CN114354583B - Electrochemiluminescence lung cancer detection kit based on metal-free light ATRP signal amplification strategy, and use method and application thereof - Google Patents
Electrochemiluminescence lung cancer detection kit based on metal-free light ATRP signal amplification strategy, and use method and application thereof Download PDFInfo
- Publication number
- CN114354583B CN114354583B CN202210016767.8A CN202210016767A CN114354583B CN 114354583 B CN114354583 B CN 114354583B CN 202210016767 A CN202210016767 A CN 202210016767A CN 114354583 B CN114354583 B CN 114354583B
- Authority
- CN
- China
- Prior art keywords
- solution
- concentration
- electrode
- bmp
- atrp
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 238000001514 detection method Methods 0.000 title claims abstract description 41
- 238000000034 method Methods 0.000 title claims abstract description 24
- 238000010560 atom transfer radical polymerization reaction Methods 0.000 title claims abstract description 21
- 230000003321 amplification Effects 0.000 title claims abstract description 17
- 238000003199 nucleic acid amplification method Methods 0.000 title claims abstract description 17
- 206010058467 Lung neoplasm malignant Diseases 0.000 title claims abstract description 16
- 201000005202 lung cancer Diseases 0.000 title claims abstract description 16
- 208000020816 lung neoplasm Diseases 0.000 title claims abstract description 16
- 101710141544 Allatotropin-related peptide Proteins 0.000 title claims abstract 4
- HWYHZTIRURJOHG-UHFFFAOYSA-N luminol Chemical compound O=C1NNC(=O)C2=C1C(N)=CC=C2 HWYHZTIRURJOHG-UHFFFAOYSA-N 0.000 claims abstract description 37
- 238000006243 chemical reaction Methods 0.000 claims abstract description 30
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims abstract description 24
- 239000010931 gold Substances 0.000 claims abstract description 21
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 18
- MBYLVOKEDDQJDY-UHFFFAOYSA-N tris(2-aminoethyl)amine Chemical compound NCCN(CCN)CCN MBYLVOKEDDQJDY-UHFFFAOYSA-N 0.000 claims abstract description 16
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims abstract description 12
- 229910052737 gold Inorganic materials 0.000 claims abstract description 12
- 239000002994 raw material Substances 0.000 claims abstract description 9
- 229910021642 ultra pure water Inorganic materials 0.000 claims abstract description 6
- 239000012498 ultrapure water Substances 0.000 claims abstract description 6
- 239000000243 solution Substances 0.000 claims description 111
- 108010036226 antigen CYFRA21.1 Proteins 0.000 claims description 38
- 230000004048 modification Effects 0.000 claims description 12
- 238000012986 modification Methods 0.000 claims description 12
- 239000011259 mixed solution Substances 0.000 claims description 9
- 238000002791 soaking Methods 0.000 claims description 9
- -1 H 2 O Substances 0.000 claims description 6
- 230000003213 activating effect Effects 0.000 claims description 6
- 238000003756 stirring Methods 0.000 claims description 6
- 238000005406 washing Methods 0.000 claims description 6
- 238000004020 luminiscence type Methods 0.000 claims description 4
- 230000015572 biosynthetic process Effects 0.000 claims description 3
- 238000001035 drying Methods 0.000 claims description 3
- 238000003786 synthesis reaction Methods 0.000 claims description 3
- 238000005498 polishing Methods 0.000 claims description 2
- 238000002360 preparation method Methods 0.000 claims description 2
- 229910001385 heavy metal Inorganic materials 0.000 abstract description 11
- 150000002500 ions Chemical class 0.000 abstract description 10
- 239000003054 catalyst Substances 0.000 abstract description 8
- 230000035945 sensitivity Effects 0.000 abstract description 5
- 239000002086 nanomaterial Substances 0.000 abstract description 3
- 102000004190 Enzymes Human genes 0.000 abstract description 2
- 108090000790 Enzymes Proteins 0.000 abstract description 2
- YXMISKNUHHOXFT-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) prop-2-enoate Chemical compound C=CC(=O)ON1C(=O)CCC1=O YXMISKNUHHOXFT-UHFFFAOYSA-N 0.000 description 32
- DKIDEFUBRARXTE-UHFFFAOYSA-N 3-mercaptopropanoic acid Chemical compound OC(=O)CCS DKIDEFUBRARXTE-UHFFFAOYSA-N 0.000 description 23
- SEACYXSIPDVVMV-UHFFFAOYSA-L eosin Y Chemical compound [Na+].[Na+].[O-]C(=O)C1=CC=CC=C1C1=C2C=C(Br)C(=O)C(Br)=C2OC2=C(Br)C([O-])=C(Br)C=C21 SEACYXSIPDVVMV-UHFFFAOYSA-L 0.000 description 19
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 13
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 description 13
- 229940098773 bovine serum albumin Drugs 0.000 description 13
- 230000035484 reaction time Effects 0.000 description 11
- 238000006116 polymerization reaction Methods 0.000 description 8
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 6
- 210000002966 serum Anatomy 0.000 description 6
- 102100025475 Carcinoembryonic antigen-related cell adhesion molecule 5 Human genes 0.000 description 4
- 238000000157 electrochemical-induced impedance spectroscopy Methods 0.000 description 4
- 239000000178 monomer Substances 0.000 description 4
- 208000002154 non-small cell lung carcinoma Diseases 0.000 description 4
- 238000005457 optimization Methods 0.000 description 4
- 150000003254 radicals Chemical class 0.000 description 4
- 239000000523 sample Substances 0.000 description 4
- 208000029729 tumor suppressor gene on chromosome 11 Diseases 0.000 description 4
- 101100537532 Rattus norvegicus Tnni3 gene Proteins 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 230000000875 corresponding effect Effects 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 230000005281 excited state Effects 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 239000003999 initiator Substances 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 238000010791 quenching Methods 0.000 description 3
- 230000000171 quenching effect Effects 0.000 description 3
- 230000004044 response Effects 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 2
- 101000914324 Homo sapiens Carcinoembryonic antigen-related cell adhesion molecule 5 Proteins 0.000 description 2
- 101000914321 Homo sapiens Carcinoembryonic antigen-related cell adhesion molecule 7 Proteins 0.000 description 2
- 101000617725 Homo sapiens Pregnancy-specific beta-1-glycoprotein 2 Proteins 0.000 description 2
- 238000004630 atomic force microscopy Methods 0.000 description 2
- 238000012512 characterization method Methods 0.000 description 2
- 238000006482 condensation reaction Methods 0.000 description 2
- 238000002484 cyclic voltammetry Methods 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 238000001378 electrochemiluminescence detection Methods 0.000 description 2
- 230000005283 ground state Effects 0.000 description 2
- 229910052751 metal Inorganic materials 0.000 description 2
- 239000002184 metal Substances 0.000 description 2
- 230000003287 optical effect Effects 0.000 description 2
- 230000037361 pathway Effects 0.000 description 2
- 229920000642 polymer Polymers 0.000 description 2
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 2
- 238000003127 radioimmunoassay Methods 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 230000002829 reductive effect Effects 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- WSLDOOZREJYCGB-UHFFFAOYSA-N 1,2-Dichloroethane Chemical compound ClCCCl WSLDOOZREJYCGB-UHFFFAOYSA-N 0.000 description 1
- XXSPGBOGLXKMDU-UHFFFAOYSA-N 2-bromo-2-methylpropanoic acid Chemical compound CC(C)(Br)C(O)=O XXSPGBOGLXKMDU-UHFFFAOYSA-N 0.000 description 1
- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 description 1
- 108010022366 Carcinoembryonic Antigen Proteins 0.000 description 1
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 102100036859 Troponin I, cardiac muscle Human genes 0.000 description 1
- 101710128251 Troponin I, cardiac muscle Proteins 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000009830 antibody antigen interaction Effects 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 125000001246 bromo group Chemical group Br* 0.000 description 1
- 208000003362 bronchogenic carcinoma Diseases 0.000 description 1
- 238000012767 chemiluminescent enzyme immunoassay Methods 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 238000003759 clinical diagnosis Methods 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 229910052802 copper Inorganic materials 0.000 description 1
- 239000010949 copper Substances 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 230000009849 deactivation Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 239000003792 electrolyte Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 125000005843 halogen group Chemical group 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000007342 radical addition reaction Methods 0.000 description 1
- 238000001959 radiotherapy Methods 0.000 description 1
- 239000000376 reactant Substances 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000027756 respiratory electron transport chain Effects 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 238000001338 self-assembly Methods 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000008719 thickening Effects 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000007704 transition Effects 0.000 description 1
- 239000000439 tumor marker Substances 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Landscapes
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
The invention discloses an electrochemiluminescence lung cancer detection kit based on a metal-free light ATRP signal amplification strategy, and a use method and application thereof, wherein the kit comprises the following raw materials: BMP, gold electrode, MPA, EDC, NHS, ab, BSA, ab2, me 6 TREN, EY, NAS, luminol, ultrapure water, H 2O2, DMSO, ethanol. The invention utilizes a metal-free photoinduced atom transfer radical polymerization (photo-ATRP) strategy, avoids the use of nano materials and biological enzymes in the current common signal amplification strategy, amplifies the signals by times, and improves the sensitivity, stability and reproducibility of detection. Meanwhile, the use of a heavy metal ion catalyst in the traditional ATRP reaction is avoided, the heavy metal ions do not need to be recovered and removed, the interference of the inherent color of the heavy metal ions on the detection result is overcome, the biotoxicity is not generated, and the method is more environment-friendly.
Description
Technical Field
The invention relates to an electrochemiluminescence lung cancer detection kit based on a metal-free photoinduced atom transfer radical polymerization (photo-ATRP) signal amplification strategy, and a use method and application thereof, and belongs to the technical field of biological analysis.
Background
CYFRA21-1 is considered to be a tumor marker mainly used for detecting lung cancer, and has important value especially for diagnosing non-SMALL CELL lung cancer (NSCLC). The likelihood of primary bronchogenic carcinoma is very high if there is an unclear circular shadow of the lung, with a serum CYFRA21-1 concentration >30 ng/mL. The positive detection rate of the CYFRA21-1 on various non-small cell lung cancers is 70-85%. The serum concentration level of CYFRA21-1 is positively correlated with clinical stage of tumor, and can also be used as an effective index for tracking early recurrence after lung cancer operation and radiotherapy and chemotherapy. Therefore, CYFRA21-1 detection is of great importance for clinical diagnosis of NSCLC. Several methods for detecting CYFRA21-1 have been reported: enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), chemiluminescent immunoassay, chemiluminescent enzyme immunoassay, etc. However, the detection process still has the defects of false positive, long time consumption, special instruments and professional operators, and the like.
The detection performance of the sensor can be improved by utilizing signal amplification technologies such as chain hybridization reaction, roller amplification reaction, nano material, polymerization reaction and the like. Among them, atom Transfer Radical Polymerization (ATRP) has the advantages of wide monomer raw materials, controllable polymerization reaction and the like, and is widely applied to the field of biosensing. However, the conventional ATRP reaction requires adding a large amount of heavy metal (e.g., copper) ions as a catalyst due to a relatively slow polymerization rate, and the heavy metal ions have a certain biotoxicity and require complicated removal and recovery processes, and the self-carried color of the heavy metal ions can interfere with the detection result of the sensor. Compared with the traditional metal catalyst, the photoinduction ATRP has the following advantages: 1) Heavy metal catalysts are not required to be added; 2) The reaction condition is mild; 3) The photoinduced polymerization process is controllable. Therefore, it is of great importance to research an ultrasensitive electrochemical sensing platform for CYFRA21-1 detection based on an optical ATRP signal amplification strategy.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide an electrochemiluminescence lung cancer detection kit based on a metal-free photoinduced atom transfer radical polymerization (photo-ATRP) signal amplification strategy, and a use method and application thereof, which avoid the use of heavy metal ion catalysts in the traditional ATRP reaction, amplify the signals in a multiplied way, and improve the detection sensitivity, stability and reproducibility.
In order to achieve the above object, one of the technical solutions of the present invention is:
An electrochemiluminescence lung cancer detection kit based on a metal-free light ATRP signal amplification strategy comprises the following raw materials: BMP, gold electrode, MPA, EDC, NHS, ab, BSA, ab2, me 6 TREN, EY, NAS, luminol, ultrapure water, H 2O2, DMSO, ethanol.
Further, when a part of the raw materials were used, a solution was prepared in which the concentration of BMP solution was 3mM, the concentration of MPA solution was 10mM, the concentration of NHS solution for activating BMP was 3mM, the concentration of EDC solution was 3mM, the concentrations of EDC and NHS in the mixed solution of EDC and NHS for activating MPA were 0.2M and 0.05M, respectively, the concentration of Ab1 solution was 1. Mu.g/mL, the concentration of BSA solution was 1%, the concentration of Ab2 solution was 1. Mu.g/mL, the concentration of Me 6 TREN solution was 10mM, the concentration of EY solution was 5mM, the concentration of NAS solution was 12mM, and the concentration of luminol solution was 15mM.
One of the technical schemes of the invention is as follows: the application method of the electrochemiluminescence lung cancer detection kit comprises the following steps:
(1) Ab2-BMP (Ab 2 x) Synthesis
① Dissolving BMP in ethanol solution to obtain BMP solution;
② Adding an NHS solution and an EDC solution into the BMP solution, and stirring to obtain a carboxyl activated BMP solution;
③ Adding the carboxyl activated BMP solution into the Ab2 solution, and stirring to obtain Ab2 solution;
(2) Electrode pretreatment
Polishing the bare gold electrode to obtain a polished mirror surface;
(3) Electrode modification
① Soaking the pretreated electrode in MPA solution, washing and drying;
② Immersing the electrode in the step ① in a mixed solution of EDC and NHS;
③ Directly dripping Ab1 solution on the surface of the electrode in the step ②, reacting, and then soaking in BSA solution;
④ Dropping a sample to be detected on the surface of the electrode in the step ③, reacting and washing;
⑤ Dropping Ab 2-x solution on the surface of the electrode in the step ④ for reaction;
⑥ Placing the electrode in the step ⑤ in a mixed solution consisting of Me 6 TREN solution, EY solution, NAS solution, H 2 O and DMSO for reaction;
⑦ Placing the electrode in the ⑥ step in a luminol solution for reaction;
⑧ The electrode of step ⑦ was placed in H 2O2 to measure the luminescence intensity of luminol.
Further, the concentration of the ethanol solution in the step (1) is 50% -80% (v/v); the volume ratio of BMP solution, NHS solution and EDC solution is 1:1:1; the concentration of the carboxyl-activated BMP solution was 1mM, the concentration of the Ab2 solution was 1. Mu.g/mL, and the volume ratio of the carboxyl-activated BMP solution to the Ab2 solution was 1:1.
Further, the stirring temperature in the step (1) is 37 ℃ and the time is 1 to 3 hours.
Further, the soaking temperature of ① in the step (2) is 37 ℃ and the time is 2-8 hours; ② The soaking temperature is 37 ℃ and the time is 1-2 h; ③ The reaction temperature is 37 ℃ and the reaction time is 1-3 h; ④ The reaction temperature is 37 ℃ and the reaction time is 1-3 h; ⑤ The reaction temperature is 37 ℃ and the reaction time is 1-3 h; ⑥ The reaction condition is room temperature, 470nm light irradiation, 2-5 h; ⑦ The reaction temperature is 37 ℃ for 1-3 h.
Further, the volume ratio of Me 6 TREN solution, EY solution, NAS solution, H 2 O, and DMSO is 5:5:1000:5000:3990.
One of the technical schemes of the invention is as follows: the application of the detection kit in CYFRA21-1 detection.
One of the technical schemes of the invention is as follows: an application of the detection kit in lung cancer detection.
The principle schematic diagram of the detection method is shown in figure 1.
The present invention employs photo-ATRP and ECL signal amplification strategies. First, MPA probes are attached to the electrode surface by self-assembled polar covalent bonds, and Ab1 is attached to the electrode surface as a capture probe by an amino condensation reaction. After blocking the residual binding sites with Bovine Serum Albumin (BSA), CYFR-1 and Ab2 * were sequentially attached to the gold electrode surface by antigen-antibody interactions. The bromo groups in BMP are recognized by the double bond of NAS, and a certain amount of NAS is grafted to the electrode surface by photo-ATRP. The monomeric NAS of photo-ATRP can provide many binding sites for the luminescent material luminol, thus significantly amplifying ECL signal. Finally, many luminol molecules are tightly linked to a large number of NAS through an amino condensation reaction.
In the metal-free light ATRP process, BMP is used as an initiator, EY is used as a catalyst, ME 6 TREN is used as a ligand, and NAS polymerization is carried out under 470nm light irradiation. The reductive quenching pathway begins with excitation by blue light of a specific wavelength, and as photons are absorbed, electrons transition from the ground state (EY) to the excited state (EY *). Thereafter, EY *, while depriving the electron donor (D, me 6 TREN), produced EY ·- and D ·+, while producing D ·+. Based on the three catalysts (EY, EY * and EY ·-), a reversible cycle was established to modulate the reduction and quenching pathways of photo-ATRP. An initiator (BMP) that activates photo-ATRP, while generating primary radicals (PO. Cndot.), halogen atoms (X -) and EY. By means of the double bond of the monomer, radical addition reaction is started to carry out chain transfer to produce polymer chain and reach the balance of activation/deactivation. By the above reduction quenching reaction, a large amount of monomer is grafted to the electrode surface. Then, the monomer was combined with luminol for ECL detection. Hydrogen peroxide is an effective co-reactant in the luminol reaction, which tends to decompose into superoxide radicals (OH ·) and superoxide anion radicals (O 2 · -). Under a certain voltage, luminol and hydrogen peroxide which are coreactants are oxidized simultaneously, then the hydrogen peroxide is rapidly decomposed to generate a high-energy free radical intermediate, the high-energy free radical intermediate participates in the oxidation reaction of luminol, the luminol is enabled to be in an excited state, and after the luminol returns to a ground state from the excited state, light with a corresponding wavelength is generated.
The invention utilizes a metal-free photoinduced atom transfer radical polymerization (photo-ATRP) strategy, avoids the use of nano materials and biological enzymes (which are easily influenced by external environment, temperature and the like) in the current common signal amplification strategy, and improves the sensitivity, stability and reproducibility of detection by multiplying the signals.
The invention adopts a metal-free photo-induced atom transfer radical polymerization (photo-ATRP) strategy, avoids the use of heavy metal ion catalysts in the traditional ATRP reaction, does not need to recover and remove heavy metal ions, overcomes the interference of the inherent color of the heavy metal ions on the detection result, does not generate biological toxicity, and is more environment-friendly.
Drawings
FIG. 1 is a schematic diagram of the detection method of the present invention.
FIG. 2A shows electrochemiluminescence intensities under different conditions. Wherein curve a is MPA free, curve b is Ab1 free, curve c is CYFRA 21-1 free, curve d is Ab2 free, curve e is NAS free, curve f is no light, curve g is EY free, curve h is Me 6 TREN free, and curve i is luminol/NAS/Ab 2/CYFRA 21-1/Ab1/MPA/Au.
Fig. 2B is an evolution process of an impedance curve of the electrode (curve a→h) after each step modification from the bare gold electrode, and the inset is an equivalent circuit diagram of the EIS. R s is solution resistance, CPE is constant phase element, Z w is Warburg impedance and R ct is charge transfer resistance.
FIG. 2C is a CV curve of the electrode (curve a.fwdarw.h) after each step modification from the bare gold electrode.
FIG. 3 is an atomic force microscope photograph of the electrode before and after the surface modification of NAS. Wherein A is before modification of NAS, and B is after modification of NAS.
Fig. 4 is a photograph of contact angles of electrode surfaces in different modification states. Wherein A is before photo-ATRP and B is after photo-ATRP.
FIG. 5 shows the optimization of photo-ATRP reaction time (A), NAS concentration (B), luminol reaction time (C), luminol concentration (D).
FIG. 6 is a graph (A) of Electrochemiluminescence (ECL) intensity versus CYFRA 21-1 concentration and corresponding linear correlation curve (B).
FIG. 7A is a graph of electroluminescent intensity versus 10pg/mL CYFRA 21-1 and BSA, CEA, cTnI at the same concentration under the same detection conditions.
FIG. 7B shows signal intensities of different concentrations of CYFRA21-1 in PBS and 10% serum samples.
Detailed Description
The following describes the embodiments of the present invention in further detail with reference to examples.
Ab1, ab2 and CYFRA 21-1 are all purchased from Beijing health science and technology Co., ltd, and the product numbers are CY11N005, CY11N007 and CS-CY211 respectively.
Example 1, kit
Electrochemiluminescence detection kit based on metal-free light ATRP signal amplification strategy comprises the following raw materials: 2-bromoisobutyric acid (BMP), gold electrode, 3-mercaptopropionic acid (MPA), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), ab1, BSA, ab2, me 6 TREN, eosin Y (EY), N-acryloxysuccinimide (NAS), luminol, ultrapure water, H 2O2, dimethyl sulfoxide (DMSO), ethanol.
When the raw materials are used, a solution is prepared, wherein the concentration of BMP solution is 3mM, the concentration of MPA solution is 10mM, the concentration of NHS solution for activating BMP is 3mM, the concentration of EDC solution is 3mM, the concentrations of EDC and NHS in the EDC and NHS mixed solution for activating MPA are 0.2M and 0.05M respectively, the concentration of Ab1 solution is 1 mug/mL, the mass concentration of BSA solution is 1%, the concentration of Ab2 solution is 1 mug/mL, the concentration of Me 6 TREN solution is 10mM, the concentration of EY solution is 5mM, the concentration of NAS solution is 12mM, and the concentration of luminol solution is 15mM.
Example 2 method of Using the kit
(1) Ab2-BMP (Ab 2 x) Synthesis
① Dissolving BMP in 60% (v/v) ethanol solution to obtain 3mM BMP solution;
② To 150. Mu.L of BMP solution were added 150. Mu.L of NHS solution (3 mM) and 150. Mu.L of EDC solution (3 mM), and the mixture was stirred at 37℃for 1 hour to obtain a 1mM carboxyl-activated BMP solution;
③ The carboxyl-activated BMP solution was added to Ab2 solution (1 μg/mL) (1:1 by volume) and stirred at 37 ℃ for 1h to give Ab2 solution.
(2) Electrode pretreatment
Bare gold electrodes (diameter 2 mm) were polished to obtain polished mirror surfaces.
(3) Electrode modification
① Immersing the pretreated electrode in 150 mu L of MPA solution (10 mM) at 37 ℃ for 8 hours, washing with ultrapure water, and drying with nitrogen;
② Immersing the electrode in the step ① in 150. Mu.L of a mixed solution of EDC (0.2M) and NHS (0.05M) at 37 ℃ for 1h;
③ Directly dropping 10 mu L of Ab1 solution (1 mu g/mL) onto the surface of the electrode (MPA/Au) of the step ②, reacting for 1h in a constant-temperature incubator at 37 ℃, and then soaking for 1h in 150 mu L of 1% BSA solution;
④ Dropping 10 mu L of a sample to be tested (containing CYFRA 21-1) on the electrode surface (Ab 1/MPA/Au) of the step ③, reacting for 2 hours at 37 ℃, and washing with ultrapure water;
⑤ Dropping 10 mu L of Ab 2-x solution on the electrode surface (CYFRA 21-1/Ab 1/MPA/Au) of the step ④, and reacting for 1h at 37 ℃;
⑥ The electrode (Ab 2. Times./CYFRA 21-1/Ab 1/MPA/Au) from step ⑤ was placed in a mixed solution of 5. Mu.L Me 6 TREN solution (10 mM), 5. Mu.L EY solution (5 mM), 1mL NAS solution (12 mM), 5mL H 2 O and 3990. Mu.L DMSO, and reacted for 3 hours under 470nm light irradiation at room temperature (photo-ATRP);
⑦ The electrode of step ⑥ (NAS/Ab 2/CYFRA 21-1/Ab 1/MPA/Au) was placed in 150. Mu.L of luminol solution (15 mM) and reacted at 37℃for 1.5h;
⑧ The electrode of step ⑦ (luminol/NAS/Ab 2 x/CYFRA 21-1/Ab 1/MPA/Au) was placed in 6mL H 2O2 (10 mm, pbs buffer ph=7.4) and the luminescence intensity of luminol was measured using an MPI-E type electrochemiluminescence detector.
Example 3 feasibility verification
To demonstrate the feasibility of establishing the assay of the invention, 9 sets of blank experiments were performed and ECL (enhanced chemiluminescence) responses were recorded. As shown in fig. 2A, in the absence of MPA (curve a), ab1 (curve b), CYFRA 21-1 (curve c), ab2 x (curve d), NAS (curve e), light (curve f), EY (curve g), me 6 TREN (curve h) or luminol (curve i), ECL signals were all very weak, whereas a significant ECL response (curve j) was clearly observed for the fully modified electrode, and the above experimental results indicate the necessity of the presence of each step of modification and the feasibility of the method to detect CYFRA 21-1.
Example 4 characterization
Electrochemical Impedance Spectroscopy (EIS) can sensitively monitor microscopic changes from the electrode surface to the electrolyte, for verification of each step of electrode modification. As shown in fig. 2B, R ct is relatively low (-240 Ω) when the electrode is unmodified; when the small molecule MPA is combined with the gold electrode, R ct is increased (401 omega), and a layer of film is formed on the surface of the electrode; after Ab1, BSA, CYFRA21-1 and Ab2 modifications, R ct continues to rise, which is related to the continual thickening of the previously formed membrane by the macromolecular structure of the protein. After photo-ATRP, R ct increased significantly (-5726Ω), which is the result of the successful grafting of large amounts of NAS onto initiator to form polymer chains. Finally, when the last step is completed, R ct increases further (-6875Ω) because of the increased space resistance of the luminol connection. These results indicate that each connection of the present invention was successful.
The surface characteristics of each fixation step of the electrode were monitored using Cyclic Voltammetry (CV) (fig. 2C). When MPA is connected to a bare electrode by self-assembly, the peak current is reduced compared to a bare gold electrode. In addition, when Ab1, BSA, CYFRA21-1, ab2, NAS, and luminol were modified in order on the electrode surface, the peak current was decreased in order, which was consistent with the EIS results described above. The results show that the preparation of the full electrode of the invention was successful.
The electrode surfaces were characterized by Atomic Force Microscopy (AFM) and Water Contact Angle (WCA). The surface thicknesses of the front and rear electrodes of the modified NAS were 12.2nm and 30.5nm, respectively, indicating the success of the photo-ATRP reaction (FIG. 3). The wettability of the electrode surface varies with the reaction and can therefore be characterized by WCA (fig. 4). The WCA was 96.6℃before polymerization of FIG. 4A and 99.9℃after polymerization of FIG. 4B. These characterization results demonstrate the success of the full electrode construction of the present invention.
Example 5 detection Condition optimization
In order to increase the sensitivity of the detection, the present invention investigated the effect of photo-ATRP reaction time and NAS concentration, and luminol reaction time and concentration on the reaction.
(1) Optimization of the photo-ATRP reaction time
FIG. 5A shows that ECL signal increases almost linearly with reaction time over the first 150 min. Gradually, the growth rate slows down and eventually reaches zero after 180min. This is due to the effects of poor electron transfer efficiency or radical termination. Thus, the optimal reaction polymerization time for photo-ATRP was 180min.
(2) NAS concentration optimization
FIG. 5B shows the variation of NAS concentration and luminescence intensity, the electroluminescent signal increases gradually with concentration and reaches a maximum at 12mM, confirming that NAS is saturated. Thus, 12mM was chosen as the optimal concentration for NAS.
(3) Reaction time and concentration of luminol
As shown in fig. 5C, the electroluminescent signal gradually increased with the reaction time of luminol within 60min and stopped increasing at 90min. Thus, the optimal time for the luminol reaction was chosen to be 90min. FIG. 5D shows that the electroluminescent signal increases with increasing luminol concentration within 15mM. Subsequently, with further increases in luminol concentration, the electroluminescent signal gradually becomes smooth. Thus, the concentration of luminol in the experiment was set at 15mM.
Example 6 analytical performance
Under optimal conditions, the limit of detection (LOD) and linear response range of CYFRA 21-1 detection kit were assessed by ECL. As shown in FIG. 6A, the ECL signal increases linearly when the concentration of CYFRA 21-1 is within the interval of 10fg/mL-1 ng/mL. FIG. 6B further shows that there is a positive correlation between the logarithmic value of I ECL and the CYFRA 21-1 concentration. The corresponding linear equation is i=1458 log (C CYFRA 21-1/pg mL-1)+3320(R2 =0.9914), and the calculated LOD is 5.8fg/mL (S/n=3).
Compared with other methods, the method provided by the invention for detecting CYFRA 21-1 has relatively low detection limit (table below) due to the combination of the sensitivity of ECL and the advantages of no metal light ATRP signal amplification. This shows that the kit has potential application value in early detection of lung cancer.
Example 7: selectivity, tamper resistance and stability of electroluminescent sensor
To verify the selectivity of this signal amplification method, the present invention selects different detection substances including cardiac troponin I (cTnI, 10 pg/mL), carcinoembryonic antigen (CEA, 10 pg/mL) and BSA (10 pg/mL) and performs a selectivity test under the same experimental conditions (CYFRA 21-1, 10 pg/mL). As can be seen from FIG. 7, the optical signal intensities of BSA, CEA and cTnI were 6.16%, 6.78% and 9.61% of CYFRA21-1, respectively. Because Ab1 on the electrode can be specifically recognized by CYFRA21-1, this allows Ab1 to be recognized and bind to CYFRA21-1. This resulted in a difference in the binding efficiency of different antigens to Ab1 in the experiment, indicating that the method of the present invention is effective in distinguishing CYFRA21-1.
To verify the stability of this signal amplification method, 5 fully modified electrodes (luminol/NAS/Ab 2 x/CYFRA 21-1/Ab 1/MPA/Au) were selected and stored in a refrigerator at 4 ℃. After three weeks, the ECL signal intensity can reach about 91% of the original signal. These results indicate that the electrode (luminol/NAS/Ab 2 x/CYFRA 21-1/Ab 1/MPA/Au) has good stability during storage.
To test the anti-interference effect of the kit, ECL was used to detect different concentrations of CYFRA21-1 (1 ng/mL, 10pg/mL, 100 fg/mL) in PBS and 10% serum samples. As shown in FIG. 7B, the signal intensities of 1ng/mL, 10pg/mL, 100fg/mL CYFRA21-1 in the 10% serum samples were 96.89%, 96.28% and 82.63% in PBS, respectively. These results indicate that the method of the invention has good anti-interference capability in the serum of complex patients and has remarkable clinical application potential.
Claims (8)
1. An electrochemiluminescence lung cancer detection kit based on a metal-free light ATRP signal amplification strategy is characterized by comprising the following raw materials: BMP, gold electrode, MPA, EDC, NHS, ab, BSA, ab2, me 6 TREN, EY, NAS, luminol, ultrapure water, H 2O2, DMSO, and ethanol;
When the raw materials are used, the raw materials are prepared into a solution, wherein the concentration of the BMP solution is 3mM, the concentration of the MPA solution is 10mM, the concentration of the NHS solution for activating BMP is 3mM, the concentration of the EDC solution is 3mM, the concentrations of EDC and NHS in the EDC and NHS mixed solution for activating MPA are 0.2M and 0.05M respectively, the concentration of the Ab1 solution is 1 mug/mL, the concentration of the BSA solution is 1%, the concentration of the Ab2 solution is 1 mug/mL, the concentration of the Me 6 TREN solution is 10mM, the concentration of the EY solution is 5mM, the concentration of the NAS solution is 12 mM, and the concentration of the luminol solution is 15 mM.
2. A method of using the electrochemiluminescent lung cancer detection kit according to claim 1, comprising the steps of:
(1) Ab2-BMP (Ab 2 x) Synthesis
① Dissolving BMP in ethanol solution to obtain BMP solution;
② Adding an NHS solution and an EDC solution into the BMP solution, and stirring to obtain a carboxyl activated BMP solution;
③ Adding the carboxyl activated BMP solution into the Ab2 solution, and stirring to obtain Ab2 solution;
(2) Electrode pretreatment
Polishing the bare gold electrode to obtain a polished mirror surface;
(3) Electrode modification
① Soaking the pretreated electrode in MPA solution, washing and drying;
② Immersing the electrode in the step ① in a mixed solution of EDC and NHS;
③ Directly dripping Ab1 solution on the surface of the electrode in the step ②, reacting, and then soaking in BSA solution;
④ Dropping a sample to be detected on the surface of the electrode in the step ③, reacting and washing;
⑤ Dropping Ab 2-x solution on the surface of the electrode in the step ④ for reaction;
⑥ Placing the electrode in the step ⑤ in a mixed solution consisting of Me 6 TREN solution, EY solution, NAS solution, H 2 O and DMSO for reaction;
⑦ Placing the electrode in the ⑥ step in a luminol solution for reaction;
⑧ The electrode of step ⑦ was placed in H 2O2 to measure the luminescence intensity of luminol.
3. The method of using an electrochemiluminescence lung cancer detection kit according to claim 2, wherein the concentration of the ethanol solution in the step (1) is 50% -80% (v/v); the volume ratio of BMP solution, NHS solution and EDC solution is 1:1:1; the concentration of the carboxyl-activated BMP solution was 1mM, the concentration of the Ab2 solution was 1. Mu.g/mL, and the volume ratio of the carboxyl-activated BMP solution to the Ab2 solution was 1:1.
4. The method of claim 2, wherein the stirring temperature in step (1) is 37 ℃ and the time is 1-3 h.
5. The method of using an electrochemiluminescence lung cancer detection kit according to claim 2, wherein the soaking temperature of ① in the step (3) is 37 ℃ and the time is 2-8 h; ② The soaking temperature is 37 ℃ and the time is 1-2 h; ③ The reaction temperature is 37 ℃ and the time is 1-3 h; ④ The reaction temperature is 37 ℃ and the time is 1-3 h; ⑤ The reaction temperature is 37 ℃ and the time is 1-3 h; ⑥ The reaction condition is room temperature, 470 nm light irradiation, 2-5 h; ⑦ The reaction temperature is 37 ℃ and 1-3 h.
6. The method of claim 2, wherein the volume ratio of Me 6 TREN solution, EY solution, NAS solution, H 2 O, and DMSO is 5:5:1000:5000:3990.
7. Use of the detection kit of claim 1 in CYFRA21-1 detection.
8. Use of the detection kit according to claim 1 for the preparation of a lung cancer detection product.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210016767.8A CN114354583B (en) | 2022-01-07 | 2022-01-07 | Electrochemiluminescence lung cancer detection kit based on metal-free light ATRP signal amplification strategy, and use method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210016767.8A CN114354583B (en) | 2022-01-07 | 2022-01-07 | Electrochemiluminescence lung cancer detection kit based on metal-free light ATRP signal amplification strategy, and use method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114354583A CN114354583A (en) | 2022-04-15 |
| CN114354583B true CN114354583B (en) | 2024-06-07 |
Family
ID=81106490
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210016767.8A Active CN114354583B (en) | 2022-01-07 | 2022-01-07 | Electrochemiluminescence lung cancer detection kit based on metal-free light ATRP signal amplification strategy, and use method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114354583B (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013028756A1 (en) * | 2011-08-22 | 2013-02-28 | Carnegie Mellon University | Atom transfer radical polymerization under biologically compatible conditions |
| CN105461850A (en) * | 2015-12-27 | 2016-04-06 | 湘潭大学 | Method for synthesizing nanometer carrier material pH response copolymer through catalysis of photoinduced trace copper |
| CN110507633A (en) * | 2019-08-25 | 2019-11-29 | 南京理工大学 | Respond pH and the drug gene carrier of ultraviolet light and preparation method thereof |
| CN110596407A (en) * | 2019-11-04 | 2019-12-20 | 鲁东大学 | Preparation method for constructing electrochemical immunosensor based on mussel-like chemistry |
| CN111175507A (en) * | 2020-03-13 | 2020-05-19 | 河南中医药大学 | Lung cancer early diagnosis kit based on signal amplification of ring-opening polymerization reaction initiated by hydroxyl functionalized graphene oxide |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20110306148A1 (en) * | 2010-06-14 | 2011-12-15 | Siemens Healthcare Diagnostics Inc. | Composition for use as an assay reagent |
| CN110628415B (en) * | 2019-09-23 | 2023-03-14 | 济南大学 | Preparation and application of aptamer-coated luminol hollow porous silica material |
-
2022
- 2022-01-07 CN CN202210016767.8A patent/CN114354583B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013028756A1 (en) * | 2011-08-22 | 2013-02-28 | Carnegie Mellon University | Atom transfer radical polymerization under biologically compatible conditions |
| CN105461850A (en) * | 2015-12-27 | 2016-04-06 | 湘潭大学 | Method for synthesizing nanometer carrier material pH response copolymer through catalysis of photoinduced trace copper |
| CN110507633A (en) * | 2019-08-25 | 2019-11-29 | 南京理工大学 | Respond pH and the drug gene carrier of ultraviolet light and preparation method thereof |
| CN110596407A (en) * | 2019-11-04 | 2019-12-20 | 鲁东大学 | Preparation method for constructing electrochemical immunosensor based on mussel-like chemistry |
| CN111175507A (en) * | 2020-03-13 | 2020-05-19 | 河南中医药大学 | Lung cancer early diagnosis kit based on signal amplification of ring-opening polymerization reaction initiated by hydroxyl functionalized graphene oxide |
Non-Patent Citations (3)
| Title |
|---|
| Application of peptide nucleic acid in electrochemical nucleic acid biosensors;Haobo Sun 等;《Biopolymers》;20210702;第112卷(第12期);第1-12页 * |
| Metal-free Photochemically Mediated ATRP for Ultrasensitive Quantification of CYFRA 21–1 for Detection of Early Stage Non-Small Cell Lung Cancer;Fuchun Si 等;《Journal of The Electrochemical Society》;20220928;第169卷(第09期);第097509页 * |
| 血清CYFRA21-1、CEA联合MMP-1和SAA在非小细胞肺癌检测中的价值分析;翟志强;《标记免疫分析与临床》;20180825;第25卷(第08期);第1159-1163，1195页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114354583A (en) | 2022-04-15 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Liu et al. | Three-dimensional electrochemical immunosensor for sensitive detection of carcinoembryonic antigen based on monolithic and macroporous graphene foam | |
| Xiang et al. | A redox cycling-amplified electrochemical immunosensor for α-fetoprotein sensitive detection via polydopamine nanolabels | |
| Wang et al. | Electrogenerated chemiluminescence determination of C-reactive protein with carboxyl CdSe/ZnS core/shell quantum dots | |
| Wang et al. | Sensitive and selective “signal-off” electrochemiluminescence sensing of prostate-specific antigen based on an aptamer and molecularly imprinted polymer | |
| Zhong et al. | Dual-wavelength responsive photoelectrochemical aptasensor based on ionic liquid functionalized Zn-MOFs and noble metal nanoparticles for the simultaneous detection of multiple tumor markers | |
| CN111004836B (en) | Bidirectional amplification ratio type electrochemical aptamer sensor and application thereof | |
| CN110823977B (en) | Hg detection method2+Preparation method of self-enhanced electrochemiluminescence aptamer sensor | |
| Chen et al. | Sensitive detection of enrofloxacin using an electrochemiluminescence immunosensor based on gold-functionalized C 60 and Au@ BSA nanoflowers | |
| Huang et al. | Electrochemical immunosensor detection for lactoferrin in milk powder | |
| CN114441616B (en) | Method for modifying new coronavirus biological probe on electrochemical biosensor | |
| Alizadeh et al. | Molecularly imprinted polymer specific to creatinine complex with copper (II) ions for voltammetric determination of creatinine | |
| CN114354583B (en) | Electrochemiluminescence lung cancer detection kit based on metal-free light ATRP signal amplification strategy, and use method and application thereof | |
| US10942127B2 (en) | Method for measuring concentration of test substance, and detection apparatus | |
| Xu et al. | Macroinitiator triggered polymerization for versatile immunoassay | |
| Wang et al. | Peroxydisulfate/oxygen system-based electrochemiluminescent immunosensing of Hg 2+ using Pt/Pd nanodendrites-thiosemicarbazide/norfloxacin as a signal enhancer | |
| CN115032247B (en) | Preparation method of electrochemical luminescence immunosensor based on boron fluoride (BODIPY) luminophor | |
| CN111175507A (en) | Lung cancer early diagnosis kit based on signal amplification of ring-opening polymerization reaction initiated by hydroxyl functionalized graphene oxide | |
| CN110257519A (en) | A kind of lung cancer early diagnosis kit causing the amplification of atom transition free radical polymerization reaction signal based on Nafion | |
| Cui et al. | Electrochemiluminescence resonance energy transfer between Ru (bpy) 32+@ Cu3 (HHTP) 2 and GO-Au composites for C-reactive protein detection | |
| CN110687178B (en) | Mycobacterium tuberculosis CFP-10 antigen immunosensor and preparation method and application thereof | |
| Akram et al. | Signal generation at an electrochemical immunosensor via the direct oxidation of an electroactive label | |
| CN113899796A (en) | High-sensitivity detection of Pb2+Ratiometric electrochemical sensors and their use | |
| CN112521930A (en) | Electrochemiluminescence immunosensor | |
| Meng et al. | Adriamycin coated silica microspheres as labels for cancer biomarker alpha-fetoprotein detection | |
| KR20200131275A (en) | Electrochemical sensor |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant |