detecting dopamine |
| CN111004622B (en) * | 2019-11-28 | 2023-08-15 | 郑州轻工业大学 | Preparation method and application of high-sensitivity fluorescent probe for detecting dopamine |
| CN112834475A (en) * | 2020-06-09 | 2021-05-25 | 郑州轻工业大学 | Aptamer, method for regulating signal intensity of aptamer and application |
| DE102020115332A1 (en) | 2020-06-09 | 2021-12-09 | Forschungszentrum Jülich GmbH | BIOMARKER SENSOR-BASED DETECTION OF DISEASE-SPECIFIC BIOMARKERS |
| CN112834475B (en) * | 2020-06-09 | 2023-03-14 | 郑州轻工业大学 | Aptamer, method for regulating signal intensity of aptamer and application |
| CN112630439A (en) * | 2020-12-11 | 2021-04-09 | 郑州轻工业大学 | Splitting aptamer sensor based on nanogold and preparation method and application thereof |
| CN112557646A (en) * | 2021-01-21 | 2021-03-26 | 天津工业大学 | Fluorescent adenosine triphosphate sensor based on circulation of aptamer and enzymatic target |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108872173B (en) | 2020-12-15 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108872173A (en) | A kind of fluorescence enhancement type aptamer sensor and its preparation method and application | |
| Liu et al. | Recent advances in sensors for tetracycline antibiotics and their applications | |
| Zhou et al. | Carbon nanodots as fluorescence probes for rapid, sensitive, and label-free detection of Hg 2+ and biothiols in complex matrices | |
| Wang et al. | Immunoassay of goat antihuman immunoglobulin G antibody based on luminescence resonance energy transfer between near-infrared responsive NaYF4: Yb, Er upconversion fluorescent nanoparticles and gold nanoparticles | |
| US8896829B2 (en) | Metal particles for surface-enhanced raman scattering and molecular sensing | |
| Zhang et al. | Protein-binding aptamer assisted signal amplification for the detection of influenza A (H1N1) DNA sequences based on quantum dot fluorescence polarization analysis | |
| Wei et al. | Highly sensitive electrochemical biosensor for streptavidin detection based on CdSe quantum dots | |
| Ma et al. | Versatile electrochemiluminescence assays for PEDV antibody based on rolling circle amplification and Ru-DNA nanotags | |
| CN104813158A (en) | Biosensor comprising metal nanoparticles | |
| Wu et al. | Ratiometric fluorescence sensor for the sensitive detection of Bacillus thuringiensis transgenic sequence based on silica coated supermagnetic nanoparticles and quantum dots | |
| Liu et al. | A novel aptamer-mediated CuInS 2 quantum dots@ graphene oxide nanocomposites-based fluorescence “turn off–on” nanosensor for highly sensitive and selective detection of kanamycin | |
| Zhang et al. | Apoferritin nanoparticle: a novel and biocompatible carrier for enzyme immobilization with enhanced activity and stability | |
| Fang et al. | Highly sensitive aptasensor for oxytetracycline based on upconversion and magnetic nanoparticles | |
| CN108535236A (en) | A method of based on dual amplification SERS signal system super sensitivity detection miRNA | |
| Luo et al. | Functional silica nanospheres for sensitive detection of H9N2 avian influenza virus based on immunomagnetic separation | |
| CN102866139A (en) | Establishment method based on surface plasma reinforcing energy transferring biosensor | |
| Chen et al. | A sandwich sensor based on imprinted polymers and aptamers for highly specific double recognition of viruses | |
| CN105136758B (en) | A kind of Eu to the residual detection of agriculture3+Mark molecule marking transducer production method | |
| Qi et al. | Multivalent glycosylated Cu: CdS quantum dots as a platform for rapid bacterial discrimination and detection | |
| CN114295694A (en) | Electrochemical luminescence aptamer sensor for breast cancer HER-2 detection and detection method thereof | |
| Zhao et al. | Fluorescent materials with aggregation-induced emission characteristics for array-based sensing assay | |
| Lenyk et al. | Dual‐Transducer Malaria Aptasensor Combining Electrochemical Impedance and Surface Plasmon Polariton Detection on Gold Nanohole Arrays | |
| Xiao et al. | Recent advances in fluorescence anisotropy/polarization signal amplification | |
| CN111007053B (en) | Fluorescent aptamer sensor for detecting silver ion concentration and preparation method and application thereof | |
| Xie et al. | Sensitive SERS detection of SARS-CoV‑2 spike protein based on Y‑shaped-aptasensor and AuNPs/COFs composites |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |