CN113552364B - High-frequency piezoelectric quartz crystal sensing system for detecting myocardial infarction marker and detection method thereof - Google Patents
High-frequency piezoelectric quartz crystal sensing system for detecting myocardial infarction marker and detection method thereof Download PDFInfo
- Publication number
- CN113552364B CN113552364B CN202110615055.3A CN202110615055A CN113552364B CN 113552364 B CN113552364 B CN 113552364B CN 202110615055 A CN202110615055 A CN 202110615055A CN 113552364 B CN113552364 B CN 113552364B
- Authority
- CN
- China
- Prior art keywords
- aptamer
- quartz crystal
- modified
- wafer
- frequency
- 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
- 239000013078 crystal Substances 0.000 title claims abstract description 129
- 239000010453 quartz Substances 0.000 title claims abstract description 120
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicon dioxide Inorganic materials O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 title claims abstract description 120
- 238000001514 detection method Methods 0.000 title claims abstract description 77
- 208000010125 myocardial infarction Diseases 0.000 title claims abstract description 32
- 239000003550 marker Substances 0.000 title claims description 17
- 239000011324 bead Substances 0.000 claims abstract description 53
- 238000000034 method Methods 0.000 claims abstract description 49
- 108010062374 Myoglobin Proteins 0.000 claims abstract description 45
- 102000036675 Myoglobin Human genes 0.000 claims abstract description 45
- 102000013394 Troponin I Human genes 0.000 claims abstract description 39
- 108010065729 Troponin I Proteins 0.000 claims abstract description 39
- 108091023037 Aptamer Proteins 0.000 claims description 124
- 235000012431 wafers Nutrition 0.000 claims description 93
- 239000000243 solution Substances 0.000 claims description 66
- 239000007853 buffer solution Substances 0.000 claims description 59
- 239000000523 sample Substances 0.000 claims description 54
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 30
- 238000006243 chemical reaction Methods 0.000 claims description 27
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 claims description 24
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 24
- 238000007789 sealing Methods 0.000 claims description 24
- 238000004140 cleaning Methods 0.000 claims description 23
- 238000011010 flushing procedure Methods 0.000 claims description 20
- WYTZZXDRDKSJID-UHFFFAOYSA-N (3-aminopropyl)triethoxysilane Chemical compound CCO[Si](OCC)(OCC)CCCN WYTZZXDRDKSJID-UHFFFAOYSA-N 0.000 claims description 15
- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 claims description 15
- HXLAEGYMDGUSBD-UHFFFAOYSA-N 3-[diethoxy(methyl)silyl]propan-1-amine Chemical compound CCO[Si](C)(OCC)CCCN HXLAEGYMDGUSBD-UHFFFAOYSA-N 0.000 claims description 15
- 238000001035 drying Methods 0.000 claims description 15
- 238000002347 injection Methods 0.000 claims description 15
- 239000007924 injection Substances 0.000 claims description 15
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 15
- 229960002685 biotin Drugs 0.000 claims description 13
- 239000011616 biotin Substances 0.000 claims description 13
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 12
- 235000020958 biotin Nutrition 0.000 claims description 12
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 12
- 229910052757 nitrogen Inorganic materials 0.000 claims description 12
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 claims description 12
- 230000005284 excitation Effects 0.000 claims description 10
- 238000011896 sensitive detection Methods 0.000 claims description 10
- 238000005406 washing Methods 0.000 claims description 10
- FALRKNHUBBKYCC-UHFFFAOYSA-N 2-(chloromethyl)pyridine-3-carbonitrile Chemical compound ClCC1=NC=CC=C1C#N FALRKNHUBBKYCC-UHFFFAOYSA-N 0.000 claims description 9
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 claims description 9
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 claims description 9
- 239000002904 solvent Substances 0.000 claims description 9
- 229940014800 succinic anhydride Drugs 0.000 claims description 9
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 claims description 8
- 230000004913 activation Effects 0.000 claims description 7
- 230000027455 binding Effects 0.000 claims description 7
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 6
- 241000252506 Characiformes Species 0.000 claims description 6
- 239000002253 acid Substances 0.000 claims description 6
- 230000003213 activating effect Effects 0.000 claims description 6
- 239000002981 blocking agent Substances 0.000 claims description 6
- 229940098773 bovine serum albumin Drugs 0.000 claims description 6
- 239000000872 buffer Substances 0.000 claims description 6
- 239000003795 chemical substances by application Substances 0.000 claims description 6
- 235000019441 ethanol Nutrition 0.000 claims description 6
- 238000010438 heat treatment Methods 0.000 claims description 6
- 238000007885 magnetic separation Methods 0.000 claims description 6
- 229960005322 streptomycin Drugs 0.000 claims description 6
- 239000000126 substance Substances 0.000 claims description 6
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 6
- 239000012498 ultrapure water Substances 0.000 claims description 6
- 230000009471 action Effects 0.000 claims description 5
- 230000008569 process Effects 0.000 claims description 5
- 108010090804 Streptavidin Proteins 0.000 claims description 4
- 239000003446 ligand Substances 0.000 claims description 4
- 239000007987 MES buffer Substances 0.000 claims description 3
- 239000008367 deionised water Substances 0.000 claims description 3
- 229910021641 deionized water Inorganic materials 0.000 claims description 3
- 238000000151 deposition Methods 0.000 claims description 3
- 238000007599 discharging Methods 0.000 claims description 3
- 238000002386 leaching Methods 0.000 claims description 3
- 239000011259 mixed solution Substances 0.000 claims description 3
- 230000010355 oscillation Effects 0.000 claims description 3
- 239000002245 particle Substances 0.000 claims description 3
- 230000006641 stabilisation Effects 0.000 claims description 3
- 238000011105 stabilization Methods 0.000 claims description 3
- 238000010025 steaming Methods 0.000 claims description 3
- 239000011550 stock solution Substances 0.000 claims description 3
- QAOWNCQODCNURD-UHFFFAOYSA-N sulfuric acid Substances OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 3
- 238000009210 therapy by ultrasound Methods 0.000 claims description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 3
- 150000001875 compounds Chemical class 0.000 claims description 2
- 238000011534 incubation Methods 0.000 claims description 2
- 230000035484 reaction time Effects 0.000 claims description 2
- 230000001678 irradiating effect Effects 0.000 claims 1
- 230000035945 sensitivity Effects 0.000 abstract description 9
- 238000005516 engineering process Methods 0.000 abstract description 6
- 238000003380 quartz crystal microbalance Methods 0.000 abstract description 6
- 230000009871 nonspecific binding Effects 0.000 abstract description 4
- 230000009870 specific binding Effects 0.000 abstract description 4
- 210000002966 serum Anatomy 0.000 description 9
- 238000002360 preparation method Methods 0.000 description 6
- 238000012360 testing method Methods 0.000 description 5
- 206010000891 acute myocardial infarction Diseases 0.000 description 4
- 238000004401 flow injection analysis Methods 0.000 description 4
- 108091008104 nucleic acid aptamers Proteins 0.000 description 4
- 108020004414 DNA Proteins 0.000 description 3
- 102000004903 Troponin Human genes 0.000 description 3
- 108090001027 Troponin Proteins 0.000 description 3
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 239000002131 composite material Substances 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000004811 liquid chromatography Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000002198 surface plasmon resonance spectroscopy Methods 0.000 description 2
- 238000010998 test method Methods 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108091034117 Oligonucleotide Proteins 0.000 description 1
- 101100537532 Rattus norvegicus Tnni3 gene Proteins 0.000 description 1
- 108020004682 Single-Stranded DNA Proteins 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000000747 cardiac effect Effects 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000006073 displacement reaction Methods 0.000 description 1
- 239000012154 double-distilled water Substances 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 150000007523 nucleic acids Chemical group 0.000 description 1
- UWJJYHHHVWZFEP-UHFFFAOYSA-N pentane-1,1-diol Chemical compound CCCCC(O)O UWJJYHHHVWZFEP-UHFFFAOYSA-N 0.000 description 1
- 238000007747 plating Methods 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 238000002331 protein detection Methods 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000002444 silanisation Methods 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 239000013076 target substance Substances 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N5/00—Analysing materials by weighing, e.g. weighing small particles separated from a gas or liquid
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/795—Porphyrin- or corrin-ring-containing peptides
- G01N2333/805—Haemoglobins; Myoglobins
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Immunology (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Hematology (AREA)
- Physics & Mathematics (AREA)
- General Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Pathology (AREA)
- General Health & Medical Sciences (AREA)
- Analytical Chemistry (AREA)
- Biomedical Technology (AREA)
- Urology & Nephrology (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a high-frequency piezoelectric quartz crystal sensing system for detecting myocardial infarction markers and a detection method thereof. The high-frequency single-sided electrodeless quartz crystal microbalance for improving the fundamental frequency of the quartz crystal by thinning the crystal is used for realizing high-sensitivity detection of a detection object, and the sensitivity is further improved by combining a magnetic bead enrichment technology under an alternating magnetic field. In addition, the resonance field amplitude modulation method is used for distinguishing nonspecific binding and specific binding, so that high-sensitivity and high-selectivity rapid detection of troponin I and myoglobin is realized.
Description
Technical Field
The invention belongs to the technical field of sensing detection, and particularly relates to a high-frequency piezoelectric quartz crystal sensing system for detecting myocardial infarction markers and a detection method thereof.
Background
Nucleic acid aptamers (aptamers) are oligonucleotide sequences or short polypeptides, typically single-stranded DNA or RNA sequences, selected from random single-stranded nucleic acid sequences by exponential enrichment ligand evolution techniques (SELEX), and have high specificity and high affinity for target substances. Nucleic acid aptamers, also known as chemical antibodies, have been widely used in the construction of sensors. Compared to antibodies, the nucleic acid aptamer has the following advantages: the specificity and the affinity are good; the nucleic acid aptamer is easy to synthesize, purify and modify, and can be flexibly designed according to different sensor requirements; and the product has stable property, is not easily influenced by external environment, and is easy to store.
Acute Myocardial Infarction (AMI) is one of the leading causes of morbidity and mortality worldwide. Sensitive detection of cardiac markers is important as a useful diagnostic tool for accurately diagnosing patients suffering from AMI symptoms. Optimal markers for AMI include troponin I (cTnI) or myoglobin (Mb). Several conventional methods have been used to detect troponin I or myoglobin, such as mass spectrometry, liquid chromatography, electrochemical and Surface Plasmon Resonance (SPR), and the like. Most of these methods show high sensitivity, but these methods are time consuming and require expensive equipment and thus cannot be applied to point of care (POC) testing.
Most of existing quartz crystal microbalance biosensors based on an aptamer use quartz crystals with gold-plated surfaces, on one hand, the quartz wafers are easy to wear, and the cost is increased; on the other hand, the mass sensitivity of the crystal is proportional to the square of the fundamental frequency of the crystal, which is inversely proportional to the thickness of the crystal, so that gold plating on the bare quartz surface reduces the sensitivity of the quartz crystal to some extent. In addition, most aptamer-based quartz crystal microbalance biosensors are amplified by DNA hybridization, enzyme-catalyzed substrate, magnetic bead enrichment, etc. to improve sensitivity, but the reaction involving DNA, enzyme, etc. requires strict control of conditions such as temperature and pH required for the reaction, whereas the bead enrichment technique based on the incremental method requires an additional washing step to remove unbound magnetic beads, adding a lot of additional time to make it impossible to detect in real time in a short time.
Existing aptamer-based quartz crystal microbalance biosensors generally employ a flow injection method to increase the probability of reaction occurrence, so as to detect low-concentration objects to be detected as much as possible, however, the flow injection method is very strict on experimental conditions, for example, bubbles and flow velocity changes generated in the detection have great influence on frequency response, so that detection failure is caused, and the amount of consumed objects to be detected required by the flow injection method is large. However, some quartz crystal microbial sensors which do not use a flow injection mode have limited detection limits, and can only detect high-concentration objects to be detected, mainly because biomolecules slowly move in a solution and most of the biomolecules cannot be combined with receptors on the surface of the crystals.
The invention is based on the aptamer detection troponin I or myoglobin as an example, realizes the sensitive detection of the detection object by using a high-frequency single-sided electrodeless quartz crystal microbalance, and further improves the sensitivity by combining an alternating magnetic field to increase the combination probability of the detection object and the probe and a magnetic bead enrichment technology. In addition, by utilizing a resonance field amplitude modulation method to distinguish nonspecific binding and specific binding, a high-sensitivity and high-selectivity biosensor is established, so that the high-efficiency and rapid protein detection can be realized.
Disclosure of Invention
The invention aims to provide a high-frequency piezoelectric quartz crystal sensing system for detecting myocardial infarction markers, which has high sensitivity, good biological selectivity and high detection speed, and a detection method thereof.
The invention relates to a high-frequency piezoelectric quartz crystal sensing system for detecting myocardial infarction markers, which comprises a system control and data acquisition device, a DDS signal generator, an electrodeless quartz crystal vibration starting coupler, a phase-sensitive detection detector, a temperature control device, an automatic sample injection device and a high-frequency single-sided electrodeless aptamer piezoelectric quartz crystal sensor, wherein the system control and data acquisition device is used for acquiring myocardial infarction markers; the system control and data acquisition device is respectively connected with the temperature control device, the automatic sample injection device, the DDS signal generator and the phase sensitive detector; the DDS signal generator is interconnected with the electrodeless quartz crystal vibration starting coupler;
the high-frequency single-sided electrodeless aptamer piezoelectric quartz crystal sensor comprises a detection tank, a heating device fixed in the detection tank, a modified high-frequency single-sided electrodeless piezoelectric quartz crystal fixed in the detection tank, and a magnetic field generating device positioned outside the detection tank; the detection pool is provided with a sample inlet and a sample outlet, the sample inlet is connected with an automatic sample injection device, the heating device is connected with a temperature control device, an excitation coil of the modified high-frequency single-sided electrodeless piezoelectric quartz crystal is connected with an electrodeless quartz crystal vibration starting coupler, and a receiving coil is connected with a phase-sensitive detector; the magnetic field generating device is connected with the system control and data acquisition device;
In the detection process, the system control and data acquisition device enables the DDS signal generator to generate an excitation signal, the excitation signal acts on the modified high-frequency single-sided electrodeless piezoelectric quartz crystal through the electrodeless quartz crystal starting coupler, and the signal is changed due to the fact that the modified high-frequency single-sided electrodeless piezoelectric quartz crystal is reacted during detection, the changed signal is detected by the phase sensitive detection detector and is transmitted to the system control and data acquisition device, and then the signal of the meter can be obtained;
The automatic sample feeding device controls the sample feeding amount and the sample feeding time, the temperature control device controls the temperature during detection, and the magnetic field generating device is used for guaranteeing the reaction process of the modified high-frequency single-sided electrodeless piezoelectric quartz crystal and the detection sample.
The detection method of the high-frequency piezoelectric quartz crystal sensor system for detecting myocardial infarction markers comprises the following steps:
1) Modifying a high-frequency single-sided electrodeless piezoelectric quartz crystal;
2) Preparing an aptamer-magnetic bead complex;
3) Preparing a standard curve: fixing the high-frequency single-sided electrodeless piezoelectric quartz crystal modified in the step 1) in a detection tank, starting a sensing system, adjusting the voltage of a DDS signal generator to be 1V, injecting buffer solution into the detection tank through an automatic sample injection device, and discharging the buffer solution after a stable baseline is obtained; then introducing the myocardial infarction marker with set concentration and the aptamer-magnetic bead compound in the step 2), generating an alternating magnetic field by a magnetic field generator to increase the reaction binding probability, wherein the reaction time is 30min, turning off the alternating magnetic field until the frequency is stable, and recording the frequency f 0 at the moment; the DDS signal generator is used for increasing the voltage to 10V, so that the vibration of the crystal surface is aggravated, nonspecific substances are removed, and after stabilization, the frequency f 1 at the moment is recorded; then the DDS signal generator is used for increasing the voltage to 20V, the occurrence frequency is greatly increased under the high pressure of 20V, the bond is broken, and after the frequency is stable, the resonance frequency f 2 after the bond is broken is recorded; calculating the frequency difference delta f=f 2-f1, obtaining a corresponding sensing signal, changing the concentration of the myocardial infarction marker, and then establishing a standard curve of the concentration and the frequency difference delta f;
4) Detecting myocardial infarction markers with unknown concentrations according to the method in the step 4) to obtain a frequency difference delta f, and then obtaining the concentrations according to a standard curve.
The myocardial infarction marker is troponin I or myoglobin.
In the step 1), 3 methods for modifying the high-frequency single-sided electrodeless piezoelectric quartz crystal are available.
The specific method for modifying the high-frequency single-sided electrodeless piezoelectric quartz crystal comprises the following steps of:
A-1, cleaning a high-frequency single-sided electrodeless piezoelectric quartz crystal in absolute ethyl alcohol, then drying by nitrogen, putting the quartz crystal in an ultraviolet irradiation cleaning machine for irradiation for 8-12 min, taking out the quartz crystal, then thoroughly flushing an electrode by absolute ethyl alcohol and ultrapure water, and drying by nitrogen; obtaining a processed wafer;
a-2, dripping 15-25 mu L of streptavidin solution to the wafer treated in the step A-1, and incubating at 3-5 ℃ overnight to obtain a wafer modified by streptomycin;
And A-3, dropwise adding 15-25 mu L of the aptamer I solution to the wafer modified by the streptomycin in the step A-2, and reacting for 2-4 hours to obtain the wafer modified by the aptamer, wherein: the concentration of the solution of the aptamer I is 1.5-2.5 mu M, and the aptamer I is a biotin-modified troponin I aptamer or a myoglobin aptamer; the sequence of the biotin modified troponin I aptamer is 5'-biotin-CGTGCAGTACGCCAACCTTTCTCATGCGCTGCCCCTCTTA-3'; the biotin modified myoglobin aptamer sequence is 5'-biotin-ATCCAGAGTGACGCAGCACAACGTGCAAATTATACCTGTTTTCCCCTTTTCCTACAAGTGCTATGGACACGGTGGCTTAGT-3';
A-4 flushing the wafer modified by the aptamer in the step A-3 by using a buffer solution I, then dripping 15-25 mu L of sealing agent, performing sealing reaction for 0.5-1.5 h, after sealing, cleaning the buffer solution I and double-distilled water respectively to obtain a high-frequency single-sided electrodeless piezoelectric quartz crystal of the modified probe, and then placing the high-frequency single-sided electrodeless piezoelectric quartz crystal at 4 ℃ for later use; wherein: buffer solution I is PBS buffer solution, pH is 7.4, and concentration is 10mM; the blocking agent was a 0.1wt% bovine serum albumin solution.
The specific method for modifying the high-frequency single-sided electrodeless piezoelectric quartz crystal comprises the following steps of:
B-1, treating the high-frequency single-sided electrodeless piezoelectric quartz crystal with piranha acid for 10-20 min, taking out, thoroughly flushing an electrode with absolute ethyl alcohol and ultrapure water, and drying with nitrogen to obtain a treated wafer; wherein: piranha acid is concentrated sulfuric acid, 30% h 2O2 = 3:1;
B-2, dropwise adding 15-25 mu L of 3-aminopropyl triethoxysilane (APTES) solution into the wafer treated in the step B-1, and fixing for 1-2 h; obtaining an APTES modified wafer; wherein: the concentration of the 3-aminopropyl triethoxysilane solution is 2-5wt% and the solvent is anhydrous toluene;
B-3, flushing the APTES modified wafer in the step B-2 by using a buffer solution I, and then dropwise adding 15-25 mu L of glutaraldehyde solution on the wafer for reaction for 0.5-2 h; obtaining a glutaraldehyde-treated wafer; wherein the concentration of glutaraldehyde solution is 2.5-5 wt%;
B-4 washing the step of washing the tripentaginal treated wafer with the buffer solution I, then dripping 20 mu L of 2 mu M aptamer I solution on the wafer, and reacting for 2-4 hours to obtain an aptamer modified wafer, wherein: the concentration of the solution of the aptamer II is 1.5-2.5 mu M, the aptamer II is amino modified troponin I aptamer or myoglobin aptamer, and the sequence of the amino modified troponin I aptamer is 5'-NH 2(CH2)6 -CGTGCAGTACGCCAACCTTTCTCATGCGCTGCCCCTCTTA-3'; amino-modified myoglobin aptamer sequence 5'-NH2(CH2)6-ATCCAGAGTGACGCAGCACAACGTGCAAATTATACCTGTTTTCCCCTTTTCCTACAAGTGCTATGGACACGGTGGCTTAGT-3';
Flushing the wafer modified by the aptamer in the B-4 by using a buffer solution I, then dripping 15-25 mu L of a sealing agent, performing a sealing reaction for 0.5-1.5 h, after sealing, respectively cleaning the buffer solution I and double steam to obtain a high-frequency single-sided electrodeless piezoelectric quartz crystal of the modified probe, and then placing the high-frequency single-sided electrodeless piezoelectric quartz crystal at 4 ℃ for later use; wherein: buffer solution I is PBS buffer solution, pH is 7.4, and concentration is 10mM; the blocking agent was a 0.1wt% bovine serum albumin solution.
The specific method for modifying the high-frequency single-sided electrodeless piezoelectric quartz crystal comprises the following steps of:
C-1, immersing a high-frequency single-sided electrodeless piezoelectric quartz crystal into a 1.5-2.5wt% 3-aminopropyl methyl diethoxy silane (3-APDES) solution, and immersing for 2-4 hours in an inert environment to obtain a 3-aminopropyl methyl diethoxy silane treated wafer; wherein: 3-APDES is absolute ethyl alcohol;
C-2, sequentially leaching the wafer treated by the 3-aminopropyl methyl diethoxy silane in the step C-1 into the solvents according to the sequence of ethanol, ethanol/acetone (1:1 (v/v)) and acetone, and carrying out ultrasonic treatment on each group of solvents for 1-2 min; then, dropwise adding 15-20 mu L of 100mM succinic anhydride solution on the wafer, and then washing with deionized water and drying to obtain a succinic anhydride modified wafer;
C-3 adding 15-25 mu L of (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) containing 3mg/ml into the succinic anhydride modified wafer in the step C-2 for activation to obtain an activated wafer;
C-4 rinsing the activated wafer of the step C-3 by using a buffer solution I, then dripping 15-25 mu L of 2 mu M aptamer I solution to the wafer, and reacting for 2-4 hours to obtain an aptamer modified wafer, wherein: buffer III is PBS buffer, pH is 7.4, and concentration is 10mM; the aptamer III is amino modified troponin I aptamer or myoglobin aptamer, and the amino modified troponin I aptamer has a sequence of 5'-NH 2(CH2)6 -CGTGCAGTACGCCAACCTTTCTCATGCGCTGCCCCTCTTA-3'; amino-modified myoglobin aptamer sequence 5'-NH2(CH2)6-ATCCAGAGTGACGCAGCACAACGTGCAAATTATACCTGTTTTCCCCTTTTCCTACAAGTGCTATGGACACGGTGGCTTAGT-3';
C-5 flushing the aptamer modified wafer of the step C-4 by using a buffer solution I, then dripping 15-25 mu LBSA for sealing, and performing sealing reaction for 0.5-1.5 h to obtain a sealed wafer, wherein: BSA concentration was 0.1wt%;
c-6, respectively cleaning the wafers sealed in the step C-5 by using a buffer solution I and double steaming, drying by using nitrogen to obtain the high-frequency single-sided electrodeless piezoelectric quartz crystal of the modified probe, and then placing the high-frequency single-sided electrodeless piezoelectric quartz crystal at the temperature of 4 ℃ for standby.
The specific method for preparing the aptamer-magnetic bead complex in the step 2) comprises the following steps:
Step one, activating magnetic beads with carboxyl groups: adding a nano magnetic bead stock solution with carboxyl into an EP tube, adding a buffer solution II for cleaning, and activating carboxyl groups on the surface of the magnetic beads by using a (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) solution and an N-hydroxysuccinimide (NHS) solution prepared by the buffer solution II to activate the carboxyl groups on the surface of the magnetic beads, and vibrating to accelerate the activation to obtain an activated magnetic bead solution;
step two, carrying out magnetic separation and cleaning on the magnetic bead solution activated in the step one: placing a magnet at the edge of the EP tube, depositing nano magnetic beads on the tube wall of the EP tube under the action of a magnetic field, sucking out the solution by using a pipetting gun, taking care not to touch the tube wall, adding a buffer solution II, and repeating the steps for three times;
step three: adding an aptamer IV solution into the magnetic bead solution treated in the step two, incubating the mixed solution at room temperature with slight shaking, repeating the magnetic separation and cleaning operation for three times, removing the aptamer I which is not bound to NMBs, and finally suspending the magnetic bead bound with the proper ligand in 200 mu L of buffer solution I.
The buffer solution II is MES buffer solution, the pH is 5-6, and the concentration is 25-100 mM; the particle size of the nano magnetic beads in the first step is 200-300 nm, and the concentration is 5mg/ml; in the second step, the concentration of (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) is 5-10 mM, in the third step, the incubation time is 4-6 h, and the sequence of the aptamer IV is related to the sequence of the aptamer I-III:
When the sequence of the aptamer I-III is biotin or amino modified troponin I aptamer, the sequence of the corresponding aptamer IV is 5'-NH 2(CH2)6 -CGCATGCCAAACGTTGCCTCATAGTTCCCTCCCCGTGTCC-3';
When the sequence of the aptamer I-III is biotin or amino modified myoglobin, the sequence of the corresponding aptamer IV is 5'-NH 2(CH2)6 -CCCTCCTTTCCTTCGACGTAGATCTGCTGCGTTGTTCCGA-3'.
In the step 3), the sample injection amount is 50 mu L each time; the detection temperature is 25-37 ℃; setting the concentration to be 0-500 ng/mL, selecting the concentration according to the requirement, and selecting at least 5 concentrations; the magnetic field intensity is B= -0.3T.
The principle of the invention is as follows: the invention adopts a resonance field amplitude modulation technology and a direct digital frequency synthesis (DDS) technology to scan and excite the quartz crystal oscillator according to the preset frequency and sine wave amplitude, and the excitation voltage is gradually increased in the scanning process, so that the oscillation of the modified wafer is gradually aggravated, the weakly-bound substances on the surface of the wafer are removed in advance, and when the oscillation of the wafer reaches a certain degree, namely the surface reaches the maximum displacement, enough mechanical energy is generated to promote the separation of the specifically-bound substances on the surface of the wafer, thereby realizing the high specificity of the biosensor.
The magnetic field reaction device can generate an alternating magnetic field for improving sensitivity, the magnetic field reaction device generates the alternating magnetic field, the aptamer modifying the magnetic beads moves to the surface of the crystal under the action of the magnetic field so as to bind the receptor on the surface of the crystal, when the direction of the magnetic field is reversed, the aptamer which is not successfully bound can leave the surface, the vacant binding site can wait for the next round of binding, the binding probability is increased, and the problem that the slow-moving biomolecules are limited in transportation in the reaction process of binding the receptor with the surface is solved.
The invention has the beneficial effects that:
(1) The invention provides a high-frequency single-sided electrodeless aptamer piezoelectric quartz crystal sensor for detecting myocardial infarction markers, which uses a single-sided electrodeless quartz crystal wafer, takes troponin I or myoglobin aptamer as a probe, fixes the probe on the wafer through streptavidin-biotin or silanization fixation to capture troponin I or myoglobin, and then couples a 5-terminal amino-modified troponin I or myoglobin aptamer with a surface-modified carboxyl magnetic bead to form a probe-target-magnetic bead composite structure. The high-frequency single-sided electrodeless quartz crystal microbalance for improving the fundamental frequency of the quartz crystal by thinning the crystal is used for realizing high-sensitivity detection of a detection object, and the sensitivity is further improved by combining a magnetic bead enrichment technology under an alternating magnetic field. In addition, the resonance field amplitude modulation method is used for distinguishing nonspecific binding and specific binding, so that high-sensitivity and high-selectivity rapid detection of troponin I and myoglobin is realized.
(2) The invention mainly breaks through the defect that the detection protein carries out complex pretreatment on an actual sample and the repeated washing process and amplifying process interfere with reproducibility, and the required volume of the to-be-detected object is very small, thereby providing a novel, rapid, high-sensitivity and high-specificity analysis detection means for the detection of troponin I and myoglobin in the sample in practical application and having important application value for medical instant detection and on-site rapid detection.
Drawings
FIG. 1 is a schematic diagram of a high-frequency piezoelectric quartz crystal sensing system for detecting myocardial infarction markers in the present invention;
FIG. 2 is a schematic flow chart of the detection of the present invention;
FIG. 3 is a schematic diagram of the detection technique of the present invention;
FIG. 4 is a standard graph of examples 2-4;
FIG. 5 is a standard graph of examples 5-7;
The system comprises a 1-system control and data acquisition device, a 2-DDS signal generator, a 3-electrodeless quartz crystal vibration starting coupler, a 4-automatic sample injection device, a 5-phase sensitive detector, a 6-temperature control device and a 7-high-frequency single-sided electrodeless aptamer piezoelectric quartz crystal sensor; 71 detection cell, 72 modification high-frequency single-sided electrodeless piezoelectric quartz crystal, 73 magnetic field generating device, 74 heater.
Detailed Description
Example 1
The high-frequency piezoelectric quartz crystal sensing system for detecting the myocardial infarction marker is shown in the figure 1, and comprises a system control and data acquisition device 1, a DDS signal generator 2, an electrodeless quartz crystal vibration starting coupler 3, a phase-sensitive detection detector 5, a temperature control device 6, an automatic sample injection device 4 and a high-frequency single-sided electrodeless aptamer piezoelectric quartz crystal sensor 7; the system control and data acquisition device 1 is respectively connected with the temperature control device 6, the automatic sample injection device 4, the DDS signal generator 2 and the phase sensitive detection detector 5; the DDS signal generator 2 is connected with the electrodeless quartz crystal vibration starting coupler 3;
The high-frequency single-sided electrodeless aptamer piezoelectric quartz crystal sensor 7 comprises a detection cell 71, a heating device 74 fixed in the detection cell, a modified high-frequency single-sided electrodeless piezoelectric quartz crystal 72 fixed in the detection cell, and a magnetic field generating device 73 positioned outside the detection cell; the detection pool is provided with a sample inlet and a sample outlet, the sample inlet is connected with an automatic sample injection device, a heating device 74 is connected with a temperature control device 6, an excitation coil of the modified high-frequency single-sided electrodeless piezoelectric quartz crystal 72 is connected with an electrodeless quartz crystal vibration starting coupler 3, and a receiving coil is connected with a phase-sensitive detection detector 5; the magnetic field generating device 73 is connected with the system control and data acquisition device 1;
In the detection process, the system control and data acquisition device 1 enables the DDS signal generator 2 to generate an excitation signal, the excitation signal acts on the modified high-frequency single-sided electrodeless piezoelectric quartz crystal 72 through the electrodeless quartz crystal vibration starting coupler 3, and as the modified high-frequency single-sided electrodeless piezoelectric quartz crystal is subjected to reaction during detection, the signal reaction is changed, the changed signal is detected by the phase sensitive detection detector 5 and is transmitted to the system control and data acquisition device 1, and the signal of the meter can be obtained;
The automatic sample feeding device 4 controls the sample feeding amount and the sample feeding time, the temperature control device 6 controls the temperature during detection, and the magnetic field generating device 73 is used for guaranteeing the reaction process of the modified high-frequency single-sided electrodeless piezoelectric quartz crystal 72 and the detection sample.
The test flow chart of the high-frequency piezoelectric quartz crystal sensing system for detecting the myocardial infarction marker is shown in figure 3, the principle is shown in figure 3, troponin I or myoglobin is used as the myocardial infarction marker, a single-sided electrodeless quartz wafer is firstly used, troponin I or myoglobin aptamer is used as a probe, the probe is fixed on the wafer through a streptavidin and/or biotin system to capture troponin I or myoglobin, and then the troponin I or myoglobin aptamer with an amino modified at the 5-end is coupled with a magnetic bead with carboxyl so as to be combined with troponin I or myoglobin, the combination probability of an object to be detected is greatly increased (a) under the action of an external alternating magnetic field, and a probe-target-magnetic bead composite structure is formed through a double-aptamer sandwich method. The vibration of the crystal surface is aggravated by increasing the voltage within a certain range, the frequency is increased (b) in a small range under the low voltage (10V), some nonspecific adsorption is removed, the frequency is increased greatly under the higher voltage (20V) (c), and specific bonds are broken, so that the nonspecific binding and specific binding are distinguished by using a resonance field amplitude modulation method, a high-frequency technology is combined, and the high-sensitivity and high-selectivity rapid detection of troponin I or myoglobin in an actual sample can be realized according to the obtained frequency signal change. Specific probe modification and detection procedures are shown in examples 2 to 7.
Example 2
1. Modified high-frequency single-sided electrodeless piezoelectric quartz crystal
A-1, cleaning a high-frequency single-sided electrodeless piezoelectric quartz crystal in absolute ethyl alcohol, then drying by nitrogen, putting the quartz crystal in an ultraviolet irradiation cleaning machine for irradiation for 8-12 min, taking out the quartz crystal, then thoroughly flushing an electrode by absolute ethyl alcohol and ultrapure water, and drying by nitrogen; obtaining a processed wafer;
a-2 dropping 20 mu L of 2mg/mL streptavidin solution to the wafer treated in the step A-1 and incubating at 4 ℃ overnight to obtain a wafer modified by streptomycin;
A-3 dropwise adding 20 mu L of 2 mu M aptamer I to the wafer modified by streptomycin in the step A-2, and reacting for 3 hours to obtain a wafer modified by the aptamer, wherein: the aptamer I is a biotin-modified troponin I aptamer, and the specific sequence is 5'-biotin-CGTGCAGTACGCCAACCTTTCTCATGCGCTGCCCCTCTTA-3';
a-4, flushing the wafer modified by the aptamer in the step A-3 by using a buffer solution I, then dripping 20 mu L of a sealing agent, performing a sealing reaction for 1h, after sealing, respectively cleaning the buffer solution I and double steam to obtain a high-frequency single-sided electrodeless piezoelectric quartz crystal of the modified probe, and then placing the high-frequency single-sided electrodeless piezoelectric quartz crystal at 4 ℃ for later use; wherein: buffer solution I is PBS buffer solution, pH is 7.4, and concentration is10 mM; the blocking agent was a 0.1wt% bovine serum albumin solution.
2. Preparation of aptamer-magnetic beads
1) Activating the magnetic beads with carboxyl groups: placing nano magnetic bead stock solution (the particle size of the nano magnetic beads is 200-300nm, the concentration is 5 mg/mL) with carboxyl groups in an EP tube, adding buffer II for cleaning (the buffer II is MES buffer, the pH is 5-6, the concentration is 50 mM), and activating the carboxyl groups on the surfaces of the magnetic beads by using prepared (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) solutions (the concentration of both of which is 3 mM), and vibrating to accelerate the activation to obtain an activated magnetic bead solution;
2) Performing magnetic separation and cleaning on the magnetic bead solution after the activation in the step 1): placing a magnet at the edge of the EP tube, depositing nano magnetic beads on the tube wall of the EP tube under the action of a magnetic field, sucking out the solution by using a pipetting gun, taking care not to touch the tube wall, adding a buffer solution II, and repeating the steps for three times;
3): adding 20 mu L of 2 mu M aptamer IV solution (the aptamer IV is an aptamer with 5-end modified amino group and the specific sequence is 5'-NH 2(CH2)6 -CGCATGCCAAACGTTGCCTCATAGTTCCCTCCCCGTGTCC-3') into the treated magnetic bead solution in the step 2), incubating the mixed solution for 5h by slight shaking at room temperature, repeating the magnetic separation washing operation for three times, removing the aptamer II which is not bound to NMBs, and finally suspending the magnetic bead bound with the proper ligand in 200 mu L of buffer solution I.
3. Testing
Fixing the high-frequency single-sided electrodeless piezoelectric quartz crystal modified in the first step in a detection pool, starting a sensing system, adjusting the voltage of a DDS signal generator to be 1V, setting the detection temperature to be 37 ℃, injecting a buffer solution I into the detection pool through an automatic sample injection device, and discharging the buffer solution after a stable base line is obtained; then 50uL of troponin I solution with set concentration (the concentration is specifically 0, 0.5, 1.0, 2.0, 3.0, 4.0 and 5.0 ng/mL) and 50uL of the aptamer-magnetic bead complex in the second step are introduced, an alternating magnetic field (the magnetic field strength is-0.3T) is generated through a magnetic field generator to increase the reaction binding probability, the reaction lasts for 30min, and after the alternating magnetic field is turned off and the frequency is stable, the frequency f 0＝ 99998325Hz at the moment is recorded; the DDS signal generator is used for increasing the voltage to 10V, so that the vibration of the crystal surface is aggravated, the frequency change in a low-voltage area is small, non-specific substances are removed, and after stabilization, the frequency f 1 at the moment is recorded; the DDS signal generator is used for increasing the voltage to 20V, the occurrence frequency is greatly increased under higher pressure, the bond is broken, and after the bond is stabilized, the resonance frequency f 2 after the bond breaking is recorded. Calculating the frequency difference delta f=f 2-f1, obtaining a corresponding sensing signal, changing the concentration of the myocardial infarction marker, and then establishing a standard curve of the concentration and the frequency difference delta f; see in particular line a in fig. 4; the equation for obtaining the standard curve by fitting is: y=168.26 x+1030r 2 =0.989.
Serum samples with unknown concentrations are detected according to the method in the standard curve in the step, the frequency difference delta f is 1131Hz, and then the concentration of troponin I is 0.60ng/mL according to the standard curve.
Example 3
1. Modified high-frequency single-sided electrodeless piezoelectric quartz crystal
B-1, treating the high-frequency single-sided electrodeless piezoelectric quartz crystal with piranha acid for 15min, taking out, thoroughly flushing an electrode with absolute ethyl alcohol and ultrapure water, and drying with nitrogen to obtain a treated wafer; wherein: piranha acid is concentrated sulfuric acid, 30% h 2O2 = 3:1;
B-2 dropwise adding 20 mu L of 3-aminopropyl triethoxysilane (APTES) solution to the wafer treated in the step B-1, and fixing for 1h; obtaining an APTES modified wafer; wherein: the concentration of the 3-aminopropyl triethoxysilane solution is 5wt%, and the solvent is anhydrous toluene;
b-3, flushing the APTES modified wafer in the step B-2 by using a buffer solution I, and then dropwise adding 20 mu L of pentanediol solution on the wafer for reaction for 1h; obtaining a glutaraldehyde-treated wafer; wherein the glutaraldehyde solution has a concentration of 5wt%;
B-4 washing the step of washing the tripentaldehyde treated wafer with the buffer solution I, then dripping 20 mu L of 2 mu M of the aptamer II solution on the wafer, and reacting for 3 hours to obtain an aptamer modified wafer, wherein: the concentration of the aptamer II solution is 2 mu M, the aptamer II is an amino modified troponin I aptamer, and the specific sequence is as follows: 5'-NH 2(CH2)6 -CGTGCAGTACGCCAACCTTTCTCATGCGCTGCCCCTCTTA-3';
B-5, flushing the wafer modified by the aptamer in the B-4 by using a buffer solution I, then dripping 20 mu L of a sealing agent, performing a sealing reaction for 1h, after the sealing is finished, respectively cleaning the buffer solution I and double steam to obtain a high-frequency single-sided electrodeless piezoelectric quartz crystal of the modified probe, and then placing the high-frequency single-sided electrodeless piezoelectric quartz crystal at 4 ℃ for later use; wherein: buffer solution I is PBS buffer solution, pH is 7.4, and concentration is 10mM; the blocking agent was a 0.1wt% bovine serum albumin solution.
2. Preparation of aptamer-magnetic beads
Same as in example 2
Third, test
The test method is the same as in example 2, the standard curve is obtained as line B in fig. 4, and the equation for obtaining the standard curve by fitting is: y=188.69 x+1086.3r 2 =0.983.
Serum samples with unknown concentrations were tested according to the method in the standard curve described above to obtain a frequency difference Δf of 1199Hz, and then the troponin concentration was obtained according to the standard curve to be 0.60ng/mL.
Example 4
1. Modified high-frequency single-sided electrodeless piezoelectric quartz crystal
C-1, immersing a high-frequency single-sided electrodeless piezoelectric quartz crystal into a 2wt% 3-aminopropyl methyl diethoxy silane (3-APDES) solution, and immersing for 3 hours in an inert environment to obtain a 3-aminopropyl methyl diethoxy silane treated wafer; wherein: 3-APDES is absolute ethyl alcohol;
C-2, sequentially leaching the wafer treated by the 3-aminopropyl methyl diethoxy silane in the step C-1 into the solvents according to the sequence of ethanol, ethanol/acetone (1:1 (v/v)) and acetone, and carrying out ultrasonic treatment on each group of solvents for 1min; then, dropwise adding 20 mu L of 100mM succinic anhydride solution on the wafer, and then washing with deionized water and drying to obtain a succinic anhydride modified wafer;
C-3 adding 20 mu L of (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide containing 3mg/ml to the succinic anhydride modified wafer in the step C-2 for activation to obtain an activated wafer;
C-4 rinsing the activated wafer of step C-3 with buffer solution I, followed by dropping 20. Mu.L of 2. Mu.M aptamer III solution to the wafer, and reacting for 3 hours to obtain an aptamer modified wafer, wherein: buffer solution I is PBS buffer solution, pH is 7.4, and concentration is 10mM; the aptamer III is an amino modified troponin I aptamer, and the specific sequence is as follows: 5'-NH 2(CH2)6 -CGTGCAGTACGCCAACCTTTCTCATGCGCTGCCCCTCTTA-3';
C-5, flushing the aptamer modified wafer in the step C-4 by using a buffer solution I, dripping 20 mu LBSA for sealing, and performing sealing reaction for 1h to obtain a sealed wafer, wherein: BSA concentration was 0.1wt%;
c-6, respectively cleaning the wafers sealed in the step C-5 by using a buffer solution I and double steaming, drying by using nitrogen to obtain the high-frequency single-sided electrodeless piezoelectric quartz crystal of the modified probe, and then placing the high-frequency single-sided electrodeless piezoelectric quartz crystal at the temperature of 4 ℃ for standby.
2. Preparation of aptamer-magnetic beads
Same as in example 2
Third, test
The test method is the same as in example 2, and the obtained standard curve is; the standard curve obtained is shown as line C in fig. 4, and the equation for obtaining the standard curve by fitting is: y=157.02 x+1037.1, r 2 =0.990.
Serum samples with unknown concentrations are detected according to the method in the standard curve in the step, the frequency difference delta f is 1130Hz, and then the troponin concentration is 0.59ng/mL according to the standard curve.
Examples 2 to 4 were tested on identical serum samples, and the concentration differences obtained from the three were small, indicating that the method of the invention can be used for the detection of troponin in blood.
Example 5
1. Modified high-frequency single-sided electrodeless piezoelectric quartz crystal
The method of the present example for modifying a high-frequency single-sided electrodeless piezoelectric quartz crystal is the same as that of example 2, except that the aptamer I is a biotin-modified myoglobin aptamer, and the specific sequence is 5'-biotin-ATCCAGAGTGACGCAGCACAACGTGCAAATTATACCTGTTTTCCCCTTTTCCTACAAGTGCTATGGACACGGTGGCTTAGT-3'.
2. Preparation of aptamer-magnetic beads
The procedure for the aptamer-magnetic beads of this example was the same as that of example 2, except that the sequence of aptamer IV was: 5'-NH 2(CH2)6 -CCCTCCTTTCCTTCGACGTAGATCTGCTGCGTTGTTCCGA-3'.
3. Detection of
The detection method of this example is the same as that of example 2, except that the concentration of myoglobin solution of the koji is 0, 25, 75, 100, 200, 300, 400 and 500ng/mL. The standard curve is shown as line a in fig. 5, and the equation for obtaining the standard curve by fitting is: y=14.157 x+1925.4, r 2 =0.991.
Serum samples with unknown concentrations are detected according to the method in the standard curve in the step, the frequency difference delta f is 4050Hz, and then the myoglobin concentration is 150.6ng/mL according to the standard curve.
Example 6
1. Modified high-frequency single-sided electrodeless piezoelectric quartz crystal
The method of the present example for modifying a high-frequency single-sided electrodeless piezoelectric quartz crystal is the same as that of example 3, except that the aptamer II is an amino-modified myoglobin aptamer, and the specific sequence is 5'-NH2(CH2)6-ATCCAGAGTGACGCAGCACAACGTGCAAATTATACCTGTTTTCCCCTTTTCCTACAAGTGCTATGGACACGGTGGCTTAGT-3'.
2. Preparation of aptamer-magnetic beads
Same as in example 5.
3. Detection of
The detection method of this example is the same as that of example 2, except that the concentration of myoglobin solution of the koji is 0, 25, 75, 100, 200, 300, 400 and 500ng/mL. The standard curve is shown as line B in fig. 5, and the equation for obtaining the standard curve by fitting is: y=13.073 x+1516.2r 2 =0.993.
Serum samples with unknown concentrations are detected according to the method in the standard curve in the step, the frequency difference delta f is 3470Hz, and then the concentration of myoglobin is 149.5ng/mL according to the standard curve.
Example 7
1. Modified high-frequency single-sided electrodeless piezoelectric quartz crystal
The method of the present example for modifying a high-frequency single-sided electrodeless piezoelectric quartz crystal is the same as that of example 4, except that the aptamer III is an amino-modified myoglobin aptamer, and the specific sequence is 5'-NH2(CH2)6-ATCCAGAGTGACGCAGCACAACGTGCAAATTATACCTGTTTTCCCCTTTTCCTACAAGTGCTATGGACACGGTGGCTTAGT-3'.
2. Preparation of aptamer-magnetic beads
Same as in example 5.
3. Detection of
The detection method of this example is the same as that of example 2, except that the concentration of myoglobin solution of the koji is 0, 25, 75, 100, 200, 300, 400 and 500ng/mL. The standard curve is shown as line C in fig. 5, and the equation for obtaining the standard curve by fitting is: y= 10.453 ×x+1941.1, r 2 =0.991
Serum samples with unknown concentrations are detected according to the method in the standard curve in the step, the frequency difference delta f is 3512Hz, and then the myoglobin concentration is 150.3ng/mL according to the standard curve.
Examples 5 to 7 were tested on the same serum samples, and the three serum samples with the unknown concentrations were tested by further liquid chromatography with the concentration of myoglobin of 150.1ng/mL, indicating that the data of the present invention were accurate, and therefore the method of the present invention can be used for the detection of myoglobin in blood.
Claims (10)
1. The high-frequency piezoelectric quartz crystal sensing system for detecting myocardial infarction markers is characterized by comprising a system control and data acquisition device, a DDS signal generator, an electrodeless quartz crystal vibration starting coupler, a phase-sensitive detection detector, a temperature control device, an automatic sample injection device and a high-frequency single-sided electrodeless aptamer piezoelectric quartz crystal sensor; the system control and data acquisition device is respectively connected with the temperature control device, the automatic sample injection device, the DDS signal generator and the phase sensitive detector; the DDS signal generator is interconnected with the electrodeless quartz crystal vibration starting coupler; the aptamer is biotin or amino modified troponin I aptamer or myoglobin aptamer, wherein the sequence of the biotin modified troponin I aptamer is 5'-biotin-CGTGCAGTACGCCAACCTTTCTCATGCGCTGCCCCTCTTA-3'; the biotin modified myoglobin aptamer sequence is 5'-biotin-ATCCAGAGTGACGCAGCACAACGTGCAAATTATACCTGTTTTCCCCTTTTCCTACAAGTGCTATGGACACGGTGGCTTAGT-3'; the amino modified troponin I aptamer has a sequence of 5 '-NH 2(CH2)6 -CGTGCAGTACGCCAACCTTTCTCATGCGCTGCCCCTCTTA-3'; amino-modified myoglobin aptamer sequence 5'-NH2(CH2)6-ATCCAGAGTGACGCAGCACAACGTGCAAATTATACCTGTTTTCCCCTTTTCCTACAAGTGCTATGGACACGGTGGCTTAGT-3';
The high-frequency single-sided electrodeless aptamer piezoelectric quartz crystal sensor comprises a detection tank, a heating device fixed in the detection tank, a modified high-frequency single-sided electrodeless piezoelectric quartz crystal fixed in the detection tank, and a magnetic field generating device positioned outside the detection tank; the detection pool is provided with a sample inlet and a sample outlet, the sample inlet is connected with an automatic sample injection device, the heating device is connected with a temperature control device, an excitation coil of the modified high-frequency single-sided electrodeless piezoelectric quartz crystal is connected with an electrodeless quartz crystal vibration starting coupler, and a receiving coil is connected with a phase-sensitive detector; the magnetic field generating device is connected with the system control and data acquisition device;
in the detection process, the system control and data acquisition device enables the DDS signal generator to generate an excitation signal, the excitation signal acts on the modified high-frequency single-sided electrodeless piezoelectric quartz crystal through the electrodeless quartz crystal starting coupler, and the signal is changed due to the fact that the modified high-frequency single-sided electrodeless piezoelectric quartz crystal is reacted during detection, the changed signal is detected by the phase sensitive detection detector and is transmitted to the system control and data acquisition device, and then the signal of the meter can be obtained;
The automatic sample feeding device controls the sample feeding amount and the sample feeding time, the temperature control device controls the temperature during detection, and the magnetic field generating device is used for guaranteeing the reaction process of the modified high-frequency single-sided electrodeless piezoelectric quartz crystal and the detection sample.
2. The detection method of a high-frequency piezoelectric quartz crystal sensor system for detecting myocardial infarction markers as set forth in claim 1, comprising the steps of:
1) Modifying a high-frequency single-sided electrodeless piezoelectric quartz crystal;
2) Preparing an aptamer-magnetic bead complex;
3) Preparing a standard curve: fixing the high-frequency single-sided electrodeless piezoelectric quartz crystal modified in the step 1) in a detection tank, starting a sensing system, adjusting the voltage of a DDS signal generator to be 1V, injecting buffer solution into the detection tank through an automatic sample injection device, and discharging the buffer solution after a stable baseline is obtained; then introducing the myocardial infarction marker with set concentration and the aptamer-magnetic bead compound in the step 2), generating an alternating magnetic field by a magnetic field generator to increase the reaction binding probability, wherein the reaction time is 30min, turning off the alternating magnetic field until the frequency is stable, and recording the frequency f 0 at the moment; the DDS signal generator is used for increasing the voltage to 10V, so that the vibration of the crystal surface is aggravated, nonspecific substances are removed, and after stabilization, the frequency f 1 at the moment is recorded; then the DDS signal generator is used for increasing the voltage to 20V, the occurrence frequency is greatly increased under the high pressure of 20V, the bond is broken, and after the frequency is stable, the resonance frequency f 2 after the bond is broken is recorded; calculating the frequency difference delta f=f 2-f1, obtaining a corresponding sensing signal, changing the concentration of the myocardial infarction marker, and then establishing a standard curve of the concentration and the frequency difference delta f;
4) Detecting myocardial infarction markers with unknown concentrations according to the method in the step 3) to obtain a frequency difference delta f, and then obtaining the concentrations according to a standard curve.
3. The method for detecting a myocardial infarction marker by using the high frequency piezoelectric quartz crystal sensor system as set forth in claim 2, wherein the myocardial infarction marker is troponin I or myoglobin.
4. The method for detecting a high-frequency piezoelectric quartz crystal sensor system for detecting myocardial infarction as set forth in claim 3, wherein in said step 1), there are 3 methods of modifying a high-frequency single-sided electrodeless piezoelectric quartz crystal.
5. The method for detecting a myocardial infarction marker by using a high frequency piezoelectric quartz crystal sensor system as set forth in claim 4, wherein the method for modifying a high frequency single-sided electrodeless piezoelectric quartz crystal comprises the steps of:
A-1, cleaning a high-frequency single-sided electrodeless piezoelectric quartz crystal in absolute ethyl alcohol, then drying by nitrogen, putting the quartz crystal in an ultraviolet irradiation cleaning machine, irradiating for 8-12 min, taking out, then thoroughly flushing an electrode by absolute ethyl alcohol and ultrapure water, and drying by nitrogen; obtaining a processed wafer;
C, dripping 15-25 mu L of streptavidin solution on the wafer treated in the step A-1, and incubating at 3-5 ℃ overnight to obtain a wafer modified by streptomycin;
And A-3, dropwise adding 15-25 mu L of aptamer I solution to the wafer modified by the streptomycin in the step A-2, and reacting for 2-4 hours to obtain the wafer modified by the aptamer, wherein: the concentration of the solution of the aptamer I is 1.5-2.5 mu M, and the aptamer I is a biotin-modified troponin I aptamer or a myoglobin aptamer; the sequence of the biotin modified troponin I aptamer is 5'-biotin-CGTGCAGTACGCCAACCTTTCTCATGCGCTGCCCCTCTTA-3'; the biotin modified myoglobin aptamer sequence is 5'-biotin-ATCCAGAGTGACGCAGCACAACGTGCAAATTATACCTGTTTTCCCCTTTTCCTACAAGTGCTATGGACACGGTGGCTTAGT-3';
A-4, flushing the wafer modified by the aptamer in the step A-3 by using a buffer solution I, then dripping 15-25 mu L of sealing agent, performing sealing reaction for 0.5-1.5 h, after sealing, cleaning the buffer solution I and double steam respectively to obtain a high-frequency single-sided electrodeless piezoelectric quartz crystal of the modified probe, and then placing the high-frequency single-sided electrodeless piezoelectric quartz crystal at 4 ℃ for later use; wherein: buffer solution I is PBS buffer solution, pH is 7.4, and concentration is 10mM; the blocking agent was a 0.1wt% bovine serum albumin solution.
6. The detection method of the high-frequency piezoelectric quartz crystal sensor system for detecting myocardial infarction markers as set forth in claim 4, characterized in that the specific method of modifying the high-frequency single-sided electrodeless piezoelectric quartz crystal comprises the following steps:
B-1, treating the high-frequency single-sided electrodeless piezoelectric quartz crystal with piranha acid for 10-20 min, taking out, thoroughly flushing an electrode with absolute ethyl alcohol and ultrapure water, and drying with nitrogen to obtain a treated wafer; wherein: piranha acid is concentrated sulfuric acid, 30% h 2O2 = 3:1;
B-2, dropwise adding 15-25 mu L of 3-aminopropyl triethoxysilane (APTES) solution into the wafer treated in the step B-1, and fixing for 1-2 hours to obtain an APTES modified wafer; wherein: the concentration of the 3-aminopropyl triethoxysilane solution is 2-5wt% and the solvent is anhydrous toluene;
B-3, flushing the wafer modified by APTES in the step B-2 by using a buffer solution I, and then dropwise adding 15-25 mu L glutaraldehyde solution on the wafer for reaction for 0.5-2 h; obtaining a glutaraldehyde-treated wafer; wherein the concentration of glutaraldehyde solution is 2.5-5wt%;
B-4 flushing the wafer treated by the glutaraldehyde in the step with a buffer solution I, then dripping a 20 mu L2 mu M aptamer II solution on the wafer, and reacting for 2-4 hours to obtain a wafer modified by the aptamer, wherein: the concentration of the solution of the aptamer II is 1.5-2.5 mu M, the aptamer II is an amino-modified troponin I aptamer or a myoglobin aptamer, and the sequence of the amino-modified troponin I aptamer is 5 '-NH 2(CH2)6 -CGTGCAGTACGCCAACCTTTCTCATGCGCTGCCCCTCTTA-3'; amino-modified myoglobin aptamer sequence 5'-NH2(CH2)6-ATCCAGAGTGACGCAGCACAACGTGCAAATTATACCTGTTTTCCCCTTTTCCTACAAGTGCTATGGACACGGTGGCTTAGT-3';
B-5, flushing the wafer modified by the aptamer in the B-4 by using a buffer solution I, then dripping 15-25 mu L of sealing agent, performing sealing reaction for 0.5-1.5 h, after sealing, cleaning the buffer solution I and double steam respectively to obtain a high-frequency single-sided electrodeless piezoelectric quartz crystal of the modified probe, and then placing the high-frequency single-sided electrodeless piezoelectric quartz crystal at 4 ℃ for later use; wherein: buffer solution I is PBS buffer solution, pH is 7.4, and concentration is 10mM; the blocking agent was a 0.1wt% bovine serum albumin solution.
7. The method for detecting a myocardial infarction marker by using a high frequency piezoelectric quartz crystal sensor system as set forth in claim 4, wherein the method for modifying a high frequency single-sided electrodeless piezoelectric quartz crystal comprises the steps of:
C-1, immersing a high-frequency single-sided electrodeless piezoelectric quartz crystal into a 1.5-2.5wt% 3-aminopropyl methyl diethoxy silane (3-APDES) solution, and immersing for 2-4 hours in an inert environment to obtain a 3-aminopropyl methyl diethoxy silane treated wafer; wherein: 3-APDES is absolute ethyl alcohol;
C-2, sequentially leaching the wafer treated by the 3-aminopropyl methyl diethoxy silane in the step C-1 into the solvents according to the sequence of ethanol, ethanol/acetone (1:1 (v/v)) and acetone, and carrying out ultrasonic treatment for 1-2 min in each group of solvents; then dropwise adding 15-20 mu L of 100mM succinic anhydride solution on the wafer, and then washing with deionized water and drying to obtain a succinic anhydride modified wafer;
C-3, adding 15-25 mu L of (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) containing 3mg/ml into the succinic anhydride modified wafer in the step C-2 for activation to obtain an activated wafer;
C-4 flushing the activated wafer in the step C-3 by using a buffer solution III, then dripping 15-25 mu L2 mu M aptamer III solution to the wafer, and reacting for 2-4 hours to obtain an aptamer modified wafer, wherein: buffer III is PBS buffer, pH is 7.4, and concentration is 10mM; the aptamer III is amino modified troponin I aptamer or myoglobin aptamer, and the amino modified troponin I aptamer has a sequence of 5 '-NH 2(CH2)6 -CGTGCAGTACGCCAACCTTTCTCATGCGCTGCCCCTCTTA-3'; amino-modified myoglobin aptamer sequence 5'-NH2(CH2)6-ATCCAGAGTGACGCAGCACAACGTGCAAATTATACCTGTTTTCCCCTTTTCCTACAAGTGCTATGGACACGGTGGCTTAGT-3';
C-5, flushing the aptamer modified wafer in the step C-4 by using a buffer solution III, dropwise adding 15-25 mu LBSA for sealing, and performing sealing reaction for 0.5-1.5 h to obtain a sealed wafer, wherein: BSA concentration was 0.1wt%;
c-6, respectively cleaning the wafers sealed in the step C-5 by using a buffer solution III and double steaming, drying by using nitrogen to obtain the high-frequency single-sided electrodeless piezoelectric quartz crystal of the modified probe, and then placing the high-frequency single-sided electrodeless piezoelectric quartz crystal at the temperature of 4 ℃ for standby.
8. The method for detecting a myocardial infarction marker by using a high frequency piezoelectric quartz crystal sensor system as set forth in any one of claims 2 to 7, wherein the specific method for preparing the aptamer-magnetic bead complex in step 2) comprises the following steps:
step one, activating magnetic beads with carboxyl groups: adding a nano magnetic bead stock solution with carboxyl into an EP tube, cleaning by adding a buffer solution II, activating carboxyl groups on the surface of the magnetic beads by using (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and an N-hydroxysuccinimide solution prepared by the buffer solution II, and vibrating to accelerate the activation to obtain an activated magnetic bead solution;
step two, carrying out magnetic separation and cleaning on the magnetic bead solution activated in the step one: placing a magnet at the edge of the EP tube, depositing nano magnetic beads on the tube wall of the EP tube under the action of a magnetic field, sucking out the solution by using a pipetting gun, taking care not to touch the tube wall, adding a buffer solution II, and repeating the steps for three times;
Step three: adding an aptamer IV solution into the magnetic bead solution treated in the step two, incubating the mixed solution by slight oscillation at room temperature, repeating the magnetic separation and cleaning operation for three times, removing the aptamer I which is not bound to NMBs, and finally suspending the magnetic beads bound with the proper ligand in a 200 mu L buffer solution I.
9. The detection method of the high-frequency piezoelectric quartz crystal sensor system for detecting myocardial infarction markers according to claim 8, wherein the buffer solution II is MES buffer solution, the pH is 5-6, and the concentration is 25-100 mM; the particle size of the nano magnetic beads in the first step is 200-300 nm, and the concentration is 5mg/ml; in the second step, the concentration of (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) is 5-10 mM, in the third step, the incubation time is 4-6 h, and the sequence of the aptamer IV is related to the sequence of the aptamer I-III:
When the sequence of the aptamer I-III is biotin or amino modified troponin I aptamer, the sequence of the corresponding aptamer IV is 5'-NH 2(CH2)6 -CGCATGCCAAACGTTGCCTCATAGTTCCCTCCCCGTGTCC-3';
When the sequence of the aptamer I-III is biotin or amino modified myoglobin aptamer, the sequence of the corresponding aptamer IV is 5'-NH 2(CH2)6 -CCCTCCTTTCCTTCGACGTAGATCTGCTGCGTTGTTCCGA-3'.
10. The method for detecting a myocardial infarction marker as set forth in claim 2, wherein in the step 3), the sample injection amount is 50 μl each time; the detection temperature is 25-37 ℃; setting the concentration to be 0-500 ng/mL, and selecting at least 5 concentrations among the concentrations according to the requirement; the magnetic field strength is-0.3 to 0.3T.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110615055.3A CN113552364B (en) | 2021-06-02 | 2021-06-02 | High-frequency piezoelectric quartz crystal sensing system for detecting myocardial infarction marker and detection method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110615055.3A CN113552364B (en) | 2021-06-02 | 2021-06-02 | High-frequency piezoelectric quartz crystal sensing system for detecting myocardial infarction marker and detection method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113552364A CN113552364A (en) | 2021-10-26 |
| CN113552364B true CN113552364B (en) | 2024-07-09 |
Family
ID=78130279
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110615055.3A Active CN113552364B (en) | 2021-06-02 | 2021-06-02 | High-frequency piezoelectric quartz crystal sensing system for detecting myocardial infarction marker and detection method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113552364B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114441362B (en) * | 2021-12-31 | 2024-06-14 | 浙江诺水科技发展有限公司 | Method for preparing quartz crystal microbalance sensor |
Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102816764A (en) * | 2011-06-07 | 2012-12-12 | 浦项工科大学校产学协力团 | DNA aptamer specifically binding to human cardiac troponin I |
| CN111521809A (en) * | 2019-02-02 | 2020-08-11 | 上海奥普生物医药股份有限公司 | Cardiac troponin T detection kit and preparation method and application thereof |
| TWI716827B (en) * | 2019-03-06 | 2021-01-21 | 國立清華大學 | Test kit for detecting cardiovascular disease and method for detecting concentration of cardiovascular disease-related biomarker |
| CN110031635A (en) * | 2019-05-16 | 2019-07-19 | 杭州普鲁米生物科技有限公司 | Flash type homogeneous chemistry luminescence technology detects cardiac muscle troponin I/T method |
| CN111830251A (en) * | 2020-06-24 | 2020-10-27 | 瑞芯智造(深圳)科技有限公司 | Biological sample detection method and detection kit |
| CN111812319B (en) * | 2020-07-21 | 2023-07-11 | 中南大学 | Micro-fluidic quartz wafer array sensing detection system and detection method thereof |
-
2021
- 2021-06-02 CN CN202110615055.3A patent/CN113552364B/en active Active
Non-Patent Citations (1)
| Title |
|---|
| 基于分子键裂的高频压电石英适配体生物传感器检测肌红蛋白;余建芳 等;《分析化学》;20220507;第50卷(第07期);第1065-1071页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113552364A (en) | 2021-10-26 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Lee et al. | ProteoChip: A highly sensitive protein microarray prepared by a novel method of protein immobilization for application of protein‐protein interaction studies | |
| Adányi et al. | Development of new immunosensors for determination of contaminants in food | |
| CN101438163B (en) | Detection of target molecules in a sample by using a magnetic field | |
| CN102859359A (en) | Preparation method of antigen-immobilized immunofluorescence slide, and immunofluorescence slide prepared thereby | |
| Yu et al. | An impedance array biosensor for detection of multiple antibody–antigen interactions | |
| UA57876C2 (en) | Method for isolating analyzed substance from a mix or determining the affinity between binding components and a device for the realization of the method | |
| US20090269858A1 (en) | Method of determining the concentration of an analyte using analyte sensor molecules coupled to a porous membrane | |
| Wang et al. | Characterizing the interaction between aptamers and human IgE by use of surface plasmon resonance | |
| CN113552364B (en) | High-frequency piezoelectric quartz crystal sensing system for detecting myocardial infarction marker and detection method thereof | |
| Feng et al. | Electrochemical immunosensor with aptamer-based enzymatic amplification | |
| Dou et al. | DNA-mediated strand displacement facilitates sensitive electronic detection of antibodies in human serums | |
| Hianik et al. | EQCM biosensors based on DNA aptamers and antibodies for rapid detection of prions | |
| CN101498719A (en) | Production method for enzyme functionalized nano immunity marker and use thereof | |
| CN114441616B (en) | Method for modifying new coronavirus biological probe on electrochemical biosensor | |
| CN112858432B (en) | Biosensor for detecting sarcosine and preparation method and application thereof | |
| Huang et al. | An electrochemical impedance sensor for simple and specific recognition of G–G mismatches in DNA | |
| CN111474224B (en) | Renewable electrochemical sensor for detecting trace kanamycin and preparation method and application thereof | |
| JP3788513B2 (en) | Method for immobilizing molecules on solid phase substrate and method for producing biosensor using the same | |
| Tang et al. | Direct and rapid detection of diphtherotoxin via potentiometric immunosensor based on nanoparticles mixture and polyvinyl butyral as matrixes | |
| Shin et al. | Quantitative label-free determination of thrombin using a chemically-modified M13 virus-electrode Interface | |
| Weiss | Customized Affinity and Specificity | |
| JP2000516706A (en) | Method for screening active compounds | |
| KR20220013928A (en) | Method and System for Detecting Target Material | |
| KR101006724B1 (en) | Ultra high-sensitivity detection method for C-reactive protein using quartz crystal microbalance immunosensor by gold nanoparticle | |
| Park et al. | Aptamer based electrochemical sensor system for protein using the generation/collection mode of scanning electrochemical microscope (SECM) |
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 |