CN112683977B - Novel coronavirus detection module based on multi-target-site antibody combination - Google Patents
Novel coronavirus detection module based on multi-target-site antibody combination Download PDFInfo
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Abstract
The invention belongs to the technical field of biological detection, and particularly discloses a new coronavirus detection module based on multi-target site antibody combination and a method thereof, wherein new coronavirus antibodies of different types and different target sites are simultaneously modified on a two-dimensional sensitive material through covalent bonds or intermolecular force to obtain a functionalized new coronavirus detection module, a sample to be detected is added into a detection module sample adding groove, whether the sample to be detected contains new coronavirus is judged through an electric signal, on one hand, the average detection time of the detection module on the new coronavirus antigen is less than 5 minutes, the sensitivity reaches-4.1 millivolts/(mol per liter), and the detection limit is 5 attomoles per liter (10 attomoles per liter) –18 mol/L), overcomes the problems of low sensitivity, poor specificity and long time consumption of the current antigen detection; on the other hand, the detection module can effectively improve the detection efficiency and reduce the detection cost by combining the traditional printed circuit board technology, and has potential practical value.
Description
Technical Field
The invention belongs to the technical field of biological detection, and particularly relates to a novel coronavirus detection module and method based on multi-target-site antibody combination.
Background
Pneumonia infectious diseases caused by the novel coronavirus (SARS-CoV-2) have a great influence on global public health and economic development. The rapid and accurate detection and diagnosis is particularly important for controlling secondary transmission, and the current subjects for clinical detection of the new coronavirus mainly comprise three types: nucleic acids, antigens and antibodies.
The transistor type sensor has the advantages of high sensitivity, low cost, fast response, no mark and the like. The sensing technology is characterized in that the conductivity of a semiconductor channel is changed by conducting and monitoring the adsorption and desorption processes of charged molecules, ions and the likeAnd thus trace detection is realized. In recent years, the application of two-dimensional sensitive materials has led to the rapid development of transistor-type sensing technology. The two-dimensional sensitive material has excellent conductivity and large specific surface area, and a channel with the atomic layer thickness is directly exposed in a detection environment and can make real-time and sensitive response to external small disturbance. Therefore, the biosensor based on the two-dimensional sensitive material has the detection sensitivity of at least femtomole per liter (10) –15 mol/L) magnitude. The advantages of high sensitivity, high specificity, no labeling, quick response and the like make the probe become an analysis tool with great potential, and the probe has great application value in the detection aspects of biomacromolecules (nucleic acid, protein, antibody and the like), small molecules (glucose, ATP and the like), cells, pathogens, gases, metal ions and the like, particularly in the instant detection aspect.
The spike protein (S protein) of the new coronavirus consists of two subunits, namely S1 and S2, wherein the S1 subunit and the receptor recognition Region (RBD) of the S protein are important targets in antigen detection. In the detection and exploration of new coronavirus antigens, a two-dimensional sensitive material transistor-type sensor based on a single antibody S1 protein monoclonal antibody (S1 protein monoclonal antibody) successfully realizes the screening of clinical positive samples, but the detection sensitivity and stability cannot meet the large-scale clinical requirements (ACS Nano 2020,14, 5135). Therefore, there is a need to develop highly effective viral antibodies to achieve accurate and reliable antigen detection.
Disclosure of Invention
In order to solve the problems of low sensitivity, poor specificity and long time consumption of the current detection of the new coronavirus antigen, the invention provides a new coronavirus detection module and a method based on multi-target-site antibody combination. The detection module prepared by the invention has the average detection time of less than 5 minutes for the new coronavirus antigen, the sensitivity reaches-4.1 millivolts/(mol liter), and the detection limit is 5 attomoles per liter (10 attomoles per liter) –18 mol/L)。
The purpose of the invention is realized by the following technical scheme:
a novel coronavirus detection module based on multi-target-site antibody combination comprises an insulating substrate, electrodes arranged on the insulating substrate and a two-dimensional sensitive material arranged on the insulating substrate and located between the electrodes, wherein the surface of the two-dimensional sensitive material modifies novel coronavirus antibodies through covalent bonds or intermolecular forces to obtain the functionalized novel coronavirus detection module.
Preferably, the insulating substrate comprises SiO 2 a/Si substrate, said electrode comprising chromium and/or gold.
Preferably, the two-dimensional sensitive material includes graphene, molybdenum disulfide, tungsten disulfide, graphene oxide, or a silicon, germanium, organic semiconductor, or other thin film.
Preferably, the new coronavirus antibody comprises one or more of a new coronavirus S1 protein monoclonal antibody, a CR3022 monoclonal antibody, an n3021 nanobody, an n3063 nanobody and an n3010 nanobody.
The insulating substrate comprises a printed circuit board base, and the printed circuit board base is provided with a sample adding slot.
A method for detecting a novel coronavirus based on multi-target-site antibody combination comprises the following steps:
step 1, processing an electrode on an insulating substrate, wherein the electrode comprises a source electrode and a drain electrode;
2, transferring the two-dimensional sensitive material to an insulating substrate, connecting the two-dimensional sensitive material between a source electrode and a drain electrode, and etching the two-dimensional sensitive material into a specific shape by utilizing a photoetching technology to obtain a device to be modified;
step 3, modifying connecting molecules in a two-dimensional sensitive material conducting channel region of a device to be modified;
step 4, connecting new coronavirus antibodies with different types and different target sites on the connecting molecules to form a two-dimensional sensitive material surface modified with several new coronavirus antibodies;
step 5, passivating the redundant connecting molecules to obtain a functionalized new coronavirus detection module;
step 6, connecting the new coronavirus detection module with an electrical test system, wherein the source electrode of the detection module is connected with the positive electrode, and the drain electrode of the detection module is connected with the negative electrode;
and 7, adding the new coronavirus specimen to be tested into a new coronavirus detection module for testing.
Preferably, the specific method for modifying the connecting molecules in the step 3 is to soak the two-dimensional sensitive material in 1-pyrenebutyric acid N-hydroxysuccinimide ester solution or dimethyl sulfoxide solution for 1-3 hours.
Preferably, the method for connecting the new coronavirus antibody in the step 4 is to soak the two-dimensional sensitive material in antibody combination solutions with different concentrations for 4 to 8 hours.
Preferably, the passivation process in step 5 is specifically performed by adopting a passivation agent or a passivation layer, soaking the two-dimensional sensitive material in an ethanolamine solution for 1-3 hours, wherein the passivation layer can be S1813 photoresist or Al 2 O 3 And (3) a layer.
Preferably, the detection of the new coronavirus specimen in step 7 is performed in two modes of current-time detection and current-grid voltage detection.
The samples to be tested for the new coronavirus comprise upper respiratory tract samples (pharynx swab, nose swab, nasopharynx extract and the like) and lower respiratory tract samples (deep expectoration, respiratory tract extract, bronchial lavage, alveolar lavage, lung tissue biopsy and the like). The specimen collection can be according to the method published by the laboratory test technical guideline for detecting the novel coronavirus pneumonia of Weijian Wei of China (fourth edition), and the method comprises the following specific steps:
(a) throat swab: the bilateral pharyngeal tonsils and the posterior pharyngeal wall were wiped with 2 plastic rod swabs of polypropylene fiber heads, the swab heads were dipped into a tube containing 3 ml of virus preservation solution (isotonic saline solution, tissue culture solution or phosphate buffer solution may also be used), the tail was discarded, the tube cap was screwed, and the packaging was done as per biosafety requirements.
(b) A nasal swab: the plastic rod swabs of 1 polypropylene fiber head are gently inserted into the nasal passage at the nasal palate, and slowly rotated and withdrawn after staying for a moment. Another plastic rod swab with a polypropylene fiber tip was used to collect the other nostril in the same manner. The two swabs are immersed in the same tube containing 3 ml of sampling liquid, the tail part is discarded, and the tube cover is screwed tightly and packaged according to the requirements of biological safety.
(c) Nasopharyngeal or respiratory tract aspirates: mucus is withdrawn from the nasopharynx or airway secretions from the trachea with a collector connected to a negative pressure pump. The collector head is inserted into the nasal cavity or trachea, negative pressure is switched on, the collector head is rotated and slowly withdrawn, the extracted mucus is collected and the collector is rinsed 1 time with 3 ml of sample solution (the collector can also be replaced by a pediatric catheter attached to a 50 ml syringe).
(d) Profound cough with sputum: after the patient was asked to cough deeply, the expectorated sputum was collected in a 50 ml screw plastic tube containing 3 ml of the sample.
(e) Bronchial lavage fluid: the collector head is inserted into the trachea (about 30cm deep) from the nostril or trachea insertion, 5 ml of normal saline is injected, negative pressure is connected, the collector head is rotated and slowly withdrawn. The withdrawn mucus was collected and the collector was rinsed 1 time with sample fluid (or a pediatric catheter attached to a 50 ml syringe instead of collection).
(f) Alveolar lavage fluid: after local anesthesia, the fiber bronchoscope is inserted into a branch tube of a right lung middle lobe or a left lung tongue segment through the pharynx through the mouth or the nose, the top end of the fiber bronchoscope is inserted into a branch opening of a bronchus, and sterilized normal saline is slowly added through a trachea biopsy hole, wherein 30-50 ml of the sterilized normal saline is added each time, the total amount is 100 plus 250 ml, and the total amount is not more than 300 ml.
The detection method can be divided into two modes of current-time detection and current-grid voltage detection according to a signal generation mechanism, and comprises the following steps:
current-time detection mode
(a) Adding a certain volume of negative standard sample into a sample adding slot of a new coronavirus detection module, and selecting a current-time mode of an electrical test system to start measurement: adjusting the output voltage of the test system to keep the source-drain current of the new coronavirus detection module constant, and detecting a sample to be detected when the variation percentage of the source-drain current is less than 0.2%;
(b) inactivating the specimen to be detected at 50-70 deg.C for 10-30 min, taking out negative standard sample with the same volume, adding the inactivated specimen with the same volume into a sample introduction tank to obtain normalized current response value (delta I) ds /I ds0 ): when Δ I ds /I ds0 When the concentration is less than 0.2%, the sample to be detected is not detected/negative; when Δ I ds /I ds0 If the concentration is more than 0.5%, the specimen to be detected is detected/positive;when Δ I ds /I ds0 Between 0.2% and 0.5%, a double check as a positive result is recommended.
Current-grid voltage detection mode
(a) Adding a certain volume of negative standard sample into a sample adding slot of a new coronavirus detection module, and selecting a current-grid voltage mode of an electrical test system to start measurement: the source-drain voltage and the grid voltage scanning range are given, and detection can be started when the threshold voltage or the Dirac point variation value is smaller than the voltage resolution of the instrument;
(b) inactivating the specimen to be detected at 50-70 deg.C for 10-30 min, taking out negative standard sample with the same volume, adding the inactivated specimen with the same volume into a sample introduction tank to obtain the absolute value (|. DELTA.V) of the Dirac point offset Dirac | that): when | Δ V Dirac When the level is less than 5 millivolts, the sample to be detected is undetected/negative; when | Δ V Dirac When the level is larger than 10 millivolts, the specimen to be detected is detected/positive; when | Δ V Dirac | between 5 mv and 10 mv, it is recommended to recheck as a positive result.
The detection and quality control method of the new coronavirus comprises the following steps:
(a) each batch of test has at least 1 part of weak positive quality control material and 2 parts of negative quality control material, and the quality control materials are randomly placed in clinical specimens;
(b) detecting the quality control product according to the method in the step 8, and if the weak positive quality control product is detected to be positive, and all the negative quality control products are detected to be negative, determining that the quality control products are in control; otherwise, the batch is out of control, the detection is invalid, and the reason needs to be analyzed and the detection needs to be carried out again.
The invention realizes the detection of the new coronavirus antigen based on the multi-target-site antibody combination and the two-dimensional sensitive material field effect transistor, and has obvious advantages compared with other detection methods: the technology for preparing the two-dimensional sensitive material by the chemical vapor deposition method is nearly mature day by day, and the obtained two-dimensional sensitive material has high quality and large area and can be used for preparing two-dimensional sensitive material biosensing devices in batches; due to the thickness and the large specific surface area of the atomic layer of the two-dimensional sensitive material, the channel region of the two-dimensional sensitive material biosensor is directly exposed to the detection environment and can carry out electrochemical reaction on a target detection objectThe signal makes an immediate and accurate response, and the detection sensitivity of the signal can reach at least femtomole (10) –15 mol/L) magnitude, the response time is less than 5 minutes; the portable high-flux new coronavirus detection module is prepared by combining with the PCB (shown in figure 1), so that the detection time is effectively shortened, and the detection cost is reduced.
The invention improves the detection sensitivity and reliability by using the combination of antibodies of different multi-target sites. Specifically, the novel coronavirus S1 protein monoclonal antibody, CR3022 monoclonal antibody, n3021 nano antibody, n3063 nano antibody and n3010 nano antibody are modified on the surface of the two-dimensional sensitive material at the same time.
The novel coronavirus antibody adopted by the invention comprises 3 types of mouse source/rabbit source, human source and full human source; the new coronavirus antibody targeting site adopted by the invention comprises 4 groups of epitopes in the S1 subunit and S protein receptor recognition Region (RBD) of the new coronavirus, and the specific information is shown in Table 1.
TABLE 1 types and targeting sites of novel coronavirus antibodies used in the invention
Wherein, CR3022 monoclonal antibody is broad-spectrum coronavirus monoclonal antibody, targets the cryptic epitope of S protein trimer of "upper" conformation, the sequence is:
the sequence of the light chain variable region (VL) of CR3022 (SEQ ID NO:1):
DIQLTQSPDSLAVSLGERATINCKSSQSVLYSSINKNYLAWYQQKPGQPPKL LIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPYTFGQG TKVEIK
the sequence of the heavy chain variable region (VH) of CR3022 (SEQ ID NO:2):
QLVQSGTEVKKPGESLKISCKGSGYGFITYWIGWVRQMPGKGLEWMGIIY PGDSETRYSPSFQGQVTISADKSINTAYLQWSSLKASDTAIYYCAGGSGISTPMD VWGQGTTVTV
the n3021 nanobody is a single-domain antibody, targets group A epitopes of the S1 subunit and S protein RBD of the new coronavirus, and has the sequence:
>n3021(SEQ ID NO:3)
EVQLVESGGGLVQPGGSLRLSCAASYFFDDYEMSWVRQAPGKGLEWIGEI NHSGSTNYNPSLKSRVTISRDNSKNTLYLQMNSLRAEDTALYYCVRDWLRFDY WGQGTLVTVSS
the n3063 nano antibody is a single domain antibody, targets B group epitope of S1 subunit and S protein RBD of the new coronavirus, and has the sequence as follows:
>n3063(SEQ ID NO:4)
EVQLVESGGGLVQPGGSLRLSCAASSFDFADYEMSWVRQAPGKALEWIGE IHHSGSTYYNPSLKSRVTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDLLPGGA DVWGQGTTVTVSS
the n3010 nano antibody is a single-domain antibody, targets C group epitopes of S1 subunits and S protein RBD of the new coronavirus, and has the sequence as follows:
>n3010(SEQ ID NO:5)
EVQLVESGGGLVQPGGSLRLSCAASDFDFYDYEMSWVRQAPGKALEWIG EIYHTGATNYNPSLKSRVTISRDNSKNTLYLQMNSLRAEDTAMYYCARHQPPDY YDSSGKPYYFDYWGQGTLVTVSS
compared with the prior art, the invention has the beneficial effects that:
the invention simultaneously modifies the multi-target-site and different types of new coronavirus antibodies on the two-dimensional sensitive material interface to obtain the functionalized new coronavirus detection module. The detection method based on the antibody combination effectively improves the antibody-antigen combination probability, thereby improving the sensitivity and stability of the detection of the new coronavirus antigen. In addition, the invention provides a detection module for rapidly detecting the new coronavirus antigen, which effectively reduces the detection cost, improves the detection efficiency and has potential social and economic values.
Drawings
FIG. 1 is a design drawing of the PCB base contained in the novel coronavirus detection module of the present invention.
FIG. 2 is a schematic diagram of the process for preparing the novel coronavirus detection module based on the combination of multi-targeted locus antibodies in example 1 of the present invention.
FIG. 3 is a schematic diagram of the structure of the detection module of the novel coronavirus based on the combination of multi-targeted site antibodies in example 1 of the present invention.
FIG. 4 shows the results of the test in the current-time detection mode in example 1 of the present invention.
FIG. 5 shows the test results in the current-gate voltage detection mode in example 2 of the present invention.
Fig. 6 shows the statistical results of the test in the current-gate voltage detection mode in example 3 of the present invention.
FIG. 7 is a schematic structural diagram of the detection module of the novel coronavirus obtained based on the single S1 protein monoclonal antibody in example 4 of the present invention.
FIG. 8 shows the statistical results of the test in the current-gate voltage detection mode in example 4 of the present invention.
Detailed Description
The present invention is described in further detail below with reference to the attached drawing figures.
A method for detecting a new coronavirus (SARS-CoV-2) based on a multi-target site antibody combination comprises the following steps:
step 1, in SiO 2 And/etching the electrode pattern after spin-coating the photoresist on the Si substrate.
And 2, evaporating and plating an electrode pattern to obtain a metal electrode (5-nanometer chromium and 50-nanometer gold), wherein the electrode comprises a source electrode and a drain electrode, the source electrode is a current input end, and the drain electrode is a current output end.
Step 3, spin-coating 8 wt.% polymethyl methacrylate (PMMA) on the two-dimensional sensitive material grown on the metal substrate, and transferring the two-dimensional sensitive material/PMMA film to Si/SiO with a metal electrode by using an electrochemical method 2 The substrate is connected between a source electrode and a drain electrode, and is sequentially cleaned by acetone/isopropanol/deionized water, and the two-dimensional sensitive material is etched into a specific shape by utilizing a photoetching technology to obtain a device to be modified (figure 2 a).
And 4, annealing the device to be modified for 5-15 minutes under the current condition of 100-500 microamperes, preferably for 10 minutes under the current condition of 300-500 microamperes.
Step 5, modifying connecting molecules in a two-dimensional sensitive material channel region (non-metal electrode part) on the device to be modified, wherein one example is as follows: soaking the two-dimensional sensitive material in 5-10 millimoles per liter of 1-pyrenebutyric acid N-hydroxysuccinimide ester (PASE) solution for 1-3 hours, preferably 5 millimoles per liter for 2 hours. Washing with absolute ethyl alcohol and deionized water.
And 6, soaking the two-dimensional sensitive material in multi-target locus antibody combination solutions with different concentrations (100 and 500 nanomoles per liter) for 4-8 hours, preferably soaking the two-dimensional sensitive material for 6 hours in 200 nanomoles per liter of S1 protein monoclonal antibody, 200 nanomoles per liter of n3021 nano antibody and 100 nanomoles per liter of CR3022 monoclonal antibody. Washed with phosphate buffered saline (1 × PBS buffer) (FIG. 2 b).
And 7, soaking the two-dimensional sensitive material in 100-500 millimoles of ethanolamine solution per liter for 1-3 hours, preferably 200 millimoles of ethanolamine per liter for 2 hours. The new coronavirus detection module was obtained after washing with absolute ethanol and 1 × PBS buffer (fig. 2 c).
And 8, connecting the new coronavirus detection module with the electrical test system through the alligator clip, wherein the source electrode of the detection module is connected with the positive electrode of the electrical test system, and the drain electrode of the detection module is connected with the negative electrode of the electrical test system.
Step 9, preparing a new coronavirus specimen to be detected:
the new coronavirus specimen to be detected applicable to the detection method comprises an upper respiratory tract specimen (a throat swab, a nose swab, a nasopharynx extract and the like) and a lower respiratory tract specimen (deep expectoration liquid, a respiratory tract extract, a bronchial lavage liquid, an alveolar lavage liquid, a lung tissue biopsy specimen and the like). The specimen collection can be according to the method published by the laboratory detection technical guideline for the novel coronavirus pneumonia of Weijian Wei of China (fourth edition), and the method comprises the following steps:
(a) throat swab: the bilateral pharyngeal tonsils and the posterior pharyngeal wall were wiped with 2 plastic rod swabs of polypropylene fiber heads, the swab heads were dipped into a tube containing 3 ml of virus preservation solution (isotonic saline solution, tissue culture solution or phosphate buffer solution may also be used), the tail was discarded, the tube cap was screwed, and the packaging was done as per biosafety requirements.
(b) A nasal swab: the plastic rod swabs of 1 polypropylene fiber head are gently inserted into the nasal passage at the nasal palate, and slowly rotated and withdrawn after staying for a moment. Another plastic rod swab with a polypropylene fiber tip was used to collect the other nostril in the same manner. The two swabs are immersed in the same tube containing 3 ml of sampling liquid, the tail part is discarded, and the tube cover is screwed tightly and packaged according to the requirements of biological safety.
(c) Nasopharyngeal aspirate or respiratory aspirate: mucus is withdrawn from the nasopharynx or airway secretions from the trachea with a collector connected to a negative pressure pump. The collector head is inserted into the nasal cavity or trachea, negative pressure is switched on, the collector head is rotated and slowly withdrawn, the extracted mucus is collected and the collector is rinsed 1 time with 3 ml of sample solution (the collector can also be replaced by a pediatric catheter attached to a 50 ml syringe).
(d) Profound cough with sputum: after the patient was asked to cough deeply, the expectorated sputum was collected in a 50 ml screw plastic tube containing 3 ml of the sample.
(e) Bronchial lavage fluid: the collector head is inserted into the trachea (about 30cm deep) from the nostril or trachea insertion, 5 ml of normal saline is injected, the negative pressure is switched on, the collector head is rotated and slowly withdrawn. The withdrawn mucus was collected and the collector was rinsed 1 time with sample fluid (or a pediatric catheter attached to a 50 ml syringe instead of collection).
(f) Alveolar lavage fluid: after local anesthesia, the fiber bronchoscope is inserted into a branch tube of a right lung middle lobe or a left lung tongue segment through the pharynx through the mouth or the nose, the top end of the fiber bronchoscope is inserted into a branch opening of a bronchus, and sterilized normal saline is slowly added through a trachea biopsy hole, wherein 30-50 ml of the sterilized normal saline is added each time, the total amount is 100 plus 250 ml, and the total amount is not more than 300 ml.
the detection method can be divided into two modes of current-time detection and current-grid voltage detection according to a signal generation mechanism, and comprises the following steps:
current-time detection mode
(a) Adding a certain volume of negative standard sample into a sample adding slot of a new coronavirus detection module, and selecting a current-time mode of an electrical test system to start measurement: and adjusting the output voltage of the test system to keep the source-drain current of the new coronavirus detection module constant, and detecting the specimen to be detected when the change percentage of the source-drain current is less than 0.2%.
(b) Inactivating the specimen to be detected at 50-70 deg.C for 10-30 min, taking out negative standard sample with the same volume, adding the inactivated specimen with the same volume into a sample introduction tank to obtain normalized current response value (delta I) ds /I ds0 ): when Δ I ds /I ds0 When the concentration is less than 0.2%, the sample to be detected is not detected/negative; when Δ I ds /I ds0 If the concentration is more than 0.5%, the specimen to be detected is detected/positive; when Δ I ds /I ds0 Between 0.2% and 0.5%, a double check as a positive result is recommended.
Current-grid voltage detection mode
(a) Adding a certain volume of negative standard sample into a sample adding slot of a new coronavirus detection module, and selecting a current-grid voltage mode of an electrical test system to start measurement: given the source-drain voltage and gate voltage scan ranges, detection can begin when the threshold voltage or dirac point variation is less than the instrument voltage resolution.
(b) Inactivating the specimen to be detected at 56 ℃ for 30 minutes, taking out a negative standard sample with the same volume, adding the inactivated specimen with the same volume into a sample introduction groove to obtain an absolute value (|. DELTA.V) of the Dirac point offset Dirac | to: when | Δ V Dirac When the level is less than 5 millivolts, the sample to be detected is undetected/negative; when | Δ V Dirac When the level is larger than 10 millivolts, the specimen to be detected is detected/positive; when | Δ V Dirac | between 5 mv and 10 mv, it is recommended to recheck as a positive result.
Step 11, detecting and controlling quality of the new coronavirus:
(a) each batch of test has at least 1 part of weak positive quality control material and 2 parts of negative quality control material, and the quality control materials are randomly placed in clinical specimens;
(b) detecting the quality control product according to the method in the step 10, and if the weak positive quality control product is determined to be positive, determining all negative quality control products to be negative, and determining the quality control products to be in control; otherwise, the batch is out of control, the detection is invalid, and the reason needs to be analyzed and the detection needs to be carried out again.
Example 1
The sequence of the S protein (40591-V08H) of the new coronavirus used in this example is:
VNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHA IHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNA TNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFL MDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPI GINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTIT DAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVF NATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVY ADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNY NYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVG YQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKK FLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQD VNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAG ICASYQTQTNSPRRARAHHHHHHHHHH
the S1 monoclonal antibody used in this example includes, but is not limited to, the following.
TABLE 2 product information of the monoclonal antibody against the S1 protein of the novel coronavirus
The CR3022 monoclonal antibody is a broad-spectrum coronavirus monoclonal antibody, targets the cryptic epitope of the S protein trimer in the "up" conformation, and has the sequence:
the sequence of the light chain variable region (VL) of CR3022 (SEQ ID NO:1):
DIQLTQSPDSLAVSLGERATINCKSSQSVLYSSINKNYLAWYQQKPGQPPKL LIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPYTFGQG TKVEIK
the sequence of the heavy chain variable region (VH) of CR3022 (SEQ ID NO:2):
QLVQSGTEVKKPGESLKISCKGSGYGFITYWIGWVRQMPGKGLEWMGIIYP GDSETRYSPSFQGQVTISADKSINTAYLQWSSLKASDTAIYYCAGGSGISTPMDV WGQGTTVTV
the n3021 nanobody is a single-domain antibody, targets group A epitopes of the S1 subunit and S protein RBD of the new coronavirus, and has the sequence:
>n3021(SEQ ID NO:3)
EVQLVESGGGLVQPGGSLRLSCAASYFFDDYEMSWVRQAPGKGLEWIGEI NHSGSTNYNPSLKSRVTISRDNSKNTLYLQMNSLRAEDTALYYCVRDWLRFDY WGQGTLVTVSS
the specific steps used in this example are as follows:
step 1, performing single polishing on an oxidized silicon wafer (comprising 300 nanometer SiO of an upper layer) 2 And the lower 500 micron P-type doped Si) is spin-coated with photoresist and then electrode patterns are etched. Wherein, the photoresist used includes both LOR3A and S1813: LOR3A is a sacrificial layer to ensure clean photoresist; s1813 is a photolithography layer for etching the electrode pattern.
And 2, evaporating and plating an electrode pattern to obtain a metal electrode (5-nanometer chromium and 50-nanometer gold), wherein the electrode comprises a source electrode and a drain electrode, the source electrode is a current input end, and the drain electrode is a current output end.
Step 3, 8 wt.% polymethyl methacrylate (PMMA) is coated on the graphene grown on the metal substrate in a spinning mode, and the graphene/PMMA thin film is transferred to Si/SiO with the metal electrode through an electrochemical method 2 The substrate is connected between a source electrode and a drain electrode, is soaked in acetone for 2 hours and then is cleaned by isopropanol/deionized water, and graphene is etched into a specific shape by utilizing a photoetching technology to obtain a device to be modified (figure 2 a).
And 4, annealing the device to be modified for 10 minutes under the condition of 500 microamperes of current.
Step 5, modifying a connecting molecule in a graphene channel region (a non-metal electrode part) on a device to be modified, wherein one example is as follows: graphene was soaked in 5 mmol/l pase (dmso) solution for 2 hours, and then washed with absolute ethanol and deionized water.
Step 6, soaking graphene in a multi-target locus antibody combination solution (200 nmol per liter of S1 protein mab, 200 nmol per liter of n3021 nanobody, and 100 nmol per liter of CR3022 mab, as shown in fig. 3) for 6 hours, and then washing with 1 × PBS buffer solution (fig. 2 b).
And 7, soaking the graphene in 200 millimoles per liter of ethanolamine solution for 2 hours, and washing with absolute ethyl alcohol and 1 multiplied by PBS buffer solution to obtain a new coronavirus detection module (figure 2 c).
And 8, connecting the new coronavirus detection module with the electrical test system through the alligator clip, wherein the source electrode of the detection module is connected with the positive electrode of the electrical test system, and the drain electrode of the detection module is connected with the negative electrode of the electrical test system.
Step 9, preparing a new coronavirus specimen to be detected:
in this embodimentThe concentration of the new coronavirus in the test specimen was rated at 5 attomoles per liter (10A/L) –18 mol/L), 5 femtomoles per liter (10) –15 mol/L), 5 picomoles per liter (10) –12 mol/L) and 5 nanomoles per liter (10) –9 mol/L)。
the present embodiment employs a current-time detection mode.
(a) Adding a certain volume of negative standard sample into a sample adding slot of a new coronavirus detection module, and selecting a current-time mode of an electrical test system to start measurement: and adjusting the output voltage of the test system to keep the source-drain current of the new coronavirus detection module constant, and detecting the specimen to be detected when the change percentage of the source-drain current is less than 0.2%.
(b) Taking out negative standard samples with the same volume, adding samples to be detected with the same volume into the sample introduction groove to obtain normalized current response value (delta I) ds /I ds0 )。
Example 2
The method for preparing the functionalized new coronavirus detection module is implemented according to the following specific embodiments, and the difference between the embodiment and the embodiment 1 is that:
in step 9, the concentration of the new coronavirus contained in the specimen to be tested in this embodiment is designated as 0 (negative standard sample), 0.5 attomole per liter, and 500 picomole per liter.
(a) adding a certain volume of negative standard sample into a sample adding slot of a new coronavirus detection module, and selecting a current-grid voltage mode of an electrical test system to start measurement: given the source-drain voltage and gate voltage scan ranges, detection can begin when the threshold voltage or dirac point variation is less than the instrument voltage resolution.
(b) Taking out negative standard samples with the same volume, adding samples to be detected with the same volume into a sample introduction groove to obtain an absolute value (|. DELTA.V) of the Dirac point offset Dirac | as shown in fig. 5.
Example 3
The method for preparing the functionalized new coronavirus detection module is implemented according to the following specific embodiments, and the difference between the embodiment and the embodiment 1 is that:
step 9, the concentration of the new coronavirus contained in the specimen to be tested in this embodiment is standardized to 5 attomoles per liter, 50 attomoles per liter, 500 attomoles per liter, 5 femtomoles per liter, 50 femtomoles per liter, 500 femtomoles per liter, 5 picomoles per liter, and 50 picomoles per liter.
(a) adding a certain volume of negative standard sample into a sample adding slot of a new coronavirus detection module, and selecting a current-grid voltage mode of an electrical test system to start measurement: given the source-drain voltage and gate voltage scan ranges, detection can begin when the threshold voltage or dirac point variation is less than the instrument voltage resolution.
(b) Taking out negative standard samples with the same volume, adding samples to be detected with the same volume into a sample introduction groove to obtain the Dirac point offset, and measuring the Delta V Dirac And (6) carrying out statistics.
The sensitivity of the new coronavirus detection module in this example reaches-4.1 millivolts/(mol/L), and the detection limit is less than 5 attomol/L, as shown in FIG. 6.
Example 4
The method for preparing the functionalized new coronavirus detection module is implemented according to the following specific embodiments, and the difference between the embodiment and the embodiment 1 is that:
step 6, preparing graphene: 200 nanomole of single S1 protein monoclonal antibody (unprocessed, 40150-R007) was soaked for 6 hours and then washed with 1 XPBS buffer (FIG. 7);
step 9, the concentration of the new coronavirus contained in the specimen to be tested in this embodiment is standardized to 5 attomoles per liter, 50 attomoles per liter, 500 attomoles per liter, 5 femtomoles per liter, 50 femtomoles per liter, 500 femtomoles per liter, 5 picomoles per liter, and 50 picomoles per liter.
(a) adding a certain volume of negative standard sample into a sample adding slot of a new coronavirus detection module, and selecting a current-grid voltage mode of an electrical test system to start measurement: the source-drain voltage and the grid voltage scanning range are given, and detection can be started when the threshold voltage or the Dirac point variation value is smaller than the voltage resolution of the instrument;
(b) taking out negative standard samples with the same volume, adding samples to be detected with the same volume into a sample introduction groove to obtain the Dirac point offset, and measuring the Delta V Dirac And (6) carrying out statistics.
The sensitivity of the novel coronavirus detection module of this example was only-2.8 millivolts per molar (FIG. 8), based on approximately 60-70% of the module obtained with the antibody combination (FIG. 6). This demonstrates that antibody combinations of different types and different targeting sites can improve the detection sensitivity of the novel coronavirus detection module.
The embodiments described above are described to facilitate an understanding and use of the invention by those skilled in the art. It will be readily apparent to those skilled in the art that various modifications to these embodiments may be made, and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above embodiments, and those skilled in the art should make improvements and modifications within the scope of the present invention based on the disclosure of the present invention.
Claims (4)
1. The novel coronavirus detection module based on the multi-targeted-site antibody combination is characterized by comprising an insulating substrate, electrodes arranged on the insulating substrate and a two-dimensional sensitive material arranged on the insulating substrate and positioned between the electrodes, wherein the surface of the two-dimensional sensitive material modifies a novel coronavirus antibody through a covalent bond or intermolecular force to obtain a functionalized novel coronavirus detection module, and the novel coronavirus antibody is a combination of a novel coronavirus S1 protein monoclonal antibody, a CR3022 monoclonal antibody and an n3021 nano antibody.
2. The module of claim 1, wherein the insulating substrate comprises SiO 2 a/Si substrate, said electrode comprising chromium and/or gold.
3. The module of claim 1, wherein the two-dimensional sensitive material comprises graphene, molybdenum disulfide, tungsten disulfide, graphene oxide, or a silicon, germanium, organic semiconductor film.
4. The module of claim 1, wherein the insulating substrate comprises a printed circuit board base, and the printed circuit board base is provided with a sample loading slot.
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| CN113791130B (en) * | 2021-08-27 | 2023-03-10 | 华中科技大学 | Ni 3 (HITP) 2 Field effect transistor biosensor and preparation method thereof |
| CN114634968A (en) * | 2022-02-28 | 2022-06-17 | 复旦大学 | Argonaute protein-based field effect transistor nucleic acid sensor and preparation method and application thereof |
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