CN110095443A - A kind of fluorescent method detecting brain natriuretic peptide based on graphene oxide/aptamer - Google Patents
A kind of fluorescent method detecting brain natriuretic peptide based on graphene oxide/aptamer Download PDFInfo
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- CN110095443A CN110095443A CN201910383373.4A CN201910383373A CN110095443A CN 110095443 A CN110095443 A CN 110095443A CN 201910383373 A CN201910383373 A CN 201910383373A CN 110095443 A CN110095443 A CN 110095443A
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- 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
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- 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
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- G—PHYSICS
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- 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
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- 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
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
- G01N33/6818—Sequencing of polypeptides
- G01N33/6821—Sequencing of polypeptides involving C-terminal degradation
Abstract
The present invention provides a kind of highly sensitive, highly selective, the inexpensive homogeneous fluorescent new detecting methods of brain natriuretic peptide (BNP).This method passes through building graphene oxide/fluorochrome label aptamer combined probe first reduces the fluorescence background of detection using graphene oxide to the fluorescence quenching of dyestuff；When the addition of BNP and its aptamer make fluorescent dye far from graphene oxide by specific molecular recognition reaction, the fluorescence signal of dyestuff is restored, and linear work curve is established in the variation based on BNP concentration and fluorescence intensity；Fluorescence intensity change value after sample is added finally by measurement simultaneously substitutes into the actual concentrations that BNP in clinical sample is calculated in working curve.The present invention overcomes various immunoassays because of the shortcomings that using antibody to identify that there are cross reactions, avoid the interference of related peptide etc., have many advantages, such as simple, quick, inexpensive, highly sensitive, highly selective, and the quantitative detection that can be used for BNP in clinically blood samples of patients, to diagnose in time, guiding treatment and prompt prognosis.
Description
Technical field
The invention belongs to technical field of medical examination, and in particular to a kind of the highly sensitive of brain natriuretic peptide, the inspection of high specific fluorescence
Survey new method.
Background technique
Cardiovascular disease is one of modern society's disease incidence and the highest disease of disability rate, seriously threatens human health.
And heart failure (Heart failure, HF) is the whole reaching advanced stages of cardiovascular disease, the death rate and readmission lead compared with
Height, but can be to avoid many death incidents as caused by heart failure, so the early stage of heart failure examines by early detection and timely treatment
It is disconnected to be of great significance.Physical signs (such as electrocardiogram, chest X-ray inspection, echocardiogram) and biochemical indicator (such as it is special
Anisotropic biomarker) monitoring be clinically diagnose heart failure main method.Compared with physical signs monitoring, biochemistry index test
Simply, accurately, it is at low cost, and be not necessarily to analysis expert, only need to be by measurement result compared with reference value.Therefore, biological marker
The accurate detection of object plays important role in the diagnosis of heart failure.Brain natriuretic peptide (Brain natriuretic peptide,
BNP) the polypeptide being made of 32 amino acid, mainly synthesizes in ventricle, exists simultaneously in Healthy People and patients with heart failure,
And recycled in peripheral blood vessel, but due to heart size expansion and Pressure Overload-induced, the BNP secretion of patients with heart failure is dramatically increased,
And it is horizontal directly proportional to heart failure severity.Therefore, BNP level can reflect cardiac function, facilitate heart failure diagnosis and
Prognosis is the goldstandard of kit for diagnosing heart failure.In addition, another polypeptide Amino terminal-pro brain natriuretic peptide (N-terminal pro-brain
Natriuretic peptide, NT-proBNP), and the marker of diagnosis heart failure, both come from plasma pro-brain natriuretic peptide levels (pro-
Brain natriuretic peptide, proBNP), proBNP is generated between the 76th and the 77th residue by cutting
The metabolin of biologically active peptide BNP and nonactive peptide NT-proBNP, these three BNP forms and BNP are existed simultaneously in blood circulation
In.Currently, detection BNP content when, the presence of related peptide is likely occurred cross reaction so that BNP assay error compared with
Greatly.
Existing document report using chemiluminescence immunoassay (Chemiluminescence immunoassay,
CLIA), enzyme linked immunosorbent assay (ELISA) (Enzyme-linked immunosorbent assay, ELISA) and immunochromatographic method
The methods of (Immunochromatography assay, ICA) detects the content of BNP, but these methods all have some disadvantages
End.Wherein, clinically most common chemiluminescence immunoassay has many advantages, such as highly sensitive, the wide range of linearity, but detects
It vulnerable to interference, and needs to introduce magnetic bead and is separated, testing cost is higher；The specificity of enzyme linked immunosorbent assay (ELISA) is good,
It is used to develop various commercial test kits, but muting sensitivity and precision limit its application；Immunochromatographic method is simple, fast
It is fast, easy to carry, it is suitable for by bed quickly detection (Point of care testing, POCT), but its sensitivity is not enough and easy
It is disturbed, so that Accurate Determining is restricted.In addition, the above method is immunoassay, these methods are same detection BNP's
When, it can also detect proBNP and BNP metabolite, because of their antibody epitopes having the same, this causes the concentration of BNP normal
Often it is overestimated.Therefore, more highly sensitive, highly selective, inexpensive detection BNP method needs to develop.
In the invention, we introduce aptamer as recognition component to avoid intersecting between BNP and related peptide
Reaction, the selectivity of improvement method.Aptamer is the aglucon phyletic evolution technology (Systematic by index concentration
Evolution of ligands by exponential enrichment, SELEX) oligonucleotide that filters out, pass through
It is folded into various conformations and specifically binds target, it is also referred to as " chemical antibody ".Compared to antibody, aptamer has
Be easy to chemical synthesis, it is at low cost, stability is good the advantages that.It has been widely used for examining by the probe that labeling nucleic acid aptamers obtain
Survey various inorganic ions, small molecule, large biological molecule, even cell and biological tissue etc..The present invention is with fluorochrome label
Aptamer is identification and signal group, constructs a kind of homogeneous fluorescent measurement BNP method.
Homogeneous determination refers to carry out in the solution, the system without being separated to component that is free and combining.Homogeneous body
The detection method of system was both simple, accurate and automation easy to accomplish.In the past decade develop and successfully have listed two kinds it is basic
Advances in Homogeneous Immunoassay, be fluorescence excitation transfer immunoassays (FETI) and fluorescent polarization immunoassay (FPIA) respectively, it
Be all based on the variation of the physical property that fluorescence labels combine Ag-Ab.As it can be seen that using fluorescent method another
Benefit can exactly develop homogeneous detection architecture, greatly reduction use cost, raising detection practicability.
The present invention is used as fluorescence quencher by introducing graphene oxide (Graphene oxide, GO), for reducing inspection
The background signal of survey realizes the highly sensitive detection to BNP.GO is a kind of novel single layer two-dimension nano materials, in recent years due to
Its unique optics and catalysis characteristics receive significant attention in fields such as bioanalysis, drug delivery, electro-catalysis.Because it has
There are biggish surface area and unique sp2(sp2/sp3) bonded network, so pi-pi accumulation and single stranded DNA flexible can be passed through
In conjunction with.In addition, GO is also used as the energy acceptor in energy transfer system, the light radiation for absorbing various fluorogens strongly causes
Fluorescent quenching occurs in it, and building is based on the sensor of fluorescence resonance energy transfer (FRET).In the presence of BNP, aptamer
And BNP forms more stable combination by specific molecular recognition reaction so that aptamer be detached from the surface GO, dyestuff it is glimmering
Light is restored.In the present invention, close to the height of BNP to the efficient quenching effect and aptamer of fluorogen by integrating GO
And power, construct the homogeneous fluorescent analyzing novel methods that highly sensitive one kind, high specific, low cost detect BNP.
Summary of the invention
For above problem and technology, the present invention provides one kind to be based on fluorochrome label aptamer/oxidation
The new method of graphene combined probe detection brain natriuretic peptide, comprising the following steps:
A kind of fluorescence detection new method of brain natriuretic peptide (hereinafter referred to as BNP), it is characterised in that: firstly, graphene oxide (with
Lower abbreviation GO) it is acted on by the aptamer (hereinafter referred to as FAM-aptamer) of pi-pi accumulation and fluorochrome label, the two
Between energy transfer lead to fluorescent quenching, background signal reduces, then in sample BNP by specific molecular recognition reaction with
FAM-aptamer is combined, and this combination can change the conformation of FAM-aptamer, make the helical structure uncoiling of aptamer, and
The interaction between aptamer and GO is destroyed, so that FAM-aptamer is released from the surface GO, the fluorescence of dyestuff is obtained
To restore, fluorescence recovery extent and BNP concentration are in a linear relationship, make corresponding standard curve, and calculate in sample accordingly
The content of BNP.
The detection method of BNP in the present invention, it is characterised in that: by GO, FAM-aptamer, the sample containing BNP in phosphate
It is reacted in (hereinafter referred to as PB) buffer, scans the maximum excitation of 200-800nm on sepectrophotofluorometer after reaction
Wavelength and maximum emission wavelength, while doing blank test；According to the fluorescence intensity change and BNP standard items at maximum emission wavelength
The working curve that concentration is established can calculate the content of BNP in sample.
As optimization, the fluorescence detection method of above-mentioned BNP, it is characterised in that: the concentration of the GO is 3-40 μ g/mL.
As advanced optimizing, the fluorescence detection method of above-mentioned BNP, which is characterized in that use following steps: first by BNP
Standard items are added to GO (20 μ g/mL) and the mixed solution mesoscale eddies of PB buffer (pH7.4) mixes, and stand 30min at room temperature；
Then it adds FAM-aptamer vortex to mix, stands 30min at room temperature；Fluorescence is measured on sepectrophotofluorometer, is arranged
Maximum excitation wavelength is 495nm, scans the fluorescence emission spectrum of 505-650nm, strong with the fluorescence at maximum emission wavelength 519nm
Degree is quantified, while doing blank test；BNP content in the sample is calculated as follows:
Y=1.787X-0.1331 (calibration curve equation)
In formula:
Y is that fluorescence restores efficiency ((F2-F1)/(F0-F1), wherein F0Indicate the fluorescence intensity of blank probe, F1It indicates to add GO
And when BNP is not added solution fluorescence intensity, F2The fluorescence intensity of solution when indicating to exist simultaneously GO and BNP)；X is BNP standard items
Concentration (unit: pg/mL).
A kind of fluorescence detection method of BNP, including blank test 1, blank test 2, sample detection and calculated result step,
It is characterized by:
(1) blank test 1:
40 μ L PB buffers (pH7.4) and 15 μ L FAM-aptamer are sequentially added in 1.5mL centrifuge tube
(100nmol/L), adding ultrapure water to be supplemented to total volume is 400 μ L, is vortexed and mixes, stands 30min at room temperature；Then in fluorescence point
It is 495nm that maximum excitation wavelength is arranged on light photometer, the fluorescence intensity of 505-650nm is scanned, by maximum emission wavelength 519nm
The fluorescence intensity at place is denoted as F0；
(2) blank test 2:
40 μ L PB buffers (pH7.4), 20 μ L GO (20 μ g/mL) and 15 μ L are sequentially added in 1.5mL centrifuge tube
FAM-aptamer (3.75nmol/L), adding ultrapure water to be supplemented to total volume is 400 μ L, is vortexed and mixes, stands 30min at room temperature；
Then maximum excitation wavelength is arranged on sepectrophotofluorometer is 495nm, scans the fluorescence intensity of 505-650nm, will be maximum
Fluorescence intensity at launch wavelength 519nm is denoted as F1；
(3) sample measures:
40 μ L PB buffers (pH7.4), 20 μ L GO (20 μ g/mL) and certain body are sequentially added in 1.5mL centrifuge tube
Sample solution of the product containing BNP, which is vortexed, to be mixed, and is stood 30min, is added 15 μ L FAM-aptamer (3.75nmol/L), add ultrapure
It is 400 μ L that water, which is supplemented to total volume, is vortexed and mixes, stands 30min at room temperature；Then it is arranged on sepectrophotofluorometer maximum
Excitation wavelength is 495nm, scans the fluorescence intensity of 505-650nm, and the fluorescence intensity at maximum emission wavelength 519nm is denoted as
F2；
(4) calculated result:
The content of BNP is calculated as follows in the sample:
Y=1.787X-0.1331 (calibration curve equation)
In formula:
Y is that fluorescence restores efficiency ((F2-F1)/(F0-F1))；X is the concentration (unit: pg/mL) of BNP.Original of the present invention
Material and reagent are commercial product.PB (Phosphate Buffer) buffer in the present invention is phosphoric acid known in the art
Salt buffer.The pre-treating method of FAM-aptamer in the present invention are as follows: by the freeze-dried powder of FAM-aptamer in 6000rpm from
Heart 5min is added the ultrapure water after 800 μ L boil and redissolves to 2 μm of ol/L.The sequence of the aptamers of BNP specificity is in the present invention
5’-H2N-TTT TTT TAA ACG CTC AAA GGA CAG AGG GTG CGT AGG AAG GGT ATT CGA CAG
GAG GCT CAC A-3’(Hye Ri Jang,Alastair W.Wark,Seung Hee Baek,Bong Hyun Chung,
Hye Jin Lee,2014,Analytical Chemistry,86,814-819)。
In clinical blood examination experiment, we compared the method for the present invention measured value and clinical diagnosis value, clinically adopt
With the BNP in double-antibody sandwich chemiluminescence immunoassay detection blood sample, it is with Siemens Type B atrial natriuretic peptide assay kit
Diagnostic kit.Kit is mainly made of labelled reagent, solid-phase reagent and measurement standard curve card.First antibody is acridinium ester
Monoclonal mouse anti-human's antibody of label, is present in labelled reagent, and for the cyclic structure of BNP, secondary antibody is and magnetic
The covalently bound monoclonal mouse anti-human's antibody of grain, is present in solid-phase reagent, for the C-terminal part of BNP.Chemiluminescence signal by
The analysis of ADVIA Centaur chemiluminescence immunoassay system obtains.
The utility model has the advantages that
The present invention reduces background signal, then the BNP in sample passes through first with the fluorescence of GO quenching FAM-aptamer
Specific molecular recognition reaction forms compound in conjunction with FAM-aptamer, so that FAM-aptamer is released from the surface GO
Come, the fluorescence of probe is restored, the influence by monitoring BNP to the fluorescence intensity of GO/FAM-aptamer combined probe, root
Restore the linear relationship between efficiency and BNP concentration according to fluorescence and establish standard curve, and quantify the BNP in sample accordingly, establishes
A kind of homogeneous fluorescent analyzing novel methods quickly, easy, inexpensive, realize highly sensitive, high specific to BNP content in sample
Detection.
Selectivity experiments have shown that in blood there are potential interfering substance such as Amino terminal-pro brain natriuretic peptide (NT-proBNP),
Interferon (Interferon, IFN), cysteine (Cysteine, Cys), glycine (Glycine, Gly), arginine
(Arginine, Arg), histidine (Histidine, His), human serum albumins (Human serum albumin, HSA) and
The influence to testing result such as bovine serum albumin(BSA) (Bovine serum albumin, BSA) is smaller, identical with BNP concentration
Chaff interferent to the fluorescence of GO/FAM-aptamer combined probe substantially without recovery, even same as kit for diagnosing heart failure goldstandard
NT-proBNP will not interference measurement, this is because aptamer is introduced into overcome in current immunoassay method and exists
Cross reaction interference, improve method choice.
The practicability of this method is stronger, when especially the concentration of BNP is within the scope of 0.074-0.56pg/mL in sample, BNP
It is in good linear relationship (r=0.9929) that content and fluorescence, which restore efficiency, in blood sample recovery testu, recovery of standard addition
For 96.44-109.46%, RSD 1.58-6.87%.In clinical blood pattern detection, collects altogether and measure 45 clinical samples
(wherein 14 women and 31 male's blood samples, age are respectively less than 16 years old), and measurement result and clinical diagnosis value are compared,
Testing result value of the present invention is lower than hospital clinical chemiluminescence immunoassay measured value, thus it is speculated that is since difference is drawn between methodology
It rises.The detection of the method for the present invention is limited to 0.045pg/mL, and the detection far below clinical chemistry luminescent immunoassay limits (2.0pg/
mL).As it can be seen that this method is sensitiveer.
In testing cost estimation, the method for the present invention buys 1g graphene oxide (GO) and spends 100 yuan, 1OD FAM-
The price of aptamer freeze-dried powder (16 μ L ultrapure waters is added to be made into the solution that concentration is 100 μm of ol/L) is 70 yuan, and one bottle of BNP freezes
400 yuan of price of dry powder (2mL reagent water is added to be made into the solution that concentration is 1482pg/mL), the measurement of a sample includes standard
The foundation of curve and the measurement of actual sample, according to each reagent dosage and corresponding concentration (GO:20 μ g/mL；FAM-aptamer:
3.75nmol/L), estimating reagent cost is about 1 yuan, and clinical using chemiluminescence immunoassay detection BNP, according to weight
City's medical payment standard is celebrated, a sample is detected and collects 180 yuan.As it can be seen that compared to clinical chemistry luminescent immunoassay, we
Another advantage of method is that homogeneous fluorescent detection substantially reduces cost.
In addition, according to clinical diagnosis result (whether heart failure) and the method for the present invention and clinical chemistry luminescent immunoassay pair
The measurement result of BNP content in clinical samples blood sample, the ability of building receiver operating curves (ROC) evaluation BNP diagnosis HF, knot
Fruit shows that two methods all have preferable diagnosis accuracy, and the threshold value of two methods is for all having 100% when diagnosing heart failure
Sensitivity, when exclusion for heart failure all has high negative predictive value (100%).But it is sent out compared to hospital clinical chemistry
Light immunoassay, the method for the present invention have higher specificity (the method for the present invention and clinical chemistry hair for the diagnosis of heart failure
The specificity of light immunoassay is respectively 66.7% and 63.6%).Because this acquisition research object sample is children's sample, the heart
The illness rate that declines is lower, in addition the non-heart failure children in part have different degrees of injury of kidney, influences BNP level in blood, this method is to mesh
Preceding children's kit for diagnosing heart failure difficulty provides new approaches, while this method has better effect in adult kit for diagnosing heart failure.
In short, the method for the present invention is quick, easy, at low cost, high sensitivity, selectivity it is good, practical, be suitable for clinic
The accurate detection of BNP content in sample, and important reference value can be provided for the diagnosis and prognosis of Patients with Cardiac Failure, it is suitable for carrying out
It promotes the use of.
Detailed description of the invention
Fig. 1 is the schematic illustration of present invention detection BNP；
Fig. 2 is spectrogram of the various concentration GO to FAM-aptamer fluorescent quenching；
Fig. 3 is the spectrogram that BNP restores GO/FAM-aptamer combined probe fluorescence；
Fig. 4 is the working curve that fluorescence restores that efficiency is fitted relative to BNP concentration；
Fig. 5 is that the selectivity of disturbance substance is investigated；
Fig. 6 is that the specificity of different sequence dna probes is investigated；
Fig. 7 is the pattern of GO (a), GO/FAM-aptamer compound (b) and GO/BNP/FAM-aptamer compound (c)
And the atomic force microscopy diagram of cross section；
Fig. 8 is that the measurement of clinical samples sample this method blood sample is dissipated with the comparison of hospital clinical Chemiluminescence immunoassay determination data
Point analysis；
Fig. 9 is the method for the present invention and subject's work (ROC) curve pair of hospital clinical Chemiluminescence immunoassay measurement BNP
Compare result.
Specific embodiment
The present invention will be further described combined with specific embodiments below, it is pointed out here that following embodiment is served only for this
Invention is described further, and should not be understood as limiting the scope of the invention, the person skilled in the art of this field can root
Some nonessential modifications and adaptations are made to the present invention according to foregoing invention content.
Instrument and material
Instrument
RF-5301PC sepectrophotofluorometer (Japanese Shimadzu) is used for the fluoremetry of sample solution；Use Dimension
Icon atomic force microscopy system (U.S.) obtains atomic force microscope (AFM) image；(big dragon has MX-S vortex mixed instrument in Beijing
Limit company) it is used to mix solution；BY-R18 high speed freezing medical centrifuge (Beijing Bai Yang Medical Devices Co., Ltd.) is used for
The centrifugation of FAM-aptamer freeze-dried powder and blood sample；Sartorius electronic balance (Germany) is used for the weighing of sample.
Material
BNP and NT-proBNP is purchased from Siemens Healthcare Diagnostics Inc. (U.S.)；It is special to BNP
Nucleic acid aptamer sequence are as follows: 5 '-H2N-TTT TTT TAA ACG CTC AAA GGA CAG AGG GTG CGT AGG AAG
GGT ATT CGA CAG GAG GCT CAC A-3'；Random sequence 1:5 '-H2N-CGG ACC GCG CAT AGT CGG ATC
TGT CTA ACT TAC CTA CGT ATC CAG TAC TGA TGT AAG TGT G-3'；Random sequence 2:5 '-H2N-GCC
AGG CGC GAA ACA GCG TAG ACA GAT AGA TTG GTT GGA AAG CCC TTG TCAACATTC ACA C-
3'；Random sequence 3:5 '-H2N-ATT TAC TCA CAA AGC CGA CCA GAC CCG ACC AAT TT-3'；Stochastic ordering
Arrange 4:5 '-H2N-ATG TAC GAC CAC AAA AAA ACC TCA CAA AGC ACA AA-3'；Random sequence 5:5 '-H2N-
(the raw work biotechnology in Shanghai is limited by AGT AAT GCC CGG TAG TTA TTC AAA GAT GAG TAG GAA AAG A-3 '
Company)；Graphene oxide (GO) is purchased from Nanjing Xian Feng Nono-material Science & Technology Ltd.；Interferon (IFN), cysteine
(Cys), glycine (Gly), human serum albumins (HSA), bovine serum albumin(BSA) (BSA), arginine (Arg) and histidine
(His) it is purchased from Shanghai Aladdin Reagent Company；Blood sample is obtained from Children's Hospital Attached to Chongqing Medical Univ.；Phosphate buffer
(0.2mol/L) is used for the acidity of regulation system；Agents useful for same is that analysis is pure, and experimental water is the ultrapure water (18.2 after boiling
MΩ·cm)。
Patient information
We have studied patient's (male/female=31/14, age are 3 hours to 16 years old) of 45 doubtful heart failures in total, they
All it is admitted to hospital because of expiratory dyspnea, edema, oliguresis or heart murmur, bradycardia, increased heart rate.In addition to age and gender, we
Fat and renal function situation is investigated, also so that their influence can be avoided as far as possible in ROC curve is analyzed, it is however generally that,
Obesity can be such that BNP level in blood declines, and renal damage rises its level.Average BMI when all patients are admitted to hospital is 15.0 ±
3.2kg/m2(normal range (NR) of BMI is 18.5-24.9kg/m2), BMI is in 25.0-29.9kg/m2When in range, to be fat, therefore
This research is not related to obese patient.We calculate the glomerular filtration rate (GFR) of each patient with simplified MDRD formula, are commented with this
Estimate their injury of kidney situation, it is however generally that, the normal value of GFR is 80-120mL/min/1.73m2, may lower than the range
There are different degrees of injury of kidney.The renal function of part HF patient is decreased obviously, and in 4 patients for being diagnosed as HF, there is 2 trouble
The GFR of person is respectively 3.7 and 6.7mL/min, and in other 41 non-patients with heart failure, the range of GFR is 24.1-181.8mL/
Min, there is also different degrees of injury of kidney for individual non-patients with heart failure.Making a definite diagnosis for HF is that expert makes according to all inspection results
Judgement, including the analysis of medical history, blood routine, physical examination, electrocardiogram, chest x-ray and echocardiogram.
The preparation and detection method of 1 solution of embodiment
The preparation of FAM-aptamer stock solution
The freeze-dried powder of FAM-aptamer is centrifuged 5min in 6000rpm, the 800 μ L of ultrapure water being added after boiling is prepared
At the stock solution of 2 μm of ol/L, saved in 4 DEG C of refrigerators.Being diluted to concentration before use is 100nmol/L.
The preparation of BNP standard items stock solution
The ultrapure water after 2mL boils is added in freeze-dried powder using liquid-transfering gun, at room temperature (20-30 DEG C) balance 15-
20min is completely dissolved freeze-dried powder, and then to uniform, the concentration of the stock solution of configuration is rotation gently/reverse reagent
1482pg/mL is saved in -20 DEG C of refrigerators, is diluted before use.
Detection method
Blank group 1: 40 μ L PB buffers (pH7.4) and 15 μ L FAM-aptamer are sequentially added in 1.5mL centrifuge tube
(3.75nmol/L), adding the ultrapure water boiled to be supplemented to total volume is 400 μ L, is vortexed and mixes, stands 30min at room temperature；Glimmering
Fluorescence intensity (the excitation wavelength: 495nm of solution is measured on light spectrophotometer；Emission spectrum scanning range: 505-650nm；It sweeps
Retouch speed: Very Fast；Slit width: ex:5nm, em:5nm), the fluorescence intensity of measurement is denoted as F0。
Blank group 2: 40 μ L PB buffers (pH7.4), 20 μ L GO (20 μ g/mL) are sequentially added in 1.5mL centrifuge tube
With 15 μ L FAM-aptamer (3.75nmol/L), adding the ultrapure water boiled to be supplemented to total volume is 400 μ L, is vortexed and mixes, room
Temperature is lower to stand 30min；Fluorescence intensity (the excitation wavelength: 495nm of solution is measured on sepectrophotofluorometer；Emission spectrum is swept
Retouch range: 505-650nm；Scanning speed: Very Fast；Slit width: ex:5nm, em:5nm), the fluorescence intensity note of measurement
For F1。
Experimental group: sequentially added in 1.5mL centrifuge tube 40 μ L PB buffers (pH7.4), 20 μ L GO (20 μ g/mL) with
Sample solution of the certain volume containing BNP, which is vortexed, to be mixed, and is stood 30min, is added 15 μ L FAM-aptamer (3.75nmol/L),
Adding the ultrapure water boiled to be supplemented to total volume is 400 μ L, is vortexed and mixes, stands 30min at room temperature；On sepectrophotofluorometer
Measure the fluorescence intensity (excitation wavelength: 495nm of solution；Emission spectrum scanning range: 505-650nm；Scanning speed: Very
Fast；Slit width: ex:5nm, em:5nm), the fluorescence intensity of measurement is denoted as F2。
Referring to embodiment 1, embodiment 2-8 is run, measures the content of BNP in sample.
2 factors influencing of embodiment
Inventor has studied influence of the GO concentration to FAM-aptamer fluorescence probe, specific as follows.
A series of GO solution of various concentrations is added separately to PB buffer (pH7.4) and FAM-aptamer
It in the mixed solution of (3.75nmol/L), then with ultrapure water is settled to 400 μ L, is vortexed and mixes, stand 30min at room temperature, by real
1 fluorescence parameter of example is applied to be measured.
When GO concentration is 0-40 μ g/mL, FAM-aptamer fluorescence intensity change is as shown in Figure 2 in system.As seen from the figure,
After GO is added, the fluorescence of FAM-aptamer is quenched rapidly, and with the increase of GO concentration, fluorescence intensity is gradually decreased directly
To complete quenching.The present invention selects 20 μ g/mL GO as subsequent detection condition.
The drafting of 3 standard curve of embodiment
A series of BNP standard solution of various concentrations is added separately to slow by 20 μ L GO (400 μ g/mL) and 40 μ L PB
In the mixed solution of fliud flushing (pH7.4) composition, it is vortexed and mixes, stand 30min at room temperature, be then added 15 into each mixed solution
μ L FAM-aptamer (3.75nmol/L), and 400 μ L are settled to ultrapure water, it is vortexed and mixes, stand 30min at room temperature, press
The fluorescence parameter that embodiment 1 is set is measured.Repetition is done tests three times.
From the figure 3, it may be seen that the fluorescence intensity of GO/FAM-aptamer combined probe is gradually recovered with the increase of BNP concentration.
Restore efficiency ((F with fluorescence2-F1)/(F0-F1)) it is ordinate, BNP concentration is abscissa, and linear is in BNP concentration
There is good linear relationship (Fig. 4) in the range of 0.074-0.56pg/mL, calibration curve equation are as follows: Y=1.787X-
0.1331 (r=0.9929).Easy, quick, high sensitivity of the invention, the detection time of average each sample are 1min, standard
Curvilinear equation is that quantifying for BNP provides foundation in sample solution.
4 selectivity experiment of embodiment
Inventor investigated in biological sample can influence of the compatible interfering substance to the detection method, as shown in figure 5,
Mainly investigate in solution that there are Amino terminal-pro brain natriuretic peptide (No. 2), interferon (No. 3), cysteines (No. 4), glycine (5
Number), it is human serum albumins (No. 6), bovine serum albumin(BSA) (No. 7), potential in the blood such as arginine (No. 8) and histidine (No. 9)
The fluorescence of system restores efficiency when interfering substance, and compared with the brain natriuretic peptide of same concentrations (No. 1) sample solution.
The results showed that chaff interferent identical with BNP concentration is basic to the fluorescence of GO/FAM-aptamer combined probe
Without recovery.Even equally as kit for diagnosing heart failure goldstandard and possessing the homologous metabolin NT-proBNP of BNP of longer half-life,
It will not interference measurement.So this method can realize highly selective detection BNP without being done by potential interference substance in blood
It disturbs.
5 probe specificity of embodiment is investigated
Inventor has also investigated the specificity of aptamer used in this experiment, has randomly selected and has all been contaminated with fluorescence
Five sections of not homotactic DNA fragmentations for expecting label, the fluorescence of each dye marker DNA is quenched with 20 μ g/mLGO, is then added identical
The BNP of concentration (0.2pg/mL) observes the change of each fluorescence probe intensity.
As a result as shown in Figure 6, it can be seen that the fluorescence of FAM-aptamer restores efficiency up to 37%, and other random sequences
The fluorescence of DNA probe restores efficiency below 7%.As it can be seen that the binding affinity of aptamer and BNP are high, specificity is good,
The introducing of aptamers improves the selectivity of detection method.
6 atomic force microscope of embodiment (AFM) characterization
The AFM:1 of following sample solution is characterized respectively) GO (20 μ g/mL)；2) GO (20 μ g/mL) and FAM-aptamer
The mixed solution of (3.75nmol/L)；3) GO (20 μ g/mL), FAM-aptamer (3.75nmol/L) and BNP (0.30pg/mL)
Mixed solution.
For characterization result as shown in fig. 7, the average thickness of GO nanometer sheet is about 1.5nm, this shows GO nanometer sheet in the solution
It is well dispersed, it is nearly all to exist in the form of single or double layer；After introducing FAM-aptamer, occur on GO nanometer sheet surface
" island " that some average heights are about 5.0nm, this illustrates that FAM-aptamer has been adsorbed on the surface GO；After BNP is added, nanometer sheet
Height be restored to 1.5nm again, and form the compound that many height are about 60nm beside nanometer sheet, show FAM-
Aptamer can be discharged with stronger active force in conjunction with BNP from the surface of GO nanometer sheet.
7 clinical samples blood sample recovery testu of embodiment
Firstly, blood preparation is centrifuged 20min in 10000rpm, supernatant liquid is taken, clinical chemistry electrochemiluminescent immunoassay is referred again to
Measured value is analyzed by each diluting blood sample certain multiple, each clinical blood sample is then divided into two groups, one group directly takes 25 μ L sample of blood to make
For sample to be tested, another group takes 25 μ L sample of blood mixed for the BNP standard solution of 0.074,0.296 and 0.393pg/mL with concentration respectively
Cooperation is sample to be tested, and sample to be tested is then added to whirlpool in GO (20 μ g/mL) and the mixed solution of PB buffer (pH7.4)
Rotation mixes, and stands 30min at room temperature, FAM-aptamer (3.75nmol/L) finally is added into each mixed solution, and with ultrapure
Water is settled to 400 μ L, is vortexed and mixes, stands 30min at room temperature, is measured by the fluorescence parameter that embodiment 1 is set.Repeat three
Secondary experiment.Fluorescence is restored into efficiency ((F2-F1)/(F0-F1)) the linear equation Y=1.787X-0.1331 being fitted is substituted into, it calculates
Concentration c after BNP blood sample mark-on outTest sample+standard items, and according to formula: recovery of standard addition (%)=(cTest sample+standard items-cTest sample)/cStandard items
× 100%；Calculate clinical samples blood sample recovery of standard addition.Measurement result is shown in Table 1.
BNP recovery testu result in 1 blood sample of table
As shown in Table 1, it is 1.58-6.87%, recovery of standard addition 96.44- that each blood sample, which is measured in parallel RSD three times,
109.46%, show that the method for the present invention is accurate and reliable, precision is high and practical.The homogeneous fluorescent detection method of exploitation is being faced
There is potential using value in bed sample blood sample, the BNP in working curve quantitative detection clinical sample can be passed through.
8 clinical blood sample B NP assay of embodiment
In view of the method for the present invention has highly sensitive, high specific, while being avoided that potential interference substance in blood
Interference, therefore further it is applied to the assay of BNP in clinical samples blood sample, and compared with hospital clinical diagnostic value
Compared with.It collects altogether and measures 45 clinical samples (wherein 14 women and 31 male's blood samples, age are respectively less than 16 years old).Blood sample
This makes in sample solution in 10000rpm centrifugation 20min referring next to clinical diagnosis value by each diluting blood sample certain multiple first
The blood sample diluted in the linear range, is added to GO (20 μ g/mL) and PB buffer (pH7.4) later by BNP theoretical content
Mixed solution mesoscale eddies mix, stand 30min at room temperature, FAM-aptamer be then added into mixed solution
(3.75nmol/L), and 400 μ L are settled to ultrapure water, it is vortexed and mixes, 30min is stood at room temperature, by 1 fluorescence parameter of embodiment
It is measured.It tests in triplicate.Fluorescence is restored into efficiency ((F2-F1)/(F0-F1)) substitute into the linear equation Y=being fitted
1.787X-0.1331 calculates the content of BNP in blood sample, and it is real to get BNP in each blood sample to be multiplied by corresponding extension rate
Border content.
Measurement result and clinical diagnosis value are compared, as a result as shown in figure 8, the measured value of the method for the present invention is below
Hospital clinical diagnostic value, but general trend is consistent, i.e., and the blood sample that clinical chemistry luminescent immunoassay is measured as high level is sent out with this
Bright method is measured to be similarly high level, and intermediate value, low value are also same, thus it is speculated that numerical values recited difference is caused by methodology difference.In addition, root
According to the measurement result of BNP content in clinical diagnosis result and clinical samples blood sample, construct receiver operating curves (ROC) (Fig. 9)
Evaluate the ability of BNP diagnosis HF.It is gradually increased because BNP content increases with the age, internal BNP content significantly rises after birth
Height, it is latter week in be down to normal level, and BNP content asexuality difference in 10 years old or less children's body, in order to exclude the age
With the influence of gender, we select the clinical samples blood sample of 7 days to 10 years old children of birth to do ROC curve analysis, and total 37 are faced
Bed sample (female/male=12/25, wherein 4 patients with heart failure, 33 normal persons).Specifically, the method for the present invention AUC=0.856
(95% confidence interval [CI], 0.689-1.000, P < 0.05) calculates facing for diagnosis by outstanding mounting index (Youden index)
Dividing value (cut-off) is 101.4pg/mL (sensibility 100%, specificity are 66.7%, diagnosis accuracy 70.3%)；Face
Bed chemiluminescence immunoassay AUC=0.864 (95% confidence interval [CI], 0.697-1.000, P < 0.05), diagnosis is most
Excellent critical value (cut-off) is that (sensibility 100%, specificity are 63.6% to 122.6pg/mL, and diagnosis accuracy is
67.6%).ROC curve and corresponding AUC analysis result illustrate that two methods all have preferable diagnosis accuracy, two methods
Threshold value the diagnosis of heart failure is all had 100% sensitivity, when exclusion for heart failure all has high negative prediction
It is worth (100%).But compared to hospital clinical chemiluminescence immunoassay, the method for the present invention has higher for the diagnosis of heart failure
Specificity, thus it is speculated that be because this method use aptamer as recognition group, avoid the friendship between BNP peptide associated therewith
Fork reaction.In addition, the aptamer that the method for the present invention introduces largely iii vitro chemicals can synthesize, is low in cost, and do not need
Beads enrichment more makes the reduction of this product actual cost.Because this acquisition research object sample is children's sample, heart failure illness rate
It is lower, in addition the non-heart failure children in part have different degrees of injury of kidney, BNP level in blood is influenced, this method gives current children's heart
The difficult diagnosis that declines provides new approaches, while this method has better effect in adult kit for diagnosing heart failure.It is worth noting that,
The 100pg/mL that the method operation instructions are recommended is greater than by the cut-off value of the ROC hospital clinical Chemiluminescence immunoassay determined,
AUC is also far below AUC value described in specification (0.919,95% [CI], 0.904-0.934), thus it is speculated that with chemiluminescence immunoassay
Method accuracy itself and interference free performance are unstable related, it is also possible to related with the clinical sample state of acquisition testing.
Claims (5)
1. a kind of based on graphene oxide/aptamer detection brain natriuretic peptide fluorescent method, which is characterized in that including following step
It is rapid:
(1) fluoremetry of the nucleic acid aptamer probe of fluorochrome label:
The nucleic acid aptamer probe solution and phosphate buffer of fluorochrome label are vortexed after mixing, detect mixed solution
Fluorescence intensity；
(2) building of stannic oxide/graphene nano piece/fluorochrome label aptamer fluorescent quenching system:
The aptamer solution of fluorochrome label and stannic oxide/graphene nano piece dispersion liquid and phosphate buffer are vortexed
After mixing, graphene oxide can adsorb nucleic acid aptamer probe by pi-pi accumulation, form graphene oxide/dye marker
Aptamer compound, the fluorescence of dyestuff is oxidized graphene quenching at this time, detects and calculates the change of mixed solution fluorescence intensity
Change the relationship between graphene oxide concentration, determines the best quenching concentration of graphene oxide；
(3) linear relationship of stannic oxide/graphene nano piece/fluorochrome label aptamer combined probe detection brain natriuretic peptide
It establishes:
To the aptamer solution and phosphate buffer solution by a certain amount of stannic oxide/graphene nano piece/fluorochrome label
Brain natriuretic peptide standard solution is added in the mixed solution of composition, is vortexed after mixing, specificity point occurs for brain natriuretic peptide and aptamer
Son identification, the conformational change of aptamer are simultaneously detached from surface of graphene oxide, and the fluorescence signal of dyestuff is restored, and detection is simultaneously
The relationship between mixed solution fluorescence intensity change and brain natriuretic peptide concentration is calculated, corresponding fluorescence is established and restores linear equation；
(4) the brain sodium in stannic oxide/graphene nano piece/fluorochrome label aptamer combined probe detection clinical sample
Peptide:
In the aptamer solution and phosphate buffer solution by a certain amount of stannic oxide/graphene nano piece/fluorochrome label
The clinical sample to be tested solution containing brain natriuretic peptide is added in the mixed solution of composition, is vortexed after mixing, detects its fluorescence intensity change
It is worth and substitutes into the linear equation of step (3) building, the content of brain natriuretic peptide in clinical sample is calculated.
2. a kind of fluorescent method that brain natriuretic peptide is detected based on graphene oxide/aptamer according to claim 1,
It is characterized in that, the method is homogeneous fluorescent measuring method.
3. a kind of fluorescent method that brain natriuretic peptide is detected based on graphene oxide/aptamer according to claim 1,
Be characterized in that, in step (2), the stannic oxide/graphene nano piece dispersion liquid the preparation method comprises the following steps: the graphite oxide of commercialization
For alkene nanometer sheet through ultrasonic disperse in ultrapure water, concentration is 0-40 μ g/mL.
4. a kind of fluorescent method that brain natriuretic peptide is detected based on graphene oxide/aptamer according to claim 1,
It is characterized in that, in step (2), the stannic oxide/graphene nano piece quenches the best dense of the aptamer of fluorochrome label
Degree is 20 μ g/mL, vortex time 1-2min.
5. a kind of fluorescent method that brain natriuretic peptide is detected based on graphene oxide/aptamer according to claim 1,
It is characterized in that, in step (3), the linear equation is Y=1.787X -0.1331, r=0.9929, the detection to brain natriuretic peptide
It is limited to 0.045pg/mL；Wherein Y is that fluorescence restores efficiency, and X is the concentration of brain natriuretic peptide, unit pg/mL.
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