CN117169492A - Detection kit with high low-end sensitivity and application thereof - Google Patents

Detection kit with high low-end sensitivity and application thereof Download PDF

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CN117169492A
CN117169492A CN202311111380.1A CN202311111380A CN117169492A CN 117169492 A CN117169492 A CN 117169492A CN 202311111380 A CN202311111380 A CN 202311111380A CN 117169492 A CN117169492 A CN 117169492A
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antibody
iso
dna
conjugate
detection
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成舜
叶平生
任丽君
曹丹
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Nanjing Puguang Biotechnology Co ltd
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Nanjing Puguang Biotechnology Co ltd
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    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
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    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
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Abstract

The invention discloses a detection kit with high low-end sensitivity and application thereof, wherein the detection kit comprises the following detection solutions: DNA 1-antibody-peroxidase conjugate, DNA 2-scaffold small molecule conjugate, AE-labeled DNA3 and GO-AOD complex; wherein the antibody is thyroxine antibody, and the scaffold small molecule is thyroxine antigen; DNA1 is complementary to DNA2 by 7 bases, and DNA3 is complementary to DNA1 and DNA2 by 8 bases, respectively. According to the invention, the ortho peroxidase is introduced into the acridine lipid homogeneous phase immunoassay detection method, so that the low-end sensitivity and stability of the detection method are remarkably improved.

Description

Detection kit with high low-end sensitivity and application thereof
Technical Field
The invention relates to the technical field of chemiluminescent immunoassay, in particular to a detection kit with high low-end sensitivity and application thereof. The application number of the kit is 202310592173.6, and the invention is a division of a detection kit and application thereof.
Background
Chemiluminescent immunoassay (chemiluminescence immunoassay, CLIA) combines a chemiluminescent assay technique with high sensitivity with a highly specific immune reaction, and is used for various antigens, haptens, small molecules, antibodies, drugs, and other analytical techniques. Depending on whether separation of free and bound species is desired, homogeneous and heterogeneous assays can be distinguished.
The resonance energy transfer effect enables a pair of DNA coupled with the antibody to be separated by immune reaction, thereby triggering DNA assembly, triggering cascade DNA assembly and generating detection signals, and realizing sensitive detection of a target detection object. The method can be carried out homogeneously, simply, rapidly and sensitively.
The patent (CN 111007239B) discloses a homogeneous immunoassay method based on ortho-strike effect and graphene oxide quenched acridine ester chemiluminescence, and a device for using the same, wherein the sensitivity and stability are deficient in actual detection, and the accuracy of a detection value cannot be ensured.
The sensitivity of a diagnostic test refers to the ability to accurately identify a patient suffering from a disease. The high sensitivity is advantageous for efficient identification of patients carrying pathogens. Because very small amounts of analyte may be of great importance in defining disease states, screening for disease, or revealing the presence of toxic substances, pollutants, infectious agents, etc. The more sensitive the test, the fewer false negative results, helping to prevent infection and to implement therapeutic measures early. Sensitivity is an important technical index, and has important significance for evaluating the kit.
Disclosure of Invention
The invention aims to: the invention aims to provide a kit for homogeneous immunoassay detection, which has high low-end sensitivity and good stability.
The technical scheme is as follows: the invention provides a detection kit, which comprises the following detection solutions: DNA 1-antibody 1-peroxidase conjugate, DNA 2-antibody 2 conjugate, AE-labeled DNA3 and GO-AOD complex; wherein antibody 1 and antibody 2 are two different troponin I antibodies; DNA1 is complementary to DNA2 by 7 bases, and DNA3 is complementary to DNA1 and DNA2 by 8 bases, respectively.
Further, the peroxidase comprises one or more of lactoperoxidase, microperoxidase, myeloperoxidase, haloperoxidase, vanadium bromoperoxidase, horseradish peroxidase, fungal peroxidase and lignin peroxidase.
The invention provides a detection kit, which comprises the following detection solutions: DNA 1-antibody 1-iron complex conjugate, DNA 2-antibody 2 conjugate, AE-labeled DNA3 and GO-AOD complex; wherein antibody 1 and antibody 2 are two different troponin I antibodies; DNA1 is complementary to DNA2 by 7 bases, and DNA3 is complementary to DNA1 and DNA2 by 8 bases, respectively.
Further, the iron complex includes hemin, mn-TPPS4.
Further, the DNA 1-peroxidase conjugate/DNA 1-antibody 1-iron complex conjugate is obtained by covalently binding the DNA1 to the amino group of the antibody 1 on the antibody 1-peroxidase conjugate/antibody 1-iron complex conjugate, and the antibody 1-peroxidase conjugate/antibody 1-iron complex conjugate is obtained by covalently binding the amino group on the antibody 1 to the carbonyl group after hydroformylation of the peroxidase/iron complex.
Further, the DNA 2-antibody 2 conjugate is obtained by covalently binding DNA2 to an amino group on antibody 2.
Further, in the DNA 1-antibody 1-peroxidase conjugate/DNA 1-antibody 1-iron complex conjugate, the nucleotide sequence of the DNA1 is shown as SEQ ID NO. 1; in the DNA 2-antibody 2 conjugate, the nucleotide sequence of the DNA2 is shown as SEQ ID NO. 2; the nucleotide sequence of the DNA3 is shown as SEQ ID NO. 3.
Further, the concentration of the DNA 1-antibody 1-peroxidase conjugate/DNA 1-antibody 1-iron complex conjugate is 400-600 ng/mL, the concentration of the DNA 2-antibody 2 conjugate is 200-400 ng/mL, the concentration of the DNA3 for marking the acridine ester AE is 0.15 mu M, and the concentration of the graphene oxide GO combined with the antioxidant AOD is 20 mu g/mL.
Further, G in the nucleotide sequences of the DNA1, the DNA2 and the DNA3 is isoG, and C is isoC.
The invention also provides application of the detection kit in detection of troponin I content.
The invention also provides an acridine lipid homogeneous immunoassay detection method based on graphene oxide-antioxidant quenching resonance energy transfer, which comprises the following detection steps:
1) Mixing a calibration solution containing different target molecule concentrations with the detection solution in the detection kit, incubating, adding a chemiluminescent substrate, detecting a chemiluminescent signal of the solution, recording a chemiluminescent value, and drawing a calibration curve according to the chemiluminescent value of the calibration solution to obtain a standard curve between the calibration solution and the chemiluminescent intensity;
2) Mixing a sample to be detected with the detection solution in the detection kit, incubating, adding a chemiluminescent substrate, and detecting a chemiluminescent signal of the sample to be detected to obtain a chemiluminescent value of the sample to be detected; and (3) bringing the chemiluminescent value of the sample to be measured into a calibration curve to obtain the concentration of the target molecule in the sample to be measured.
When no target protein exists, DNA3 is adsorbed on the surface of GO through pi-pi stacking, and chemiluminescent substrate H is added 2 O 2 Later, DNA 3-terminally labeled AE failed to oxidize and emit light due to the presence of antioxidants on GO. In the presence of the protein of interest, the antibodies on the two antibody-DNA complexes achieve a double antibody sandwich, bringing DNA1 and DNA2 close enough to form an orthotopic complex and capable of hybridizing with DNA 3. The complex is hardly adsorbed by GO, the AOD coupled on graphene does not influence AE luminescence on the complex, and because the ortho peroxidase/iron complex is introduced into the complex, a chemiluminescent substrate H is added 2 O 2 More sheets can then be created around the complexAnd the linear oxygen enhances the luminous intensity of AE.
Further, the concentration of the calibration solution ranges from 0.01 to 50ng/mL.
Further, the DNA3 also comprises 5 '-end modified NH2C6 which is connected with a primer 5' -sugar ring through a B-cyanoethyl chemical reaction; DNA3-AE was then formed by addition of the acridinium ester.
Specifically, the DNA1 contains 54 bases, the DNA2 contains 52 bases, and the DNA3 contains 21 bases; the modification of the NH2C6 at the DNA 35 ' end (5 ' Aminolinker (C6)) was added to the 5' sugar ring of the primer by B-cyanoethyl chemistry in the form of an amine phosphite in the last step of the synthesis cycle, rather than to the last base.
Further, the test sample includes whole blood// serum/plasma containing the test target molecule.
Further, the incubation conditions are incubation at 37℃for 5-10 minutes.
Further, the chemiluminescent substrate is hydrogen peroxide at 100mM ph=10.
The invention also provides a detection kit, which comprises the following detection solutions: DNA 1-antibody-peroxidase conjugate, DNA 2-scaffold small molecule conjugate, AE-labeled DNA3 and GO-AOD complex; wherein the antibody is thyroxine antibody, and the scaffold small molecule is thyroxine antigen; DNA1 is complementary to DNA2 by 7 bases, and DNA3 is complementary to DNA1 and DNA2 by 8 bases, respectively.
Further, the peroxidase comprises one or more of lactoperoxidase, microperoxidase, myeloperoxidase, haloperoxidase, vanadium bromoperoxidase, horseradish peroxidase, fungal peroxidase and lignin peroxidase.
The invention provides a detection kit, which comprises the following detection solutions: DNA 1-antibody-iron complex conjugate, DNA 2-scaffold small molecule conjugate, AE-labeled DNA3 and GO-AOD complex; wherein the antibody is thyroxine antibody, and the scaffold small molecule is thyroxine antigen; DNA1 is complementary to DNA2 by 7 bases, and DNA3 is complementary to DNA1 and DNA2 by 8 bases, respectively.
Further, the iron complex includes hemin, mn-TPPS4.
Further, the DNA 1-antibody-peroxidase conjugate/DNA 1-antibody-iron complex conjugate is obtained by covalently binding DNA1 to an amino group of an antibody on the antibody-peroxidase conjugate/antibody-iron complex conjugate, and the antibody-peroxidase conjugate/antibody-iron complex conjugate is obtained by covalently binding the amino group on the antibody to a carbonyl group after hydroformylation of the peroxidase/iron complex.
Further, the DNA 2-scaffold small molecule conjugate is obtained by covalently binding DNA2 with amino groups on a scaffold small molecule.
Further, in the DNA 1-antibody-peroxidase conjugate/DNA 1-antibody-iron complex conjugate, the nucleotide sequence of the DNA1 is shown as SEQ ID NO. 1; in the DNA 2-bracket small molecule conjugate, the nucleotide sequence of the DNA2 is shown as SEQ ID NO. 2; the nucleotide sequence of the DNA3 is shown as SEQ ID NO. 3.
Further, the concentration of the DNA 1-antibody-peroxidase conjugate/DNA 1-antibody-iron complex conjugate is 400-600 ng/mL, the concentration of the DNA 2-scaffold small molecule conjugate is 200-400 ng/mL, the concentration of the DNA3 for marking the acridine ester AE is 0.15 mu M, and the concentration of the graphene oxide GO combined with the antioxidant AOD is 20 mu g/mL.
Further, G in the nucleotide sequences of the DNA1, the DNA2 and the DNA3 is isoG, and C is isoC.
The invention also provides application of the detection kit in detecting the content of serum free thyroxine.
The invention also provides an acridine lipid homogeneous immunoassay detection method based on graphene oxide-antioxidant quenching resonance energy transfer, which comprises the following detection steps:
1) Mixing a calibration solution containing different target molecule concentrations with the detection solution in the detection kit, incubating, adding a chemiluminescent substrate, detecting a chemiluminescent signal of the solution, recording a chemiluminescent value, and drawing a calibration curve according to the chemiluminescent value of the calibration solution to obtain a standard curve between the calibration solution and the chemiluminescent intensity;
2) Mixing a sample to be detected with the detection solution in the detection kit, incubating, adding a chemiluminescent substrate, and detecting a chemiluminescent signal of the sample to be detected to obtain a chemiluminescent value of the sample to be detected; and (3) bringing the chemiluminescent value of the sample to be measured into a calibration curve to obtain the concentration of the target molecule in the sample to be measured.
In the absence of the small molecule of interest, the DNA 1-antibody-peroxidase conjugate/DNA 1-antibody-iron complex conjugate binds to the DNA 2-scaffold small molecule conjugate, allowing DNA1 and DNA2 to be in sufficient proximity to form an ortho complex and to hybridize to DNA 3. The compound is not adsorbed by GO, the AOD coupled on graphene hardly affects AE luminescence on the compound, and because the compound introduces the ortho peroxidase/iron compound, the substrate solution H is added 2 O 2 More singlet oxygen can be generated around the compound, and the luminous intensity of AE is enhanced. In the presence of the target small molecule, the DNA 1-antibody-peroxidase conjugate/DNA 1-antibody-iron complex conjugate is competitively combined, so that part of the DNA 1-antibody-peroxidase conjugate/DNA 1-antibody-iron complex conjugate cannot be combined with the DNA 2-scaffold small molecule conjugate, part of the DNA3 is adsorbed on the GO surface through pi-pi stacking effect, and luminescence is quenched.
Further, the concentration of the calibration solution is in the range of 0.5 to 100pmol/L.
Further, the DNA3 also comprises 5 '-end modified NH2C6 which is connected with a primer 5' -sugar ring through a B-cyanoethyl chemical reaction; DNA3-AE was then formed by addition of the acridinium ester.
Specifically, the DNA1 contains 54 bases, the DNA2 contains 52 bases, and the DNA3 contains 21 bases; the modification of the NH2C6 at the DNA 35 ' end (5 ' Aminolinker (C6)) was added to the 5' sugar ring of the primer by B-cyanoethyl chemistry in the form of an amine phosphite in the last step of the synthesis cycle, rather than to the last base.
Further, the test sample includes whole blood// serum/plasma containing the test target molecule.
Further, the incubation conditions are incubation at 37℃for 5-10 minutes.
Further, the chemiluminescent substrate is hydrogen peroxide at 100mM ph=10.
The beneficial effects are that: compared with the prior art, the invention has the following remarkable advantages: according to the invention, the ortho peroxidase or the iron complex is introduced into the acridine lipid homogeneous immunoassay detection method, so that the low-end sensitivity and stability of the detection method are remarkably improved.
Drawings
FIG. 1 is a schematic diagram of the detection method of the present invention when the target molecule to be detected is a protein molecule;
FIG. 2 is a complementary pairing diagram of DNA3 and DNA1, DNA2 when the target molecule to be measured is a protein molecule;
FIG. 3 is a schematic diagram of the detection method of the present invention when the target molecule to be detected is a small molecule;
FIG. 4 is a diagram showing complementary pairing of DNA3 and DNA1, DNA2 when the target molecule to be measured is a small molecule.
Detailed Description
The technical scheme of the invention is further described below with reference to the accompanying drawings.
EXAMPLE 1 kit containing DNA 1-antibody 1-peroxidase conjugate for detection of troponin I in serum
1. Preparation of antibody 1-HRP conjugates
1. Weighing 5mg of HRP (horseradish peroxidase purchased from Shanghai Xueman biotechnology Co., ltd., specification: RZ >3, > 300U/mg) and dissolving in 1mL of distilled water to obtain an HRP solution;
2. adding 0.2mL of newly prepared 0.1M NaIO into HRP solution 4 Solution, at room temperature protected from lightStirring for 20 minutes;
3. the solution was packed in a dialysis bag, 1mM sodium acetate buffer (specifically, 0.2M NaAc (1.361 g/50 mL) 3.7mL;0.2M HAc (0.601 mL/50 mL) 6.3mL; distilled water was added to 2,000 mL) was added for dialysis at 4℃overnight;
4. 20. Mu.L of 0.2M carbonate buffer (the carbonate buffer is specifically Na) with pH9.5 is added 2 CO 3 0.32g；NaHCO 3 0.586g; distilled water to 50 mL) was added to raise the pH of the above hydroformylation HRP to 9.0-9.5, followed immediately by the addition of 10mg of antibody 1 (purchased from Hytest corporation under clone No. 7b9 cc) in 1mL of 0.01m carbonate buffer (carbonate buffer specifically Na 2 CO 3 0.32g；NaHCO 3 0.586g; distilled water is added to 1L), and the mixture is gently stirred at room temperature for 2 hours in a dark place;
5. adding 0.1mL of newly prepared 4mg/mL NaBH 4 Mixing the solutions, and standing at 4deg.C for 2 hr;
6. the solution was placed in a dialysis bag, and 2-3L of 0.15M PH7.4 PBS was added for dialysis at 4℃overnight to obtain an antibody 1-HRP conjugate.
2. Preparation of AE-modified DNA3
1. Preparing a DNA3 solution: 20. Mu.M DNA3 (purchased from Kirschner Biotech Co., ltd., 5' -NH2C6- iso C iso GAT iso CT iso CA iso G iso CAA iso CT iso CA iso G iso CA iso G iso C iso G-3') was dissolved by adding 1mL of purified water.
2. Preparing NHS-AE solution: 4mg of acridine ester (NSP-DMAE-NHS) (available from Suzhou subfamily technologies Co., ltd., CAS number 194357-64-7) was weighed and dissolved in 1mL of purified water.
3. Coupling: mu.L of NHS-AE solution was taken, 10. Mu.L of DNA3 solution was added, mixed well and incubated for 30min at 37 ℃.
4. And (3) dialysis: sucking out the coupled DNA3 from the EP tube, adding into a dialysis bag (specification 5 kd), putting the pricked dialysis bag into a beaker filled with 2-3L TE solution (10mM Tris,1mM EDTA,PH =8.0), dialyzing (soaking the dialysis bag in advance), changing the dialyzate for 2-3 hours for one time, dialyzing for three times, collecting the liquid in the dialysis bag after the dialysis is completed, putting the liquid into a centrifuge tube, and storing at 2-8 ℃ for standby.
3. Preparation of DNA 1-antibody 1-HRP conjugate
1. Preparing a BS3 solution: 10mg of bis-succinimidyl suberate sodium salt (BS 3) (purchased from Shanghai Ala Biotechnology Co., ltd., product No. S304724) was weighed out and dissolved in 1mL of purified water.
2. Activating antibody 1-HRP conjugate and conjugating DNA1: taking out the split charged antibody 1-HRP conjugate, thawing, centrifuging and mixing uniformly. mu.L of BS3 solution was added to each 1mg of antibody 1-HRP conjugate, and 6.5. Mu.L of DNA1 (purchased from Kirschner Biotech Co., ltd., 5' -A) was added iso C iso G iso CT iso GA iso GTTAT iso CAA iso C iso GA iso CTTTTTTTAT iso CA iso CAT is o CA iso G iso G iso CT iso CTA iso G iso C iso GTAT iso G iso CTATT iso G-NH2C 7-3'), mixing uniformly, and incubating at 37 ℃ for 30min to obtain the DNA 1-antibody 1-HRP conjugate.
3. And (3) dialysis: sucking out the DNA 1-antibody 1-HRP conjugate from the EP tube, adding into a dialysis bag (specification 100 kd), putting the pricked dialysis bag into a beaker filled with 2-3L 0.15M PH7.4 PBS solution, dialyzing (soaking the dialysis bag in advance), changing the dialyzate for 2-3 hours for one time, dialyzing for three times, collecting the liquid in the dialysis bag after the dialysis is completed, putting into a centrifuge tube, and storing at 2-8 ℃ for later use.
4. Preparation of DNA 2-antibody 2 conjugates
1. Preparing a BS3 solution: 10mg of bis-succinimidyl suberate sodium salt (BS 3) (purchased from Shanghai Ala Biotechnology Co., ltd., product No. S304724) was weighed out and dissolved in 1mL of purified water.
2. Activating the antibodies and coupling DNA2: the packaged antibody 2 (purchased from Hytest company, clone number RecChim20C 6) was removed, thawed, and centrifuged and mixed. mu.L of BS3 solution was added to 1mg of antibody 2, and 6.5. Mu.L of DNA2 (purchased from Kirschner Biotech Co., ltd., 5' -NH2C 6-TA) was added iso C iso GT iso C iso CA iso GAA iso CTTTA iso C iso CAAA is o C iso CA iso CA iso C iso C iso CTTTTTTT iso GT iso C iso GTT iso G iso G iso CT iso GA iso GATT iso -C6-SH-3'), mixing uniformly, and incubating at 37 ℃ for 30min to obtain the DNA 2-antibody 2 conjugate.
3. And (3) dialysis: sucking out the DNA 2-antibody 2 conjugate in the step 2 from the EP tube, adding the solution into a dialysis bag (specification 100 kd), putting the pricked dialysis bag into a beaker filled with 2-3L 0.15M PH7.4 PBS solution, dialyzing (soaking the dialysis bag in advance), changing the dialyzate for 2-3 hours for one time, dialyzing for three times, collecting the liquid in the dialysis bag after the dialysis is completed, putting the liquid into a centrifuge tube, and storing at 2-8 ℃ for later use.
5. Preparation of DNA 1-antibody 1 conjugates
1. Preparing a BS3 solution: 10mg of bis-succinimidyl suberate sodium salt (BS 3) (purchased from Shanghai Ala Biotechnology Co., ltd., product No. S304724) was weighed out and dissolved in 1mL of purified water.
2. Activating the antibodies and coupling DNA1: the packaged antibody 1 (purchased from Hytest Co., clone No. 7b9 cc) was removed, thawed, and centrifuged and mixed. mu.L of BS3 solution was added to 1mg of antibody, and 6.5. Mu.L of DNA1 (purchased from Kirschner Biotech Co., ltd., 5' -A) was added iso C iso G iso CT iso GA iso GTTAT iso CAA iso C iso GA iso CTTTTTTTAT iso CA iso CAT iso CA iso G iso G iso CT is o CTA iso G iso C iso GTAT iso G iso CTATT iso G-NH2C 7-3'), mixing well, and incubating at 37 ℃ for 30min.
3. And (3) dialysis: sucking out the antibody coupled in the step 2 from an EP tube, adding the antibody into a dialysis bag (specification is 100 kd), putting the pricked dialysis bag into a beaker filled with 2-3L 0.15M PH7.4 PBS solution, dialyzing (soaking the dialysis bag in advance), replacing the dialyzate for 2-3 hours for three times, collecting the liquid in the dialysis bag after dialysis, and storing the liquid in a centrifuge tube at 2-8 ℃ for later use.
6. Actual sample detection
1. Preparing a detection reagent A: the DNA 1-antibody 1 conjugate, DNA 2-antibody 2 conjugate, AE-modified DNA3, GO-AOD were mixed to give final concentrations of 400ng/mL, 0.15. Mu.M and 20. Mu.g/mL, respectively.
2. Preparing a detection reagent B: DNA 1-antibody 1-HRP conjugate, DNA 2-antibody 2 conjugate, AE-modified DNA3, GO-AOD were mixed to final concentrations of 400ng/mL, 0.15. Mu.M and 20. Mu.g/mL, respectively.
3. mu.L of calibration solutions (0.01, 0.1, 1, 10, 50 ng/mL) of different concentrations were mixed with 200. Mu.L of detection solution A or detection solution B, respectively, and incubated at 37℃for 5-10 minutes. After incubation, 200 μl of 100mM PH=10H was added by HSCL-5000 chemiluminescence apparatus 2 O 2 As a chemiluminescent substrate, the chemiluminescent signal of the solution was immediately detected by a photomultiplier tube (PMT) for 3 seconds. From the recorded chemiluminescent values (RLU), a calibration curve of cTnI was obtained.
4. mu.L of serum sample containing troponin I (supplied by Jiangsu Ning research biomedical Co., ltd.) was mixed with 200. Mu.L of each of the test solutions A and B, and incubated at 37℃for 5 to 10 minutes. After incubation, 200. Mu.L of chemiluminescent substrate was added by HSCL-5000 chemiluminescent instrument and the chemiluminescent signal of the solution was detected immediately by photomultiplier tube (PMT) for 3s. And obtaining the concentration of cTnI in the sample to be tested according to the recorded chemiluminescence value (RLU). The concentration of troponin I in the actual sample was obtained by bringing into a calibration curve, and the specific results are shown in table 1. Control group was the result of detecting cTnI using a yabac axym immunofluorescence analyzer for the actual samples.
Table 1 comparison table of detection values of the present example and the yaban detection method
The results show that the detection limit of the cardiac troponin of this example is 0.01-50ng/mL. The error of the test reagent A and the Yaban test value in this example was 5015%, and the error of the test reagent B and the Yaban test value was-1.73%. The detection method B of the embodiment has higher accuracy, and the low-end sensitivity is obviously higher than that of the detection reagent A.
Example 2 detection of free T4 (FT 4) in serum Using a kit comprising a DNA 1-antibody 1-peroxidase conjugate
1. The second, third and fifth preparation methods were the same as in example 1, and antibody 1 was purchased from Nanj Oukai biotechnology Co., ltd, clone number K88a6.
And fourthly, preparing the DNA 2-bracket small molecule conjugate.
1. Preparing a BS3 solution: 10mg of bis-succinimidyl suberate sodium salt (BS 3) (purchased from Shanghai Ala Biotechnology Co., ltd., product No. S304724) was weighed out and dissolved in 1mL of purified water.
2. Activating the scaffold small molecule conjugate and coupling to DNA2: taking out the split-charged bracket small molecule conjugate (purchased from Shanghai Hanzun biotechnology company, clone number is T4-BSA-HT-020561), thawing, centrifuging and mixing. 3 mu L of BS3 solution is added into each 1mg of bracket small molecule conjugate, 6.5 mu L of DNA2 is added, the mixture is uniformly mixed, and the mixture is incubated at 37 ℃ for 30min to obtain the DNA 2-bracket small molecule conjugate.
3. And (3) dialysis: sucking out the DNA 2-bracket micromolecular conjugate from an EP tube, adding the mixture into a dialysis bag (specification is 100 kd), putting the pricked dialysis bag into a beaker filled with 2-3L 0.15M PH7.4 PBS solution, dialyzing (soaking the dialysis bag in advance), replacing dialyzate for 2-3 hours for one time, dialyzing for three times, collecting the liquid in the dialysis bag after the dialysis is completed, putting the liquid into a centrifuge tube, and storing the liquid at 2-8 ℃ for later use.
6. Actual sample detection
1. Preparing a detection reagent A: the DNA 1-antibody conjugate, DNA 2-scaffold small molecule conjugate, AE-modified DNA3, GO-AOD were mixed to give final concentrations of 600ng/mL, 200ng/mL, 0.15. Mu.M and 20. Mu.g/mL, respectively.
2. Preparing a detection reagent B: DNA 1-antibody-HRP conjugate, DNA 2-scaffold small molecule conjugate, AE-modified DNA3, GO-AOD were mixed to final concentrations of 600ng/mL, 200ng/mL, 0.15. Mu.M and 20. Mu.g/mL, respectively.
3. mu.L of calibration solutions (0.5, 1, 5, 10, 50, 100 pmol/L) of different concentrations were mixed with 200. Mu.L of detection solution A or detection solution B, respectively, and incubated at 37℃for 5-10 minutes. After incubation, 200 μl of 100mM PH=10H was added by HSCL-5000 chemiluminescence apparatus 2 O 2 As a chemiluminescent substrate, the chemiluminescent signal of the solution was immediately detected by a photomultiplier tube (PMT) for 3 seconds. From the recorded chemiluminescent values (RLU), a calibration curve for FT4 was obtained.
4. mu.L of serum sample containing free T4 (supplied by Jiangsu Ning research biomedical Co., ltd.) was mixed with 200. Mu.L of the detection solution A or the detection solution B, respectively, and incubated at 37℃for 5-10 minutes. After incubation, 200. Mu.L of chemiluminescent substrate was added by HSCL-5000 chemiluminescent instrument and the chemiluminescent signal of the solution was detected immediately by photomultiplier tube (PMT) for 3s. And obtaining the concentration of FT4 in the sample to be tested according to the recorded chemiluminescence value (RLU). The concentration of free T4 (FT 4) in the actual sample was obtained by bringing into the calibration curve, and the specific results are shown in table 2. The control group was the result of detection of FT4 using the Roche FT4 kit (LOT: 54716803) for the actual samples.
Table 2 is a table showing comparison of detection values of the present example and the Roche detection method
As a result, the detection limit of the serum FT4 in this example was 0.5 to 100pmol/L. The error between the detection reagent a and the rogowski test value in the embodiment is 858.22% and the error between the detection reagent B and the rogowski test value is 13%, which indicates that the detection reagent B of the embodiment has higher accuracy and lower-end sensitivity is obviously higher than that of the detection reagent a.
EXAMPLE 3 kit containing DNA 1-antibody 1-iron Complex for detection of troponin I in serum
1. Preparation of antibody 1-Hemin conjugates
1. Weighing 1mg of Hemin (Hemin, purchased from Jiangsu moxa Kang Shengwu medical research and development Co., ltd., product number AK00HYW 6) and dissolving in 1mL of distilled water to obtain a Hemin solution;
2. adding 0.2mL of newly prepared 0.1M NaIO to the solution in the step 1 4 Stirring the solution for 20 minutes at room temperature in a dark place;
3. the solution was packed in a dialysis bag, 1mM sodium acetate buffer (specifically, 0.2M NaAc (1.361 g/50 mL) 3.7mL;0.2M HAc (0.601 mL/50 mL) 6.3mL; distilled water was added to 2,000 mL) was added for dialysis at 4℃overnight;
4. add 20. Mu.L of 0.2M carbonate buffer pH9.5 (specifically Na 2 CO 3 0.32g；NaHCO 3 0.586g; distilled water was added to 50 ml) to raise the pH of the above hydroformylation HRP to 9.0-9.5, then 10mg of antibody 1 (purchased from Hytest Co., clone No. 7b9 cc) was immediately added to 1mL of 0.01M carbonate buffer, and gently stirred at room temperature in the absence of light for 2 hours;
5. adding 0.1mL of newly prepared 4mg/mL NaBH 4 Mixing the solutions, and standing at 4deg.C for 2 hr;
6. the solution was packed in a dialysis bag, and dialyzed against 0.15M PH7.4 PBS, at 4℃overnight, to give an antibody 1-Hemin conjugate.
2. Preparation of AE-modified DNA3
1. Preparing a DNA3 solution: 20. Mu.M DNA3 (purchased from Kirschner Biotech Co., ltd., 5' -NH2C6- iso C iso GAT iso CT iso CA iso G iso CAA iso CT iso CA iso G iso CA iso G iso C iso G-3') was dissolved by adding 1mL of purified water.
2. Preparing NHS-AE solution: 4mg of acridine ester (NSP-DMAE-NHS) (available from Suzhou subfamily technologies Co., ltd., CAS number 194357-64-7) was weighed and dissolved in 1mL of purified water.
3. Coupling: mu.L of NHS-AE solution was taken, 10. Mu.L of DNA3 solution was added, mixed well and incubated for 30min at 37 ℃.
4. And (3) dialysis: sucking out the coupled DNA3 from the EP tube, adding into a dialysis bag (specification 5 kd), putting the pricked dialysis bag into a beaker filled with 2-3L TE solution (10mM Tris,1mM EDTA,PH =8.0), dialyzing (soaking the dialysis bag in advance), changing the dialyzate for 2-3 hours for one time, dialyzing for three times, collecting the liquid in the dialysis bag after the dialysis is completed, putting the liquid into a centrifuge tube, and storing at 2-8 ℃ for standby.
3. Preparation of DNA 1-antibody 1Hemin conjugate
1. Preparing a BS3 solution: 10mg of bis-succinimidyl suberate sodium salt (BS 3) (purchased from Shanghai Ala Biotechnology Co., ltd., product No. S304724) was weighed out and dissolved in 1mL of purified water.
2. Activating antibody 1-Hemin conjugate and coupling DNA1: taking out the split charged antibody 1-Hemin conjugate, thawing, centrifuging and mixing. mu.L of BS3 solution was added to 1mg of antibody 1-Hemin conjugate 1, and 6.5. Mu.L of DNA1 (purchased from Kirschner Biotech Co., ltd., 5' -A) was added iso C iso G iso CT iso GA iso GTTAT iso CAA iso C iso GA iso CTTTTTTTAT iso CA iso CAT iso CA iso G iso G iso CT iso CTA iso G iso C iso GTAT iso G iso CTATT iso G-NH2C 7-3'), mixing uniformly, and incubating at 37 ℃ for 30min to obtain the DNA 1-antibody 1Hemin conjugate.
3. And (3) dialysis: sucking out the DNA 1-antibody 1Hemin conjugate from the EP tube, adding into a dialysis bag (specification 100 kd), putting the pricked dialysis bag into a beaker filled with 2-3L 0.15M PH7.4 PBS solution, dialyzing (soaking the dialysis bag in advance), changing the dialyzate for 2-3 hours for one time, dialyzing for three times, collecting the liquid in the dialysis bag after the dialysis is completed, putting into a centrifuge tube, and storing at 2-8 ℃ for later use.
4. Preparation of DNA 2-antibody 2 conjugates
1. Preparing a BS3 solution: 10mg of bis-succinimidyl suberate sodium salt (BS 3) (purchased from Shanghai Ala Biotechnology Co., ltd., product No. S304724) was weighed out and dissolved in 1mL of purified water.
2. Activating the antibodies and coupling DNA2: the packaged antibody 2 (purchased from Hytest company, clone number RecChim20C 6) was removed, thawed, and centrifuged and mixed. mu.L of BS3 solution was added to 1mg of antibody, and 6.5. Mu.L of DNA2 (purchased from Kirschner Biotech Co., ltd., 5' -NH2C 6-TA) was added iso C iso GT iso C iso CA iso GAA iso CTTTA iso C iso CAAA is o C iso CA iso CA iso C iso C iso CTTTTTTT iso GT iso C iso GTT iso G iso G iso CT iso GA iso GATT iso -C6-SH-3'), mixing uniformly, and incubating at 37 ℃ for 30min to obtain the DNA 2-antibody 2 conjugate.
3. And (3) dialysis: sucking out the DNA 2-antibody 2 conjugate in the step 2 from the EP tube, adding the solution into a dialysis bag (specification 100 kd), putting the pricked dialysis bag into a beaker filled with 2-3L 0.15M PH7.4 PBS solution, dialyzing (soaking the dialysis bag in advance), changing the dialyzate for 2-3 hours for one time, dialyzing for three times, collecting the liquid in the dialysis bag after the dialysis is completed, putting the liquid into a centrifuge tube, and storing at 2-8 ℃ for later use.
5. Preparation of DNA 1-antibody 1 conjugates
1. Preparing a BS3 solution: 10mg of bis-succinimidyl suberate sodium salt (BS 3) (purchased from Shanghai Ala Biotechnology Co., ltd., product No. S304724) was weighed out and dissolved in 1mL of purified water.
2. Activating antibody and coupling DNA1: the packaged antibody 1 (purchased from Hytest Co., clone No. 7b9 cc) was removed, thawed, and centrifuged and mixed. mu.L of BS3 solution was added to 1mg of antibody, and 6.5. Mu.L of DNA1 (purchased from Kirschner Biotech Co., ltd., 5' -A) was added iso C iso G iso CT iso GA iso GTTAT iso CAA iso C iso GA iso CTTTTTTTAT iso CA iso CAT iso CA iso G iso G iso CT iso CTA iso G iso C iso GTAT iso G iso CTATT iso G-NH2C 7-3'), mixing well, and incubating at 37 ℃ for 30min.
3. And (3) dialysis: sucking out the antibody coupled in the step 2 from an EP tube, adding the antibody into a dialysis bag (specification is 100 kd), putting the pricked dialysis bag into a beaker filled with 2-3L 0.15M PH7.4 PBS solution, dialyzing (soaking the dialysis bag in advance), replacing the dialyzate for 2-3 hours for three times, collecting the liquid in the dialysis bag after dialysis, and storing the liquid in a centrifuge tube at 2-8 ℃ for later use.
6. Actual sample detection
1. Preparing a detection reagent A: the DNA 1-antibody 1 conjugate, DNA 2-antibody 2 conjugate, AE-modified DNA3, GO-AOD were mixed to give final concentrations of 400ng/mL, 0.15. Mu.M and 20. Mu.g/mL, respectively.
2. Preparing a detection reagent B: DNA 1-antibody 1-Hemin conjugate, DNA 2-antibody 2 conjugate, AE-modified DNA3, GO-AOD were mixed to final concentrations of 400ng/mL, 0.15. Mu.M and 20. Mu.g/mL, respectively.
3. mu.L of calibration solutions (0.01, 0.1, 1, 10, 50 ng/mL) of different concentrations were mixed with 200. Mu.L of detection solution A or detection solution B, respectively, and incubated at 37℃for 5-10 minutes. After incubation, 200 μl of 100mM PH=10H was added by HSCL-5000 chemiluminescence apparatus 2 O 2 As a chemiluminescent substrate, the chemiluminescent signal of the solution was immediately detected by a photomultiplier tube (PMT) for 3 seconds. From the recorded chemiluminescent values (RLU), a calibration curve of cTnI was obtained.
4. mu.L of serum sample containing troponin I (supplied by Jiangsu Ning research biomedical Co., ltd.) was mixed with 200. Mu.L of each of the test solutions A and B, and incubated at 37℃for 5 to 10 minutes. After incubation, 200. Mu.L of chemiluminescent substrate was added by HSCL-5000 chemiluminescent instrument and the chemiluminescent signal of the solution was detected immediately by photomultiplier tube (PMT) for 3s. And obtaining the concentration of cTnI in the sample to be tested according to the recorded chemiluminescence value (RLU). The concentration of troponin I in the actual sample was obtained by bringing into the calibration curve, and the specific results are shown in table 3. Control group was the result of detecting cTnI using a yabac axym immunofluorescence analyzer for the actual samples.
TABLE 3 comparison Table of detection values of the present example and the Atlantic detection method
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The results show that the detection limit of the cardiac troponin of this example is 0.01-50ng/mL. The error of the test reagent A and the Yaban test value of this example was 5015%, and the error of the test reagent B and the Yaban test value was-23%. The detection method B of the embodiment has higher accuracy, and the low-end sensitivity is obviously higher than that of the detection reagent A.
EXAMPLE 4 detection of free T4 (FT 4) in serum Using the kit comprising DNA 1-antibody 1-iron complex
1. The second, third and fifth antibodies were prepared in the same manner as in example 3, and antibody 1 was purchased from Nanj Oukai biotechnology Co., ltd., clone number K88a6.
And fourthly, preparing the DNA 2-bracket small molecule conjugate.
1. Preparing a BS3 solution: 10mg of bis-succinimidyl suberate sodium salt (BS 3) (purchased from Shanghai Ala Biotechnology Co., ltd., product No. S304724) was weighed out and dissolved in 1mL of purified water.
2. Activating the scaffold small molecule conjugate and coupling to DNA2: taking out the split-charged bracket small molecule conjugate (purchased from Shanghai Hanzun biotechnology company, clone number is T4-BSA-HT-020561), thawing, centrifuging and mixing. 3 mu L of BS3 solution is added into each 1mg of bracket small molecule conjugate, 6.5 mu L of DNA2 is added, the mixture is uniformly mixed, and the mixture is incubated at 37 ℃ for 30min to obtain the DNA 2-bracket small molecule conjugate.
3. And (3) dialysis: sucking out the DNA 2-bracket micromolecular conjugate from an EP tube, adding the mixture into a dialysis bag (specification is 100 kd), putting the pricked dialysis bag into a beaker filled with 2-3L 0.15M PH7.4 PBS solution, dialyzing (soaking the dialysis bag in advance), replacing dialyzate for 2-3 hours for one time, dialyzing for three times, collecting the liquid in the dialysis bag after the dialysis is completed, putting the liquid into a centrifuge tube, and storing the liquid at 2-8 ℃ for later use.
6. Actual sample detection
1. Preparing a detection reagent A: the DNA 1-antibody conjugate, DNA 2-scaffold small molecule conjugate, AE-modified DNA3, GO-AOD were mixed to give final concentrations of 600ng/mL, 200ng/mL, 0.15. Mu.M and 20. Mu.g/mL, respectively.
2. Preparing a detection reagent B: DNA 1-antibody-Hemin conjugate, DNA 2-scaffold small molecule conjugate, AE-modified DNA3, GO-AOD were mixed to final concentrations of 600ng/mL, 200ng/mL, 0.15. Mu.M and 20. Mu.g/mL, respectively.
3. mu.L of calibration solutions (0.5, 1, 5, 10, 50, 100 pmol/L) of different concentrations were mixed with 200. Mu.L of detection solution A or detection solution B, respectively, and incubated at 37℃for 5-10 minutes. After incubation, 200 μl of 100mM PH=10H was added by HSCL-5000 chemiluminescence apparatus 2 O 2 As a chemiluminescent substrate, the chemiluminescent signal of the solution was immediately detected by a photomultiplier tube (PMT) for 3 seconds. From the recorded chemiluminescent values (RLU), a calibration curve for FT4 was obtained.
4. mu.L of serum sample containing free T4 (supplied by Jiangsu Ning research biomedical Co., ltd.) was mixed with 200. Mu.L of the detection solution A or the detection solution B, respectively, and incubated at 37℃for 5-10 minutes. After incubation, 200. Mu.L of chemiluminescent substrate was added by HSCL-5000 chemiluminescent instrument and the chemiluminescent signal of the solution was detected immediately by photomultiplier tube (PMT) for 3s. And obtaining the concentration of FT4 in the sample to be tested according to the recorded chemiluminescence value (RLU). The concentration of free T4 in the actual sample was obtained by substituting the calibration curve, and the specific results are shown in table 4. The control group was the result of detection of FT4 using the Roche FT4 kit (LOT: 54716803) for the actual samples.
Table 4 shows a comparison table of the detection values of the present example and the Roche detection method
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As a result, the detection limit of the serum FT4 in this example was 0.5 to 100pmol/L. The error between the detection reagent a and the rogowski test value in the embodiment is 858.22%, and the error between the detection reagent B and the rogowski test value is 77%, which indicates that the detection reagent B of the embodiment has higher accuracy, and the low-end sensitivity is significantly higher than that of the detection reagent a.

Claims (10)

1. The detection kit with high low-end sensitivity is characterized by comprising the following detection solutions: DNA 1-antibody-peroxidase conjugate, DNA 2-scaffold small molecule conjugate, AE-labeled DNA3 and GO-AOD complex; wherein the antibody is thyroxine antibody, and the scaffold small molecule is thyroxine antigen; DNA1 is complementary to DNA2 by 7 bases, and DNA3 is complementary to DNA1 and DNA2 by 8 bases, respectively.
2. The test kit of claim 1, wherein the peroxidase comprises one or more of lactoperoxidase, microperoxidase, myeloperoxidase, haloperoxidase, vanadium bromoperoxidase, horseradish peroxidase, fungal peroxidase, lignin peroxidase.
3. The detection kit according to claim 1, wherein the DNA 1-antibody-peroxidase conjugate is obtained by covalently binding the DNA1 to an amino group of an antibody on the antibody-peroxidase conjugate, and the antibody-peroxidase conjugate is obtained by covalently binding the amino group on the antibody to a carbonyl group after hydroformylation of peroxidase.
4. The kit of claim 1, wherein the DNA 2-scaffold small molecule conjugate is obtained by covalently binding DNA2 to an amino group on a scaffold small molecule.
5. The detection kit according to claim 1, wherein the nucleotide sequence of the DNA1 is shown in SEQ ID NO. 1; in the DNA 2-bracket small molecule conjugate, the nucleotide sequence of the DNA2 is shown as SEQ ID NO. 2; the nucleotide sequence of the DNA3 is shown as SEQ ID NO. 3.
6. The detection kit according to claim 1, wherein the concentration of the DNA 1-antibody-peroxidase conjugate is 400-600 ng/mL, the concentration of the DNA 2-scaffold small molecule conjugate is 200-400 ng/mL, the concentration of the DNA3 labeled with acridinium ester AE is 0.15. Mu.M, and the concentration of the graphene oxide GO combined with the antioxidant AOD is 20. Mu.g/mL.
7. Use of the detection kit according to any one of claims 1 to 6 for detecting thyroxine content.
8. The homogeneous immunoassay detection method of the acridine lipid based on graphene oxide-antioxidant quenching resonance energy transfer is characterized by comprising the following detection steps:
1) Mixing a calibration solution containing different target molecule concentrations with a detection solution in the detection kit of claim 1, incubating, adding a chemiluminescent substrate, detecting a chemiluminescent signal of the solution, recording a chemiluminescent value, and drawing a calibration curve according to the chemiluminescent value of the calibration solution to obtain a standard curve between the calibration solution and the chemiluminescent intensity;
2) Mixing a sample to be detected with the detection solution in the detection kit in claim 1, incubating, adding a chemiluminescent substrate, and detecting a chemiluminescent signal of the sample to be detected to obtain a chemiluminescent value of the sample to be detected; and (3) bringing the chemiluminescent value of the sample to be measured into a calibration curve to obtain the concentration of the target molecule in the sample to be measured.
9. The method according to claim 8, wherein the concentration of the calibration solution is in the range of 0.5 to 100pmol/L.
10. The method of claim 8, wherein the chemiluminescent substrate is hydrogen peroxide.
CN202311111380.1A 2023-05-24 2023-05-24 Detection kit with high low-end sensitivity and application thereof Pending CN117169492A (en)

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