CN110794145B - Rapid detection system for early kidney injury urine marker neutrophil gelatinase related lipocalin, preparation method and application - Google Patents
Rapid detection system for early kidney injury urine marker neutrophil gelatinase related lipocalin, preparation method and application Download PDFInfo
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Abstract
The invention discloses a rapid detection system for neutrophil gelatinase related lipocalin as an early kidney injury urine marker, a preparation method and application thereof, wherein the detection system comprises a polydopamine nanometer microsphere-modified FAM fluorescein aptamer sensor and deoxyribonuclease; the sensor is formed by adding an aptamer for modifying NGAL specific binding of FAM fluorescein to a polydopamine nanometer microsphere. According to the invention, the polydopamine nanometer microsphere with excellent quenching effect is prepared, a PDANS-modified fluorescein aptamer detection sensor is constructed through non-covalent bond combination, DNase-I is added, an aptamer to be detected is hydrolyzed, and the detection system is free and subjected to a cyclic reaction, so that fluorescent signal amplification is realized, and the sensitivity of the detection system is improved. The detection system provided by the invention has the advantages of simple preparation method, mild reaction conditions, low cost and easiness in batch preparation, and provides a new tool and a new method for quick detection of NGAL.
Description
Technical Field
The invention belongs to the field of biomedical detection, relates to detection of early-stage acute kidney injury urine markers, and in particular relates to a rapid detection system for neutrophil gelatinase-associated lipocalin (NGAL) of the early-stage kidney injury urine markers, and a preparation method and application thereof.
Background
Acute Kidney Injury (AKI) refers to a clinical syndrome that occurs as a result of rapid decline in renal function caused by a variety of etiologies. According to statistics, the AKI incidence rate of the hospitalized patient reaches 1% -7%; the incidence rate of patients in intensive care units is up to 30% -50%. AKI can lead to loss of kidney function within hours, days or weeks. Furthermore, AKI is also a significant cause of chronic kidney disease and chronic renal failure. The current clinical kidney injury standard diagnosis method mainly depends on measurement of serum creatinine (Scr) and urea nitrogen (BUN), and has no obvious change and poor sensitivity in early kidney injury; when the renal filtration function drops by more than 50%, the concentration of SCr and BUN changes significantly. Therefore, the exploration of early biomarkers of AKI is of great importance for the timely diagnosis and treatment of AKI. In recent years, a variety of urine markers have been explored and discovered for use in early diagnosis of AKI, including interleukin-18 (IL-18), kidney injury molecule-1 (KIM-1), cystatin C, and neutrophil gelatinase-associated lipocalin (NGAL), among others. NGAL is one of the most promising biomarkers of high sensitivity, specificity, predictability for early AKI. NGAL is an apolipoprotein linked to neutrophil gelatinase and has a molecular weight of 25kDa. NGAL is expressed in low concentrations in human tissues such as kidney, lung, stomach and colon, and NGAL can be significantly expressed in damaged tubular when kidney is ischemic or toxic damaged, and can be detected in urine about 2 hours after injury, an important biomarker for early diagnosis of AKI.
At present, the detection of the early marker NGAL of urine is mainly determined by methods such as western blotting (Western Immunoblot-WB), immunostaining (immunoregulation), gel zymogram (Gelatin Zymographic), electrochemistry, enzyme-linked immunosorbent assay (ELISA) and the like, and the methods have the defects of tedious steps, long time consumption, high detection cost and the like, so that the clinical application of NGAL is limited. Therefore, development of a new tool and a new method for detecting urine NGAL rapidly, specifically, sensitively and simply is needed.
Disclosure of Invention
The invention aims to: aiming at the problems existing in the prior art, the invention provides a rapid detection system for neutrophil gelatinase-associated lipocalin (NGAL) as an early-stage kidney injury urine marker, which can rapidly, simply, conveniently, sensitively and selectively detect the early-stage kidney injury urine marker NGAL.
The invention also provides a preparation method and application of the rapid detection system for the neutrophil gelatinase-associated lipocalin (NGAL) as an early-stage kidney injury urine marker.
The technical scheme is as follows: in order to achieve the above object, the invention provides a rapid detection system for neutrophil gelatinase-associated lipocalin, which is an early-stage kidney injury urine marker, comprising a polydopamine nanoparticle-modified FAM fluorescein aptamer sensor and a deoxyribonuclease; the sensor is formed by adding NGAL-aptamer-FAM (NGAL-aptamer-FAM) specifically binding to modified FAM fluorescein into polydopamine nanometer microsphere formation (PDANS) and adding deoxyribonuclease (DNase-I).
Wherein, the nucleotide sequence of the aptamer (NGAL-aptamer-FAM) for modifying the NGAL specific binding of FAM fluorescein is shown in SEQ ID NO. 1: 5'-FAM-cggagggcggaagcaaagcgtaacagaaagccaacacgcg-3'.
The polydopamine nanometer microsphere is synthesized from dopamine hydrochloride, tris-HCl buffer solution, isopropanol and ammonia water. Preferably, the kit mainly comprises 100mg of dopamine hydrochloride, 100mL of Tris-HCl buffer (10 mM pH 7.4), 40mL of isopropanol and ammonia water, and the pH is 8.0.
The invention relates to a preparation method of a rapid detection system for neutrophil gelatinase-associated lipocalin as an early kidney injury urine marker, which comprises the following steps:
(1) Adding ammonia water into Tris-HCl buffer solution and isopropanol to adjust pH; stirring to fully mix the solution;
(2) Dissolving dopamine hydrochloride in ultrapure water, adding the solution prepared in the step (1), and continuing stirring in a dark place;
(3) Centrifuging the solution obtained in the step (2) to remove supernatant, dissolving and precipitating with ultrapure water, uniformly mixing, centrifuging, removing supernatant, and repeating until the supernatant is clear;
(4) After centrifugal purification, the precipitate is polydopamine nanometer microsphere (PDANS), and the polydopamine nanometer microsphere is frozen and dried into powder for standby;
(5) Adding an aptamer for NGAL specific binding of modified FAM fluorescein into the polydopamine nanometer microsphere solution, and uniformly mixing to form a polydopamine nanometer microsphere-modified fluorescein aptamer sensor;
(6) Adding NGAL and deoxyribonuclease (DNase-I) into a PDANS-aptamers sensor for reaction, specifically binding the NGAL and the aptamers, dissociating the aptamers from the surface of the PDANS, selectively hydrolyzing the aptamers by DNase-I, releasing the NGAL freely, and carrying out the next round of cyclic reaction;
(7) And (3) reading the fluorescence value by an enzyme-labeled instrument, and establishing a linear relation between the concentration of NGAL and the fluorescence value to obtain the rapid detection system of the neutrophil gelatinase-associated lipocalin of the early-stage kidney injury urine marker.
Wherein the concentration of the polydopamine nanometer microsphere (PDANS) solution in the step (5) is 0.2-0.6mg/mL, and the concentration of the ligand for modifying the NGAL specific binding of FAM fluorescein is 300-600nM.
Preferably, the concentration of polydopamine nanoparticle (PDANS) solution in step (5) is 0.3mg/mL and the concentration of aptamer that modifies NGAL-specific binding of FAM fluorescein is 600nM.
Wherein the DNase-I in the step (6) is 5-20U, the reaction temperature is 37 ℃, the reaction time is 1-2h, and the reaction buffer is 5-10mMCaCl 2 、2-5mM MgCl 2 。
Preferably, the deoxyribonuclease (DNase-I) in step (6) is 10U, the reaction temperature is 37 ℃, the reaction time is 1h, and the reaction buffer is 5mM CaCl 2 、2mM MgCl 2 。
Further, in the step (7), the fluorescent signal is read by using an enzyme-labeled instrument, and the linear relationship between the NGAL concentration and the fluorescence intensity in the step (7) is: when the concentration of NGAL is in the range of 12.5-400 pg/mL, the concentration accords with a linear regression equation Y=0.2731X+14.65, R 2 = 0.9954, where Y is the microplate reader reading and X is NGAL concentration.
The invention relates to an application of a rapid detection system for neutrophil gelatinase-associated lipocalin (NGAL) as an early kidney injury urine marker in preparation of a reagent or a tool for detecting neutrophil gelatinase-associated lipocalin (NGAL).
The principle of the method of the invention is explained as follows:
according to the invention, a polydopamine nanoparticle-modified fluorescein nucleic acid aptamer nanosensor is constructed based on a FRET effect, PDANS quenches fluorescein fluorescence, NGAL and NGAL aptamer are specifically combined and then dissociated from the surface of PDNAS, and fluorescence is recovered. And deoxyribonuclease-I (DNase-I) is added, hydrolysis aptamer and NGAL are dissociated and react circularly, so that fluorescent signal amplification is realized, and the sensitivity of a detection system is improved. (see FIG. 1).
In order to solve the defects of complex operation, long time, expensive instrument and the like in the detection process of the conventional NGAL, the invention provides a novel system and a novel method for detecting the NGAL by circulating amplification. The PDANS is used as a novel material widely applied, can adsorb single-stranded DNA (ssDNA) and protect the ssDNA from being hydrolyzed by enzyme, and has the advantages of simple synthesis and preparation, high-efficiency quenching fluorescence performance and the like. The FAM fluorescein is modified at the 5' -end of an aptamer chain specifically combined with NGAL, and is added into PDANS, and non-public bonds are combined to form a nano sensor, so that fluorescence is quenched. When NGAL exists in the detection system, the specific binding capacity of the aptamer and the NGAL is far greater than the adsorption capacity of PDANS on the aptamer, so that the aptamer adsorbed on the surface of the PDNAS can be competed, and fluorescence can be recovered. In order to further improve the sensitivity of the detection system, DNase-I is used for selectively hydrolyzing the aptamers, and the dissociation of NGAL and the aptamers can be carried out for the next round of cyclic reaction, so that the fluorescent signal amplification effect is realized.
In a first aspect, DNase-I is selected as an endonuclease capable of digesting single-stranded or double-stranded DNA, and is capable of selectively hydrolyzing an aptamer and a target protein-bound aptamer without the need for a specific recognition site, and without hydrolyzing an aptamer adsorbed on the surface of a PDANS, thereby achieving a target protein cycling reaction.
In the second aspect, the selected polydopamine nanometer microsphere is a novel material, and the synthesis method is simple and has high-efficiency fluorescence quenching performance. Meanwhile, PDANS can adsorb aptamer on the surface through pi-pi stacking, hydrogen bonding and other actions, so that fluorescence quenching is realized; in addition, the aptamers adsorbed on the surface of the PDANS can be protected by the PDANS and not hydrolyzed by enzymes, and only the aptamers desorbed from the surface of the PDANS can be hydrolyzed to release target proteins, so that fluorescent signal cycle amplification is realized.
The invention provides a rapid detection system for an early kidney injury urine marker NGAL. The system firstly prepares the polydopamine nanometer microsphere (PDANS) with good quenching effect. PDANA constitutes a PDANS-aptamer nanosensor by non-covalent bond binding to a aptamer that modifies fluorescein. PDANS quenches fluorescein fluorescence based on FRET effect. When NGAL exists in the detection system, the NGAL and the aptamer can be specifically combined, the aptamer is dissociated from the surface of the PDNAS, the FRET effect is weakened, and the fluorescence is recovered. In order to improve the detection sensitivity, deoxyribonuclease-I (DNase-I) is added into the system, hydrolyzed aptamer and NGAL are dissociated, and the aptamer adsorbed on the PDANA surface is circularly combined to realize the circular amplification of fluorescent signals. The method has the characteristics of rapidness, sensitivity, simplicity, convenience and the like, and can realize detection of the early marker NGAL of the AKI.
The beneficial effects are that: compared with the prior art, the invention has the following advantages:
the rapid detection system of early renal injury urine marker NGAL of the invention is based on DNase-I enzyme amplification effect, improves detection sensitivity of NGAL protein, and spontaneously reacts at 37 ℃ without additional harsh reaction conditions; NGAL and aptamer are specific for detection and do not have good fluorescence recovery for other proteins. Aptamer in the detection system binds to PDANS: the fluorescent dye has the advantages of high efficiency, stability, large specific surface area of PDANS, low cost, capability of adsorbing a large amount of aptamers on the surface of the PDANS, and further increase of fluorescent amplification effect.
The detection system provided by the invention is used for preparing polydopamine nanometer microspheres (PDANS) with excellent quenching effect, and constructing a PDANS-modified fluorescein aptamer nanometer sensor through non-covalent bond combination. Based on Fluorescence Resonance Energy Transfer (FRET) effect PDANS, the fluorescence of fluorescein is quenched, NGAL of the object to be detected is specifically combined with aptamer, the aptamer is dissociated from the surface of PDNAS, the FRET effect is weakened, and the fluorescence is recovered. And deoxyribonuclease-I (DNase-I) is added, an aptamer is hydrolyzed, and the to-be-detected object is dissociated and reacts circularly, so that fluorescent signal amplification is realized, and the sensitivity of a detection system is improved. NGAL in 12.5 ~ 400pg/mL range linear relation is good, detection limit is 6.25pg/mL. The method has the characteristics of rapidness, sensitivity, simplicity, convenience and the like.
The detection system provided by the invention has the advantages of simple preparation method, mild reaction conditions, low cost and easiness in batch preparation, and provides a novel tool and a novel method for rapid detection of neutrophil gelatinase-associated lipocalin (NGAL) as an early kidney injury urine marker.
Drawings
FIG. 1 is a schematic diagram showing rapid detection of early kidney injury urine marker NGAL of the present invention;
FIG. 2 is a schematic representation of a PDANS transmission electron microscope prepared in example 1;
FIG. 3 is a graph showing the variation of the dynamic light scattering particle size of PDANS prepared in example 1;
FIG. 4 is a plot of PDANS concentration versus fluorescence quenching rate plotted in example 2;
FIG. 5 is a graph of a linear plot of the detection of NGAL of example 3;
FIG. 6 is a schematic diagram of the results of a selectivity assay for identifying NGAL targets in example 4;
FIG. 7 is a graph showing the results of examining changes in NGAL content in urine of example 5;
Detailed Description
The invention is further described below with reference to specific embodiments and figures.
In the following embodiments, the detection of the enzyme-labeled instrument is: a Siemens femtoxidate reader; the dopamine hydrochloride and Tris-HCl used for synthesis and DNase-I enzyme used for detection are as follows: beijing Soy Bao technology Co., ltd; aptamers used for detection, aptamer to which NGAL modified FAM fluorescein specifically binds (NGAL-aptamer-FAM) were synthesized by the division of bioengineering (Shanghai); NGAL is available from Sigma.
Example 1
Verification of success of synthesis of polydopamine nanospheres
Preparing polydopamine nanometer microspheres:
(1) 100mL of 10mM Tris-HCl (pH 7.4) is prepared by ultrapure water, tris-HCl and isopropanol (40 mL) are added into a beaker, ammonia water is diluted, a small amount of the diluted solution is added into the beaker for multiple times, pH test paper is used for detecting, the pH value of the final solution is about 8.0, and a magnetic stirrer is used for slowly stirring for 30min, so that the solution is fully and uniformly mixed.
(2) Then, 100mg of dopamine hydrochloride is weighed and dissolved in 2mL of ultrapure water, the dissolved dopamine hydrochloride is slowly added into the solution obtained in the step (1), stirring is continued for 48 hours in a dark place, and stirring is stopped.
(3) Transferring the solution obtained in the step (2) into a centrifuge tube, centrifuging at 10000rpm for 10min, discarding the supernatant, dissolving and precipitating with ultrapure water, mixing uniformly by vortex, centrifuging, discarding the supernatant, and repeating for several times until the supernatant is clear.
(4) And (3) after centrifugal purification, precipitating to obtain polydopamine nanometer microspheres (PDANS), and freeze-drying to obtain powder for later use.
Fig. 2 shows a schematic representation of the transmission electron microscope of the polydopamine nanospheres prepared in example 1.
FIG. 3 shows a schematic representation of dynamic light scattering particle size characterization of the polydopamine nanospheres prepared in example 1.
As can be seen from fig. 2 and 3, TEM shows that the prepared polydopamine nanospheres are spherical, uniform in size and uniform in dispersion, and Dynamic Light Scattering (DLS) shows that the average particle size of the synthesized polydopamine nanospheres is 312.6nm, which indicates that the polydopamine nanospheres are successfully synthesized.
Example 2
Construction of PDANS-aptamers nanosensors
1. The 5' -end of NGAL-9-aptamer is modified by FAM fluorescein to obtain NGAL-aptamer-FAM, and the sequence is synthesized by Shanghai Biotechnology company.
| Name of the name | Nucleotide sequence SEQ ID NO.1 |
| NGAL-aptamer-FAM | 5’-FAM-cggagggcggaagcaaagcgtaacagaaagccaacacgcg-3’ |
2. The construction of the PDANS-aptamers nanosensor comprises the following steps:
(1) Preparing a sample: preparing NGAL-aptamer-FAM standard solution with concentration of 12 mu M for standby; the concentration of the standard solution of PDANS is 2mg/mL.
(2) The reaction is carried out: adding NGAL-aptamer-FAM standard solution to a final concentration of 600nM; then adding PDANS to make the final concentration of the mixture be 0.01mg/mL, 0.05mg/mL, 0.15mg/mL, 0.10mg/mL, 0.2mg/mL, 0.3mg/mL and 0.40mg/mL respectively; buffer concentration was 5mM CaCl 2 ，2mM MgCl 2 The method comprises the steps of carrying out a first treatment on the surface of the The reaction system was 200. Mu.L to be measured, and the buffer solvent was water. And DNase-I to form a rapid detection system.
(3) Reading: the microplate reader reads excitation 488nm and emits fluorescence values at 520 nm.
(4) Drawing a fluorescence quenching curve: quench rate, QE (%) = (F) was calculated according to the formula 0 -F)/F 0 ×100％，F 0 Represents the fluorescence value of 600nM NGAL-aptamer-FAM, F represents the fluorescence value after addition of PDANSFluorescence value. And drawing a fluorescence quenching curve by taking the concentration of the PDANS as an abscissa and the calculated quenching rate as an ordinate.
Fig. 4 shows a plot of PDANS concentration versus fluorescence quenching rate.
As can be seen from fig. 4, as the concentration of PDANS increases, the fluorescence quenching efficiency thereof becomes higher, and gradually becomes gentle when the concentration of PDANS reaches 0.3mg/mL, the quenching plateau is reached, and the quenching efficiency reaches 90% or more, so that 0.3mg/mL PDANS is selected as the concentration for the subsequent experiment. .
Example 3
Investigation of sensitivity to NGAL detection experiments
1. Sequences required for this experiment (example 2):
2. the sensitivity of the NGAL protein standard solution based on the deoxyribonuclease-I amplification system is examined, and the method comprises the following steps:
(1) Preparing a sample: preparing a non-concentration NGAL solution, wherein the concentration is 0.25ng/mL,0.5ng/mL,1ng/mL,2ng/mL,4ng/mL and 8ng/mL respectively; DNase-I enzyme 2U/. Mu.L was prepared with ultrapure water.
(2) The reaction is carried out: sequentially adding NGAL-aptamer-FAM to a final concentration of 600nM, and then adding PDANS to final concentrations of 0.3mg/mL, buffer concentration of 5mM CaCl 2 ，2mM MgCl 2 10 mu L of NGAL solution (final concentration is 12.5pg/mL,25pg/mL,50pg/mL,100pg/mL,200pg/mL and 400pg/mL respectively) is respectively added in the step (1), 5 mu L of 2U/mu L DNase-I enzyme is added, the volume of the reaction system is complemented by 200 mu L by ultrapure water, the whole detection system is formed, and the reaction system is subjected to standing incubation for 1h at 37 ℃ and detected by an enzyme label instrument.
(3) Reading: the microplate reader reads excitation 488nm and emits fluorescence values at 520 nm.
(4) Drawing a standard curve: and drawing an NGAL standard curve by taking the concentration of the NGAL standard solution as an abscissa and taking a fluorescence value at 520nm as an ordinate.
FIG. 5 shows the results of detecting NGAL.
As can be seen from FIG. 5, R meets the linear equation Y=0.2731X+14.65 when the concentration of NGAL is in the range of 12.5-400 pg/mL 2 0.9963 where Y is the microplate reader reading and X is the NGAL concentration, availableDetection of NGAL protein.
Example 4
Examine NGAL specialization
1. Random chain and other proteins required for this experiment: random was synthesized by Shanghai Biotechnology (5 '-FAM-nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnmnn-3' sequence was randomly different from NGAL), and Scr, FBS was obtained by Sigma company.
2. Examine the determination of NGAL protein specificity based on deoxyribonuclease-I amplification system, comprising the steps of:
(1) Preparing a sample: the Random strand concentration and the protein were formulated at the same concentration, i.e., 12. Mu.M Random strand and 1200ng/mL Scr, FBS, NGAL, respectively.
(2) The reaction is carried out: sequentially adding NGAL-aptamer-FAM to a final concentration of 600nM, and then adding PDANS to final concentrations of 0.3mg/mL, buffer ion concentration of 5mM CaCl 2 ，2mM MgCl 2 After mixing, 10. Mu.L of 12. Mu.M Random chain, 1200ng/mL of Scr, FBS and NGAL are added respectively to make the final concentration 600nM and 60ng/mL, 5. Mu.L of 2U/mu.L of DNase-I enzyme is added, and the volume of the reaction system is complemented with 200. Mu.L of ultrapure water, so that the whole detection system is formed. And (3) standing and incubating for 1h at 37 ℃ and detecting by using an enzyme-labeled instrument.
(3) Reading: the microplate reader reads excitation 488nm and emits fluorescence values at 520 nm.
(4) Drawing a bar chart: bar graphs were plotted with random strand and each protein name on the abscissa and fluorescence values at 520nm on the ordinate, respectively.
Fig. 6 shows the results of the investigation of the selectivity of NGAL identified in this example.
As can be seen from fig. 6, NGAL only binds to specific aptamers and fluorescence is restored; the random strand or other proteins cannot bind to the aptamer and can not restore fluorescence, and the results prove that the detection system has good specificity.
Example 5
Changes in NGAL content in urine of mice after cisplatin-induced acute kidney injury were examined.
1. The mice were intraperitoneally injected with cisplatin at a dose of 20mg/kg, and urine from the mice was collected by bladder squeezing at 0h,2h,4h,6h, and 12h after administration.
2. The detection probe system is examined to determine the NGAL content in urine environment, and the steps are as follows:
(1) Preparing a sample: urine samples were collected and diluted 10-fold all for use.
(2) Marking the curve along with the line: the formulation and experimental methods were identical to those of example 3.
(3) The reaction is carried out: sequentially adding NGAL-aptamer-FAM to a final concentration of 600nM, and then adding PDANS to final concentrations of 0.3mg/mL, buffer ion concentration of 5mM CaCl 2 ，2mM MgCl 2 And (3) respectively adding 10 mu L of the urine sample diluted in the step (1), and adding 10U of DNase-I enzyme to form an ultrapure water supplementing and mixing system of the whole detection system, so that the total volume of the whole reaction system is 200 mu L. And (3) standing and incubating for 1h at 37 ℃ and detecting by using an enzyme-labeled instrument.
(4) Reading: the microplate reader reads excitation 488nm and emits fluorescence values at 520 nm.
(5) And (3) content measurement: and (3) calculating the content of NGAL in the urine after the urine is added by taking the following working curve obtained in the step (2) as a standard curve.
(6) The calculation method comprises the following steps: substituting the measured value of the enzyme-labeled instrument into a standard curve fitting formula to obtain the measured concentration C, thus obtaining the NGAL content.
(7) Drawing a bar chart: and drawing a line graph by taking different time after administration as an abscissa and taking the NGAL content values in urine of different groups of mice as an ordinate.
Fig. 7 shows the results of the assay described in this application for NGAL content changes in urine samples.
As can be seen from fig. 7, after the cisplatin molding of the acute kidney injury model, the NGAL content in urine increases significantly after 2 hours, and continues for 12 hours, compared with the traditional kidney injury marker, the NGAL reacts to the condition of kidney injury earlier, and continues to increase in a certain period of time, which indicates that NGAL can be used as an early marker of kidney injury to provide a rapid, accurate and reliable new method for diagnosing kidney injury clinically and timely, and the detection system of the invention provides a new tool and a new method for rapid detection of neutrophil gelatinase-associated lipocalin (NGAL) as an early urine marker of kidney injury.
Example 6
Example 6 the entire detection system was constructed as in example 3, except that the polydopamine nanomicrosphere (PDANS) solution concentration was 0.2mg/mL and the aptamer concentration for the NGAL-specific binding of the modified FAM fluorescein was 300nM. DNase-I was 5U, the reaction temperature was 37℃and the reaction time was 2h, reaction buffer 5mM CaCl 2 、2mM MgCl 2 。
Example 7
Example 7 the entire detection system was constructed as in example 3, except that the concentration of polydopamine nanospheres (PDANS) solution was 0.6mg/mL and the concentration of aptamer that modified FAM fluorescein specifically bound to NGAL was 500nM. DNase-I was 20U, the reaction temperature was 37℃and the reaction time was 1h, the reaction buffer 10mM CaCl 2 、5mM MgCl 2 。
Sequence listing
<110> university of Chinese medical science
<120> early kidney injury urine marker neutrophil gelatinase related lipocalin rapid detection system, preparation method and application
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 40
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 1
cggagggcgg aagcaaagcg taacagaaag ccaacacgcg 40
Claims (2)
1. A method for rapidly detecting neutrophil gelatinase-associated lipocalin, a marker of early kidney injury in a non-diagnostic manner, comprising the steps of:
(1) Preparing a sample: preparing NGAL solutions with different concentrations, wherein the concentrations are respectively 0.25ng/mL,0.5ng/mL,1ng/mL,2ng/mL,4ng/mL and 8ng/mL; preparing DNase-I enzyme 2U/. Mu.L by ultrapure water;
(2) The reaction is carried out: sequentially adding NGAL-aptamer-FAM to a final concentration of 600nM, and then adding PDANS to final concentrations of 0.3mg/mL, buffer concentration of 5mM CaCl 2 ，2mM MgCl 2 Respectively adding 10 mu L of NGAL solution prepared in the step (1), wherein the final concentration is respectively 12.5pg/mL,25pg/mL,50pg/mL,100pg/mL,200pg/mL and 400pg/mL, adding 5 mu L of 2U/mu L DNase-I enzyme, and the volume of the reaction system is 200 mu L, so as to form the whole detection system, standing and incubating at 37 ℃ for 1h, and detecting by an enzyme-labeled instrument;
(3) Reading: reading and exciting 488nm by an enzyme label instrument, and emitting fluorescence values at 520 nm;
(4) Drawing a standard curve: drawing an NGAL standard curve by taking the concentration of the NGAL standard solution as an abscissa and taking a fluorescence value at 520nm as an ordinate;
the NGAL-aptamer-FAM is an aptamer for modifying NGAL specific binding of FAM fluorescein, and the nucleotide sequence of the aptamer is shown in SEQ ID NO. 1: 5'-FAM-cggagggcggaagcaaagcgtaacagaaagccaacacgcg-3';
the PDANS is a polydopamine nanometer microsphere, and the preparation method comprises the following steps:
(a) Preparing 100mL of 10mM Tris-HCl by ultrapure water, adding 40mL of Tris-HCl and isopropanol into a beaker, diluting with ammonia water, adding into the beaker, and slowly stirring for 30min to ensure that the pH value of the final solution is 8.0, and fully and uniformly mixing the solution;
(b) Then weighing 100mg of dopamine hydrochloride and dissolving the dopamine hydrochloride into 2mL of ultrapure water, slowly adding the dissolved dopamine hydrochloride into the solution obtained in the step (a), continuing stirring for 48 hours in a dark place, and stopping stirring;
(c) Transferring the solution obtained in the step (b) into a centrifuge tube, centrifuging at 10000rpm for 10min, discarding the supernatant, dissolving and precipitating with ultrapure water, mixing uniformly by vortex, centrifuging, discarding the supernatant, and repeating for several times until the supernatant is clear;
(d) And (c) after centrifugal purification, precipitating to obtain the polydopamine nanometer microsphere PDANS, and freeze-drying to obtain powder for later use.
2. The method according to claim 1, characterized in thatCharacterized in that the linear relationship between NGAL concentration and fluorescence intensity is: when the concentration of NGAL is in the range of 12.5-400 pg/mL, the concentration accords with a linear regression equation Y=0.2731X+14.65, R 2 = 0.9954, where Y is the microplate reader reading and X is NGAL concentration.
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