CN108976286A - It is a kind of using basic amino acid as the anchor polypeptide of anchored end and its application - Google Patents

It is a kind of using basic amino acid as the anchor polypeptide of anchored end and its application Download PDF

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CN108976286A
CN108976286A CN201810569324.5A CN201810569324A CN108976286A CN 108976286 A CN108976286 A CN 108976286A CN 201810569324 A CN201810569324 A CN 201810569324A CN 108976286 A CN108976286 A CN 108976286A
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胡继明
李昕翼
周晓东
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Wuhan University WHU
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
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    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4712—Muscle proteins, e.g. myosin, actin, protein

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Abstract

The invention discloses a kind of anchor polypeptide RRFPDD, replace traditional cysteine to be anchored using basic amino acid, achieve the purpose that Fast Load；Using acid aspartic acid, to provide a large amount of negative electrical charge, go enhancing anchor polypeptide to the protective value of gold nanoparticle；By regulating and controlling the type of hydrophobic amino acid, promote the self assembly of the polypeptide.The protection end and gold nanoparticle that target-probe is connected to anchor polypeptide are in pH=7.4 phosphate buffer; 37 DEG C are cultivated 100 seconds; biological detection reagent can be obtained; biological detection reagent can detecte the concentration of target detection thing by calibration curve method; when detecting cardiac troponin as biological detection reagent prepared by target-probe in particular with the polypeptide that sequence is FYSHSFHENWPS; the range of linearity is 0.05-500ng/mL, and detection limit is up to 0.45ng/mL.

Description

It is a kind of using basic amino acid as the anchor polypeptide of anchored end and its application
Technical field
It is fast on gold nanoparticle using basic amino acid as the anchor polypeptide of anchored end and one kind that the present invention relates to a kind of Speed efficiently and controllably loads polypeptide or the method for DNA.
Background technique
The technology that biomolecule (such as polypeptide, DNA) is carried on gold nanoparticle surface has dramatically pushed nanometer The rapid development in the fields such as biotechnology, biosensor and medical diagnosis.For this purpose, largely coming into being, such as raw Object sensing, medical diagnosis on disease and medical separation etc..
Common carrying method be using pentapeptide CALNN as anchor polypeptide, by tail end connecting peptides or DNA, And it is bonded using the sulfydryl in its cysteine with nanoparticle, realizes modification.Moreover, the asparagine in CALNN With a large amount of negative electrical charge, the stability of gold nanoparticle can be maintained in loading process.Therefore, the carrying method is since it is steady The fixed, advantages such as active force is strong, are widely used in various fields, as ion detection, Bacteria Detection and drug convey.But this biography Demand of the load means of system to anchor polypeptide is big, and incubation time is long (24 hours), and load efficiency is low, and occupies gold nano The surface site of particle is unfavorable for the functionalization in later period.
Summary of the invention
In order to improve the problem that cysteine load efficiency is low and time-consuming in conventional method, the present invention selects basic amino acid Form polypeptide anchored end, to replace traditional pentapeptide CALNN, under the mediation of the anchor polypeptide, realize it is quick, efficiently with And polypeptide or DNA are loaded controllably on gold nanoparticle, and enhance the stability of gold nanoparticle.
Technical solution provided by the invention is specific as follows:
A kind of anchor polypeptide, anchored end are basic amino acid, and middle section is hydrophobic central amino acid, and protection end is acid ammonia Base acid.Further: anchored end contains 2 basic amino acids, and protection end contains 2 acidic amino acids；The basic amine group Acid is arginine, and the acidic amino acid is aspartic acid.
Preferably: the sequence of anchor polypeptide is RRFPDD.
A method of biological detection reagent is prepared using above-mentioned anchor polypeptide, comprising the following steps:
(1) target-probe is connected to the protection end of above-mentioned anchor polypeptide, is obtained to anchor probe；
(2) NaH will be added to anchor probe2PO4-Na2HPO4In buffer solution, pH to 7.4 is adjusted, obtains visiting wait be anchored Needle solution；
(3) golden nanometer particle dispersion liquid is prepared, NaH then is added to it2PO4-Na2HPO4Buffer solution, adjust pH to 7.4, obtain AuNPs- phosphate dispersion liquid；
(4) it will be added in AuNPs- phosphate dispersion liquid to anchor probe solution, 37 DEG C of cultures try to get to biological detection Agent.
The target-probe is polypeptide or DNA, and adjusting reagent used in pH is solution of potassium carbonate, the time of 37 DEG C of cultures It is 100 seconds.
A kind of biological detection reagent, is prepared by the above method.
Application of the above-mentioned biological detection reagent in non-treatment purpose field of biological detection.
A method of cardiac troponin is detected using above-mentioned biological detection reagent, comprising the following steps:
The target detection thing of various concentration is added into above-mentioned biological detection reagent, is captured by ultraviolet specrophotometer poly- Collect signal, prepares standard curve；The target detection thing of unknown concentration is added, aggregation signal is captured by ultraviolet specrophotometer, The concentration of target detection thing is calculated according to signal strength and calibration curve formula.
The target detection thing is cardiac troponin；The target-probe is polypeptide, and sequence is FYSHSFHENWPS。
Compared with the existing technology, the present invention has the following advantages that and technical effect:
(1) present invention, which completes the load of polypeptide or DNA on gold nanoparticle, only needs 100 seconds, and the use of anchor polypeptide Amount is few.
(2) anchor series that the present invention designs have excellent protective value, after being supported on gold nanoparticle, system Have good resistance to acid and alkali, heat resistance, salt tolerance and resistant storage properties.
(3) the modification ratio of polypeptide or DNA are controllable in the present invention, and its bioactivity still retains after the completion of modification.
(4) carrying method of the invention will not impact the functionalization in gold nanoparticle later period.
Detailed description of the invention
When Fig. 1 illustrates not homopolypeptide and is supported on gold nanoparticle surface, fluorescence intensity with load time variation feelings Condition；
The number that Fig. 2 illustrates polypeptide both ends soda acid amino acid compares the influence of polypeptide-gold nanoparticle system stability；
Fig. 3 illustrates the hydrophobic amino acid type in polypeptide middle section to the influence of polypeptide-gold nanoparticle system stability；
Fig. 4 is the structural formula of anchor polypeptide RRFPDD；
Fig. 5 illustrates the comparative situation of anchor polypeptide RRFPDD and traditional polypeptide on load time；
After Fig. 6 illustrates modification anchor polypeptide, the investigation of system resistance to acid and alkali；
Fig. 7 illustrates the case where fluorescence kinetics optimization anchor polypeptide usage amount；
Fig. 8 illustrates the modification ratio that polypeptide is investigated by the export ratio of fluorescence signal；
Fig. 9 illustrates the modification ratio that DNA is investigated by the export ratio of fluorescence signal；
Figure 10 illustrates the hydrolysis efficiency of polypeptide with the situation of change of peptide modified ratio；
Figure 11 illustrates the investigation situation of DNA sandwich hybridization；
Figure 12 illustrates the load of anchor polypeptide RRFPDD and traditional peptide C ALNN to gold nanoparticle later period functionalization It influences.Wherein, curve a is the gold nanoparticle system of RRFPDD modification, and curve b is the gold nanoparticle system of CALNN modification, Curve c is unmodified gold nanoparticle system.
Figure 13 illustrates the Line Chart of detection cardiac troponin.
Specific embodiment
Technical solution of the present invention is described in detail With reference to embodiment.TMR generation in following embodiment Table tetramethylrhodamine, FAM represent 6- Fluoresceincarboxylic acid.
Instrument used in following embodiment: ultraviolet-visible spectrophotometer (UV-2550, Japanese Shimadzu Corporation), thoroughly Penetrate electron microscope (JEM-2100, day island proper electricity company), acceleration voltage 200KV.It is copolymerized burnt microscopic Raman instrument (HR-800, day This Horiba-Jobin-Yvon company), laser: He-Ne Lasers, excitation wavelength 632.8nm, camera lens: 50 times of focal length of Olympus Camera lens, the time for exposure: 5 seconds, integral number of times: 2 times.
Ultraviolet-visible absorption spectroscopy detection: sample cell: 50 μ L quartz colorimetric utensils, reference: deionized water (18.2M Ω). SERS spectra detection: taking 10 μ L fluid samples and aluminium foil, be placed on objective table, then adjusts focal length and acquires SERS signal.It is glimmering Light spectral detection: excitation wavelength: 495nm；Acquire wavelength: 530-560nm.
Embodiment 1
(1) design of anchor polypeptide
As shown in Figure 1, three kinds of design are the polypeptide sequence of anchored end with basic amino acid (arginine, lysine, histidine) Arrange (sequence 1, sequence 2, sequence 3), and protect terminal modified upper fluorophor 6- Fluoresceincarboxylic acid at it, three kinds of polypeptide sequences and its It is respectively indicated after 6- Fluoresceincarboxylic acid in modification as follows:
Serial number Using basic amino acid as the polypeptide sequence of anchored end Polypeptide after modifying 6- Fluoresceincarboxylic acid
Sequence 1 RRFPDD RRFPDD-FAM
Sequence 2 KKFPDD KKFPDD-FAM
Sequence 3 HHFPDD HHFPDD-FAM
(2) preparation of gold nanoparticle (AuNPs)
The aqueous solution of chloraurate that 100mL concentration is 0.01% (w/w) is added in flask and is heated to boiling, is acutely stirring Mix it is lower 1.5mL trisodium citrate aqueous solution (33.8mM) is rapidly added wherein, after solution colour reddens, continue to heat and protect It holds fluidized state 20 minutes, then by flask cooled to room temperature, obtains AuNPs dispersion liquid, gold nanoparticle diameter is about For 30nm.
(3) peptide modified gold nanoparticle
By the sodium dihydrogen phosphate (NaH of 100mM2PO4) aqueous solution and 100mM disodium hydrogen phosphate (Na2HPO4) aqueous solution with The volume ratio of 20:80 mixes, the NaH that obtained pH value is 7.4, concentration is 100mM2PO4-Na2HPO4Buffer solution.By NaH2PO4- Na2HPO4Buffer solution is added in AuNPs dispersion liquid, until the ultimate density of phosphate radical is 10mM, is then adjusted with solution of potassium carbonate The pH to 7.4 of AuNPs dispersion liquid obtains AuNPs- phosphate dispersion liquid.It is glimmering that prepared by embodiment 1 three kinds are modified with 6- carboxyl The polypeptide of light element is dissolved in NaH respectively2PO4-Na2HPO4In buffer solution, pH to 7.4, the polypeptide-that preparation concentration is 50 μM are adjusted FAM solution；Then three kinds of polypeptide-FAM solution are separately added into AuNPs- phosphate dispersion liquid, 37 DEG C are cultivated 100 seconds, are obtained AuNPs- polypeptide-FAM dispersion liquid.
Embodiment 2: the optimization of anchor polypeptide
(1) it is anchored the selection of terminal amino acid
The fluorescence of three kinds of AuNPs- polypeptide-FAM dispersion liquids of the preparation of embodiment 1 in different time points is obtained by luminoscope Signal makes fluorescence kinetic profiles.It, can be according to three kinds of AuNPs- since gold nanoparticle has Fluorescence quenching effect The fluorescence kinetics of polypeptide-FAM dispersion liquid change, and can filter out most efficient basic amino acid.As a result as shown in Figure 1, three kinds Arginine (R) can bring faster stronger fluorescence signal variation in basic amino acid, that is, bring higher polypeptide load efficiency.Cause This, we select anchored end of the arginine as polypeptide, to promote the modification efficiency of polypeptide.
(2) selection of amino acid number
In addition it designs 8 kinds of polypeptide sequences to be compared with sequence 1, to determine anchored end and protect the ratio of terminal amino acid. Anchored end of the arginine as polypeptide, to promote the modification efficiency of polypeptide；The other end of polypeptide is made using acid aspartic acid For protection end, to provide a large amount of negative electrical charge, and then enhance anchor polypeptide to the protective value of gold nanoparticle, and by flat Weigh soda acid amino acid number ratio, regulates and controls the distribution of charges of polypeptide.
Serial number Polypeptide sequence
Sequence 4 RFPD
Sequence 5 RFPDD
Sequence 6 RFPDDD
Sequence 7 RFPDDDD
Sequence 8 RRFPD
Sequence 9 RRFPDDD
Sequence 10 RRRFPD
Sequence 11 RRRFPDD
The amino acid classes for controlling its polypeptide middle section are consistent with number, change soda acid ammonia in its anchored end and protection end respectively The number ratio of cardinal extremity.As shown in Fig. 2, the stability of the AuNPs- polypeptide-FAM dispersion liquid by comparison 9 kinds of polypeptides preparation, screening Optimal soda acid amino acid number ratio (R:D) out, i.e. 2:2.
(3) selection of middle section amino acid
For the optimal hydrophobic neutral amino acid of optimization, we devise 9 kinds of polypeptides and sequence 1 is compared:
Serial number Polypeptide sequence
Sequence 12 RRAFDD
Sequence 13 RRAIDD
Sequence 14 RRALDD
Sequence 15 RRAPDD
Sequence 16 RRAVDD
Sequence 17 RRFLDD
Sequence 18 RRILDD
Sequence 19 RRLADD
Sequence 20 RRVLDD
Sequence 21 RRDD
Amino acid classes and the number for controlling its anchored end and protection end are certain, change the hydrophobic neutral amino in polypeptide middle section The type of acid is selected best by comparing the light absorption value of the AuNPs- polypeptide-FAM dispersion liquid prepared by above-mentioned 10 kinds of polypeptides Neutral amino acid combine hydrophobic centers as polypeptide, to promote the self assembly (Fig. 3) of the polypeptide.According to the above screening knot Fruit, optimal polypeptide sequence are sequence 1, i.e. RRFPDD (its structure such as Fig. 4).Test display, RRFPDD can be in 100 seconds to gold Nanoparticle surface Rapid Modification (Fig. 5), while the stability of gold nanoparticle system can be also greatlyd improve, so that system is existed It still keeps stablizing (Fig. 6) in the environment of pH4-12.
(4) determination of polypeptide dosage
The protection end of anchor polypeptide (sequence 1:RRFPDD) is connected to the target with 6- Fluoresceincarboxylic acid fluorophor On polypeptide (sequence 22:VVVV, sequence 23:SSSS), RRFPDD-VVVV-FAM and RRFPDD-SSSS-FAM are obtained, by the two point NaH is not dissolved in it2PO4-Na2HPO4In buffer solution (pH=7.4), the anchoring for preparing various concentration (50 μM, 70 μM, 90 μM) is more Peptide-target polypeptides-FAM solution.Then above-mentioned anchor polypeptide-target polypeptides-FAM solution is separately added into AuNPs- phosphoric acid salinity In dispersion liquid, 37 DEG C are cultivated 100 seconds, obtain AuNPs- anchor polypeptide-target polypeptides-FAM dispersion liquid of various concentration.Pass through fluorescence Spectrophotometer obtains the fluorescence letter of AuNPs- anchor polypeptide-target polypeptides-FAM dispersion liquid of various concentration in different time points Number, make fluorescence kinetic profiles.As shown in fig. 7, fluorescence fall off rate is most fast when anchor polypeptide is when dosage is 50 μM, And decline degree highest.Should the result shows that, 50 μM of anchor polypeptide usage amount, can reach highest speed under load and be anchored it is more Peptide utilization rate.
(5) investigation of ratio is modified
By target polypeptides (VVVV-FAM and SSSS-TMR) and target with 6- Fluoresceincarboxylic acid or tetramethylrhodamine DNA (sequence 24: ATTTACCACTTACTTCCGGA-FAM) is connected to the protection end of anchor polypeptide (sequence 1:RRFPDD), obtains RRFPDD- polypeptide (RRFPDD-VVVV-FAM, RRFPDD-SSSS-TMR), RRFPDD-DNA (RRFPDD- ATTTACCACTTACTTCCGGA-FAM).Relevant mixed solution is prepared using above-mentioned RRFPDD- polypeptide or RRFPDD-DNA, Make molar ratio (RRFPDD-VVVV-FAM:RRFPDD-TMR or the RRFPDD-VVVV-FAM:RRFPDD- of RRFPDD- polypeptide SSSS-TMR) it is respectively 1:1,2:1,3:1,4:1,5:1, or makes the molar ratio (RRFPDD- of RRFPDD-DNA:RRFPDD It ATTTACCACTTACTTCCGGA-FAM:RRFPDD-TMR) is respectively 1:1,2:1,3:1,4:1,5:1.Then, by specific mole The RRFPDD- polypeptide or RRFPDD-DNA of ratio are dissolved in NaH respectively2PO4-Na2HPO4In buffer solution (pH=7.4), anchoring is made Polypeptide-object-fluorophor solution.Then above-mentioned anchor polypeptide-object-fluorophor solution is separately added into AuNPs- In phosphate dispersion liquid, 37 DEG C are cultivated 100 seconds, obtain the polypeptide-AuNPs dispersion liquid of various concentration.It is completed wait load, centrifugation three Secondary removal extra target polypeptides or target dna.1.0M dithiothreitol (DTT) is added in the system completed to modification, placement overnight Afterwards, the target polypeptides or target dna of having modified on gold nanoparticle surface are replaced to get off.Then detect the fluorescence letter of its output Whether number ratio further determines that these target polypeptides or target dna according to certain with the modification ratio reflected between polypeptide Proportional load is on gold nanoparticle.As a result as shown in Figure 8 and Figure 9, when target polypeptides or target dna are with certain molar ratio (1:1,2:1,3:1,4:1,5:1) be added gold nanoparticle system when, modification ratio also be respectively 1:1,2:1,3:1,4:1, 5:1.Phenomenon explanation, these target polypeptides or target dna can be loaded according to a certain percentage.Therefore, more with the anchoring The modification that peptide RRFPDD is anchored is controllable.By regulating and controlling the modification ratio of polypeptide, hydrolysis efficiency can reach 85%.
(6) modification of Raman dyestuff
By the polypeptide (sequence 1:RRFPDD) and polypeptide that are dissolved in the phosphate buffer solution of pH=7.4 (sequence 25: CALNN it) is separately added into AuNPs- phosphate dispersion liquid, making its ultimate density is respectively 50 μM and 1.38mM, and at 37 DEG C It cultivates 100 seconds and 8 hours respectively, to obtain RRFPDD-AuNPs dispersion liquid and CALNN-AuNPs dispersion liquid.It then, will be dense Degree is that mercaptobenzoic acid-ethanol solution of 1.0mM is separately added into RRFPDD-AuNPs dispersion liquid and CALNN-AuNPs dispersion liquid In, be stored at room temperature 24 hours under the conditions of being protected from light, then three times to reaction solution centrifugation, be resuspended to original volume, with remove it is unmodified The mercaptobenzoic acid on gold nanoparticle surface.
The functionalization ability in gold nanoparticle later period is investigated using the finger print information that Raman spectrum provides, by poly- The hot spot-effect for collecting induction, obtains and compares RRFPDD-AuNPs dispersion liquid and sulfydryl benzene first is added in CALNN-AuNPs dispersion liquid Raman signal intensity after acid-ethanol solution, wherein the characteristic signal peak of mercaptobenzoic acid is in 1076cm-1And 1580cm-1.Knot Fruit is as shown in figure 12, and gold nanoparticle is modified after anchor polypeptide RRFPDD, has no effect on the later period functionalization of gold nanoparticle. And after gold nanoparticle modifies upper CALNN, the characteristic signal peak peak height of mercaptobenzoic acid is substantially reduced, the result explanation Traditional anchor polypeptide CALNN after anchoring can the later period functionalization to gold nanoparticle generate certain interference.
Embodiment 3
(1) it will be connected with the anchor polypeptide RRFPDD of target dna, i.e. DNA-RRFPDD is dissolved in NaH2PO4-Na2HPO4It is slow It rushes in solution (pH=7.4), DNA-RRFPDD concentration is 50 μM；It is then added into AuNPs- phosphate dispersion liquid, 37 DEG C Culture is centrifuged after 100 seconds and removes extra DNA-RRFPDD three times, obtains the DNA- gold nanoparticle mediated by anchor polypeptide.
(2) investigation of bioactivity:
The hydrolysis property of polypeptide: selecting one section of target polypeptides with fluorescent dye, (sequence 26:RCFRGGDD, passes through anchor Determine polypeptide RRFPDD to be supported on gold nanoparticle.After the completion of load in 100 seconds, centrifugation is resuspended, and 100nM is added into system Trypsase, culture detected fluorescence after 30 minutes.Changed by enzyme hydrolysis bring fluorescence signal, whether verifies its bioactivity In the presence of.Then by adjusting the modification ratio (10,20,40,60,80,100%) of the polypeptide, the optimal hydrolysis efficiency of optimization. The results are shown in Figure 10, when the modification ratio of polypeptide is 20%, hydrolysis efficiency highest.
(3) cross performance of DNA
Using the anchor polypeptide at the specific DNA of gold nanoparticle area load (probe 2, probe 3), then in system It is added target dna (probe 1), constitutes sandwich hybridization.Assemble the presence or absence of signal, the hybridity of validating DNA by ultraviolet detection It can whether there is.As a result as shown in figure 11, the aggregation of gold nanoparticle can be caused by target dna being added, and be illustrated more by the anchoring The cross performance of DNA still remains after peptide progress DNA modification.
Embodiment 4: detection cardiac troponin
Target polypeptides (sequence 27:FYSHSFHENWPS) energy specific recognition cardiac troponin (cTnI), design is connected in The target polypeptides of anchor polypeptide RRFPDD protection end, i.e. RRFPDD-FYSHSFHENWPS.By 50 μM of RRFPDD- FYSHSFHENWPS is supported on gold nanoparticle surface.The cardiac troponin of various concentration is added, passes through uv-spectrophotometric Meter capture aggregation signal.Acquisition data simultaneously optimize (reaction time, NaH to each parameter2PO4-Na2HPO4Buffer solution is dense Degree, gold nanometer particle grain size, peptide modified ratio etc.), the final range of linearity and detection limit for obtaining target detection thing.As a result As shown in figure 13, when carrying out the detection of cardiac troponin using this method, range of linearity 0.05-500ng/mL, detection It is limited to 0.45ng/mL.
Sequence table
<110>Wuhan University
<120>a kind of using basic amino acid as the anchor polypeptide of anchored end and its application
<160> 27
<170> SIPOSequenceListing 1.0
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Arg Phe Pro Asp
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Arg Phe Pro Asp Asp
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Arg Arg Phe Pro Asp
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Arg Arg Phe Pro Asp Asp Asp
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Arg Arg Arg Phe Pro Asp
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Arg Arg Arg Phe Pro Asp Asp
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<210> 12
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Arg Arg Ala Phe Asp Asp
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Arg Arg Ala Ile Asp Asp
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Arg Arg Ala Leu Asp Asp
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<210> 15
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<400> 15
Arg Arg Ala Pro Asp Asp
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<210> 16
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Arg Arg Ala Val Asp Asp
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<210> 17
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Arg Arg Phe Leu Asp Asp
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<210> 18
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Arg Arg Ile Leu Asp Asp
1 5
<210> 19
<211> 6
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 19
Arg Arg Leu Ala Asp Asp
1 5
<210> 20
<211> 6
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 20
Arg Arg Val Leu Asp Asp
1 5
<210> 21
<211> 4
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<213>artificial sequence (Artificial Sequence)
<400> 21
Arg Arg Asp Asp
1
<210> 22
<211> 4
<212> PRT
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<400> 22
Val Val Val Val
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<211> 4
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<400> 23
Ser Ser Ser Ser
1
<210> 24
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 24
atttaccact tacttccgga 20
<210> 25
<211> 5
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 25
Cys Ala Leu Asn Asn
1 5
<210> 26
<211> 8
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 26
Arg Cys Phe Arg Gly Gly Asp Asp
1 5
<210> 27
<211> 12
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 27
Phe Tyr Ser His Ser Phe His Glu Asn Trp Pro Ser
1 5 10

Claims (10)

1. a kind of anchor polypeptide, it is characterised in that: anchored end is basic amino acid, and middle section is hydrophobic amino acid, and protection end is acid Acidic amino acid.
2. anchor polypeptide according to claim 1, it is characterised in that: anchored end contains 2 basic amino acids, and protection end contains There are 2 acidic amino acids.
3. anchor polypeptide according to claim 1 or 2, it is characterised in that: the basic amino acid is arginine, described Acidic amino acid be aspartic acid.
4. anchor polypeptide according to claim 1, it is characterised in that: the sequence of the anchor polypeptide is RRFPDD.
5. a kind of method of the anchor polypeptide preparation biological detection reagent using claim 1, which is characterized in that including following step It is rapid:
(1) protection end that target-probe is connected to anchor polypeptide described in claim 1, obtains to anchor probe；
(2) NaH will be added to anchor probe2PO4-Na2HPO4In buffer solution, pH to 7.4 is adjusted, is obtained molten to anchor probe Liquid；
(3) nanogold dispersion liquid is prepared, NaH then is added to it2PO4-Na2HPO4Buffer solution adjusts pH to 7.4, obtains AuNPs- phosphate dispersion liquid；
(4) it will be added in AuNPs- phosphate dispersion liquid to anchor probe solution, 37 DEG C are cultivated to get biological detection reagent is arrived.
6. according to the method described in claim 5, it is characterized by: the target-probe be polypeptide or DNA, adjust pH used in Reagent be solution of potassium carbonate, 37 DEG C culture times be 100 seconds.
7. a kind of biological detection reagent, it is characterised in that: be prepared by method described in claim 5 or 6.
8. biological detection reagent as claimed in claim 7 is in the application of non-treatment purpose field of biological detection.
9. a kind of method using biological detection reagent detection cardiac troponin as claimed in claim 7, which is characterized in that packet Include following steps:
The target detection thing of various concentration is added into biological detection reagent according to any one of claims 8, passes through uv-spectrophotometric Meter capture aggregation signal, prepares standard curve；The target detection thing of unknown concentration is added, is captured by ultraviolet specrophotometer poly- Collect signal, according to the concentration for calculating target detection thing on standard curve with signal strength.
10. according to the method described in claim 9, it is characterized by: the target detection thing is cardiac troponin；It is described Target-probe be polypeptide, sequence FYSHSFHENWPS.
CN201810569324.5A 2018-06-05 2018-06-05 It is a kind of using basic amino acid as the anchor polypeptide of anchored end and its application Pending CN108976286A (en)

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CN112824878A (en) * 2019-11-21 2021-05-21 北京大学 Anchoring method of target biological molecules, expansion microscopic imaging method and application thereof

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EP2240782A2 (en) * 2008-02-04 2010-10-20 Ulive Enterprises Limited Nanoparticle conjugates
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112824878A (en) * 2019-11-21 2021-05-21 北京大学 Anchoring method of target biological molecules, expansion microscopic imaging method and application thereof
CN112824878B (en) * 2019-11-21 2021-12-03 北京大学 Anchoring method of target biological molecules, expansion microscopic imaging method and application thereof

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Application publication date: 20181211