CN112159837A - Microbial diagnosis sensor based on gold-binding polypeptide label functionalized botulinum toxin - Google Patents

Microbial diagnosis sensor based on gold-binding polypeptide label functionalized botulinum toxin Download PDF

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CN112159837A
CN112159837A CN202011129831.0A CN202011129831A CN112159837A CN 112159837 A CN112159837 A CN 112159837A CN 202011129831 A CN202011129831 A CN 202011129831A CN 112159837 A CN112159837 A CN 112159837A
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万逸
宋凤阁
申媛媛
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Hainan University
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Abstract

The timely detection of pathogenic microorganisms is very critical to the management and control and treatment of diseases, and the work aims to develop a microbial diagnosis sensor based on gold-combined polypeptide label functionalized botulinum toxin. The sensor assembles amplification products of unequal length PCR and the carnotoxin functionalized nano-gold nucleic acid probe to form a carnotoxin functionalized compound, and the SNAPtide probe is combined to convert a detection signal into a fluorescent signal or a visible light signal to realize the ultra-sensitive detection of pathogenic microorganisms. The innovation of this work is represented by: the sensor is realized by combining common PCR amplification with a botulinum toxin functionalized nano-gold nucleic acid probe, the design is simple and convenient, pathogenic microorganisms can be directly detected through dual signals of fluorescence and visible light, and a new strategy is provided for the design of a nano-gold based colorimetric detection sensor.

Description

Microbial diagnosis sensor based on gold-binding polypeptide label functionalized botulinum toxin
Technical Field
The invention provides a microbial diagnosis sensor based on gold-conjugated polypeptide label functionalized botulinum toxin, which aims to meet the demand that people hope to obtain accurate detection results by a simple and sensitive detection method.
Background
The medical epidemic and food-borne pathogenic microorganisms, including protists, viruses, bacteria and the like, are various and rapidly mutated, constitute a serious public health safety hazard in recent years, and pose a great threat and loss to human health and property safety. The rapid detection technology for pathogenic microorganisms is not only a precondition for effective prevention and control, but also a basis for scientific medicine application.
There are many detection methods for pathogenic microorganisms, and the traditional detection methods include pathogen culture, serum detection, nucleic acid detection and the like, but these methods often have the disadvantages of relatively long detection time, incapability of using some newly discovered pathogenic microorganisms such as new coronavirus, requirement of expensive instrument consumables and the like. In recent years, more and more sensor-based pathogenic microorganism detection methods have been developed. Wherein the signal reporter is an important component of the sensor. Current signal markers can be divided into three main categories: an enzyme-based signal marker, a fluorescent functional molecule-based signal marker, and a nanomaterial-based signal marker. Although the enzyme-based signal marker can greatly shorten the detection time, the enzyme-based signal marker has the weak point of strong background signal; although the signal marker based on the fluorescent functional molecule provides possibility for in-situ detection of pathogenic microorganisms, the phenomenon of instability exists; the signal marker based on the nano material has the characteristics of small volume, capability of real-time measurement in cells, no damage or micro damage to the cells and the like, but the operation is complex and experimenters are required to have related experimental experiences. Therefore, finding a sensitive signal reporter with a low signal-to-noise ratio becomes a key to constructing a pathogenic microorganism detection sensor.
The large outbreak of COVID-19 in 2019 shows that 1, a single detection method is difficult to meet the detection requirement, and multiple detection methods are often required to be combined in practical application; 2. a detection method that can meet the demand with a small amount of reagents and instruments needs to be established; 3. the detection application relying on the interdisciplinary is a big trend. In summary, the pathogen detection methods are relatively numerous, and all the methods have advantages and disadvantages, and in order to make up for the defects of the corresponding detection methods, it is necessary to further improve the sensitivity and specificity while using a plurality of methods in combination.
Botulinum toxin is a neurotoxin protein produced by botulinum in the reproductive process, is one of the most toxic natural substances, and is one of the most toxic proteins in the world. According to the difference of antigenicity, the botulinum toxin can be divided into 8 types A-H. Among them, botulinum toxin type a, which is the most toxic and the most common type, inhibits acetylcholine vesicle release at the neuromuscular junction by cleaving human SNAP-25 protein, blocks impulse conduction at the neuromuscular junction, and causes muscle paralysis to cause diseases. The SNAPtide (FITC/DABCYL) quenching probe pair is an equivalent substitute for the botulinum toxin type A action substrate SNAP-25 protein, and is capable of generating a fluorescent signal (λ ex: 490 nm; λ em: 523 nm) upon cleavage by BoNT/A. Therefore, it is suggested that the most toxic botulinum toxin type A is used as a signal reporter to convert the detection signal into a fluorescent signal, thereby improving the detection sensitivity of the sensor.
In summary, the present work is intended to develop a microbial diagnostic sensor-botulin neuron type A light chain (BoNT/A LC) activated complex assay (BACA) based on gold-binding polypeptide tag functionalized botulinum toxin. Further cutting SNAPtide quenching fluorescence pair by an amplification product of unequal length PCR and an assembly product of the carnitine functionalized nanogold nucleic acid probe so as to convert a detection signal into a fluorescence signal. Meanwhile, PCR amplification is carried out by the forward primer modified by the spacer and the reverse primer modified by the biotin in the invention, so that the nano-gold complex product can be separated by streptavidin magnetic beads, and pathogenic microorganisms can be directly detected by dual signals of fluorescence and visible light, and the sensitivity is very high.
Disclosure of Invention
The invention aims to improve the current situation that a high-sensitivity and high-accuracy detection method is lacked in the field of microbial diagnosis at present and provides an ultra-sensitive detection strategy based on functionalized botulinum toxin.
In order to achieve the purpose, the invention adopts the technical scheme that:
1. a microbial diagnostic sensor based on gold-binding polypeptide tag functionalized botulinum toxin.
2. Specifically, the microbial diagnosis sensor combines unequal-length PCR nucleic acid amplification and botulinum toxin functionalized nanogold nucleic acid probes.
3. The above-described microbial diagnostic sensor employs a quenching probe pair of a botulinum toxin action substrate.
4. The gold-conjugated polypeptide tag functionalized botulinum toxin comprises A type botulinum toxin, B type botulinum toxin, C type botulinum toxin, D type botulinum toxin, E type botulinum toxin, F type botulinum toxin, G type botulinum toxin and H type botulinum toxin.
5. The gold-binding polypeptide tag functionalized botulinum toxin comprises a light chain domain of a botulinum toxin type a-H.
6. The primer pair used in the unequal length PCR is a forward primer containing spacer modification PCR and a reverse primer containing biotin modification.
7. The quenching probe pairs include SNAPtide (FITC/DABCYL), SNAPtide (o-Abz/Dnp), VAMPtide (FITC/DABCYL), VAMPtide (Pya/Nop), SNAP tide (o-Abz/Dnp) and SYNTAXtide (o-Abz/Dnp).
8. The spacer includes one or more C18.
The invention has the advantages that:
1. the sensor is realized by combining common PCR amplification with a botulinum toxin functionalized nano-gold nucleic acid probe, is simple and convenient to design, and can directly detect pathogenic microorganisms through fluorescent signals.
2. The invention utilizes the toxic cutting effect of the botulinum toxin protein on the SNAP-25 protein as a signal generator, not only converts nucleic acid detection signals of pathogenic microorganisms into fluorescent signals, but also further improves the detection sensitivity, can achieve the detection on the aM level, meets the requirements of high selectivity and high sensitivity in the field of microbial detection, and simultaneously provides new insight for virus diagnosis.
3. The invention uses the forward primer containing spacer modification to obtain PCR products with different double-strand lengths through common PCR amplification, thereby providing a viscous end for combining with a detection probe.
4. According to the invention, the amplification product is obtained by using a biotin-modified reverse primer through common PCR, so that the amplification product can be completely separated by using streptavidin-modified magnetic beads.
5. The sensor is realized by combining common PCR amplification with a nanogold nucleic acid probe, is simple and convenient to design, and can be used for qualitatively detecting pathogenic microorganisms by directly observing a colorimetric method with naked eyes.
Description of the drawings:
FIG. 1 is a schematic diagram of the detection process of a microbial diagnostic sensor based on gold-binding polypeptide tag functionalized botulinum toxin.
FIG. 2 botulinum toxin and SNAP-25 protein purification. Among them, Lane 1 is, Lane 2 is, and Lane 3 is.
FIG. 3 is a diagram of a microbial diagnostic sensor based on gold-binding polypeptide tag functionalized botulinum toxin detecting artificially synthesized Pseudomonas aeruginosa target DNA at different concentrations.
FIG. 4A microbiological diagnostic sensor based on gold-binding polypeptide tag functionalized botulinum toxin detects different concentrations of Pseudomonas aeruginosa microorganisms.
FIG. 5A microbial diagnostic sensor based on gold-binding polypeptide tag functionalized botulinum toxin detects 6 different microorganisms.
The specific implementation mode is as follows:
example 1 botulinum toxin and SNAP-25 protein purification and assay of botulinum toxin protein Activity
pET-22b (+) -Trx-SNAP25 and pTIG-Trx-BoNT-ALC plasmids are respectively transformed into BL21(DE3) pLysS escherichia coli expression competence, positive monoclonals are screened and amplified, and the bacteria are shaken at the temperature of 37 ℃ and the rpm of 180 ℃ until the OD600 is about 0.6. Then inducing expression with 0.5 mM IPTG inducer at 25 deg.C and 16 deg.C for 16 h, centrifuging at 8000 rpm for 10 min, collecting thallus, and using lysis buffer (20 mM Na)3PO4, 500 mM NaCl, 40 mM imidazole, pH 7.4) were resuspended and sonicated. And centrifuging the ultrasonically crushed lysate for 30 min at the temperature of 4 ℃ by 10,000 rpm, collecting supernatant, filtering the supernatant by using a filter membrane of 0.22 mu m, and taking the filtered supernatant as a protein purification sample. With at least 5 column volumes of equilibration solution (20 mM Na)3PO4, 500 mM NaCl, 40 mM imidazole, pH 7.4) Balancing the Bio-Scale ™ Mini Nuvia ™ antibodyIMAC Ni-Charged Cartridges at flow rate of 5 mL/min until stable baseline wash-out; loading the sample to a balanced nickel column at a flow rate of 2 mL/min; washing the column with at least 10 column volumes of impurity washing liquid at a flow rate of 5 mL/min; eluent (20 mM Na)3PO4, 500 mM NaCl, 500 mM imidazole, pH 7.4) linear elution of the nickel column, a flow rate of 2 mL/min, and collection of the sample; and (4) performing western detection. Positive samples were dialyzed against 200 mM NaCl, 50 mM HEPES (pH 7.4) and then frozen at-80 ℃. SDS-PAGE and western were used to examine the protein purification effect. As can be seen from FIG. 2, both BoNT/A LC and SNAP25 proteins were well purified. The quadrbit Protein Assay Kits detects the Protein concentration. The activity of the BoNT/A LC protein was detected by SNAPtide FRET and SDS-PAGE methods.
Example 2 microbial diagnostic sensor based on gold-binding polypeptide tag functionalized botulinum toxin to detect different concentrations of artificially synthesized streptococcus pyogenes target and microbial concentrations
And (3) detecting the sensitivity of the artificially synthesized target:
pseudomonas aeruginosa target DNA (10) artificially synthesized by 30 muL (2 mg/mL) staphylococcus aureus capture probe streptavidin functionalized magnetic beads and 40 muL9aM) is used as a template to carry out double-stranded product reaction of unequal length PCR for 30 min. The reaction system was washed 3 times with PB buffer (10 mM PB pH 7.4), reacted for 30 min with 70 μ L Staphylococcus aureus functionalized gold nanoprobe (2.0 nM), washed 2 times with buffer (50 mM HEPES, pH 7.4, 500 mM NaCl, 0.05% TWEEN20) and resuspended in 36 μ L buffer (50 mM HEPES, pH 7.4, 2.3M TMAO, 0.05% TWEEN 20). mu.L of the successfully constructed sandwich model was mixed with 1. mu.L of SNAPtide (FITC/DABCYL) (final concentration 5. mu.M), the mixture was added to an ELISA plate, and the plate was placed in a BioTek H1 ELISA reader to collect fluorescence signals at 37 ℃ and λ ex: 490 nm and λ em: 523 nm over a period of 180 min. Meanwhile, the color change of the reaction system after the magnetic adsorption separation is observed under the condition of visible light, and the absorbance of the supernatant at 520 nm is detected by using a microplate reader.
30 mu L of staphylococcus aureus capture probe streptavidin functionalized magnetic bead (2 mg/m)L) and 40 mu L of artificially synthesized pseudomonas aeruginosa target DNA (10)8aM) is used as a template to carry out double-stranded product reaction of unequal length PCR for 30 min. The reaction system was washed 3 times with PB buffer (10 mM PB pH 7.4), reacted for 30 min with 70 μ L Staphylococcus aureus functionalized gold nanoprobe (2.0 nM), washed 2 times with buffer (50 mM HEPES, pH 7.4, 500 mM NaCl, 0.05% TWEEN20) and resuspended in 36 μ L buffer (50 mM HEPES, pH 7.4, 2.3M TMAO, 0.05% TWEEN 20). mu.L of the successfully constructed sandwich model was mixed with 1. mu.L of SNAPtide (FITC/DABCYL) (final concentration 5. mu.M), the mixture was added to an ELISA plate, and the plate was placed in a BioTek H1 ELISA reader to collect fluorescence signals at 37 ℃ and λ ex: 490 nm and λ em: 523 nm over a period of 180 min. Meanwhile, the color change of the reaction system after the magnetic adsorption separation is observed under the condition of visible light, and the absorbance of the supernatant at 520 nm is detected by using a microplate reader.
Pseudomonas aeruginosa target DNA (10) artificially synthesized by 30 muL (2 mg/mL) staphylococcus aureus capture probe streptavidin functionalized magnetic beads and 40 muL7aM) is used as a template to carry out double-stranded product reaction of unequal length PCR for 30 min. The reaction system was washed 3 times with PB buffer (10 mM PB pH 7.4), reacted for 30 min with 70 μ L Staphylococcus aureus functionalized gold nanoprobe (2.0 nM), washed 2 times with buffer (50 mM HEPES, pH 7.4, 500 mM NaCl, 0.05% TWEEN20) and resuspended in 36 μ L buffer (50 mM HEPES, pH 7.4, 2.3M TMAO, 0.05% TWEEN 20). mu.L of the successfully constructed sandwich model was mixed with 1. mu.L of SNAPtide (FITC/DABCYL) (final concentration 5. mu.M), the mixture was added to an ELISA plate, and the plate was placed in a BioTek H1 ELISA reader to collect fluorescence signals at 37 ℃ and λ ex: 490 nm and λ em: 523 nm over a period of 180 min. Meanwhile, the color change of the reaction system after the magnetic adsorption separation is observed under the condition of visible light, and the absorbance of the supernatant at 520 nm is detected by using a microplate reader.
Pseudomonas aeruginosa target DNA (10) artificially synthesized by 30 muL (2 mg/mL) staphylococcus aureus capture probe streptavidin functionalized magnetic beads and 40 muL6aM) is used as a template to carry out double-stranded product reaction of unequal length PCR for 30 min.The reaction system was washed 3 times with PB buffer (10 mM PB pH 7.4), reacted for 30 min with 70 μ L Staphylococcus aureus functionalized gold nanoprobe (2.0 nM), washed 2 times with buffer (50 mM HEPES, pH 7.4, 500 mM NaCl, 0.05% TWEEN20) and resuspended in 36 μ L buffer (50 mM HEPES, pH 7.4, 2.3M TMAO, 0.05% TWEEN 20). mu.L of the successfully constructed sandwich model was mixed with 1. mu.L of SNAPtide (FITC/DABCYL) (final concentration 5. mu.M), the mixture was added to an ELISA plate, and the plate was placed in a BioTek H1 ELISA reader to collect fluorescence signals at 37 ℃ and λ ex: 490 nm and λ em: 523 nm over a period of 180 min. Meanwhile, the color change of the reaction system after the magnetic adsorption separation is observed under the condition of visible light, and the absorbance of the supernatant at 520 nm is detected by using a microplate reader.
Pseudomonas aeruginosa target DNA (10) artificially synthesized by 30 muL (2 mg/mL) staphylococcus aureus capture probe streptavidin functionalized magnetic beads and 40 muL5aM) is used as a template to carry out double-stranded product reaction of unequal length PCR for 30 min. The reaction system was washed 3 times with PB buffer (10 mM PB pH 7.4), reacted for 30 min with 70 μ L Staphylococcus aureus functionalized gold nanoprobe (2.0 nM), washed 2 times with buffer (50 mM HEPES, pH 7.4, 500 mM NaCl, 0.05% TWEEN20) and resuspended in 36 μ L buffer (50 mM HEPES, pH 7.4, 2.3M TMAO, 0.05% TWEEN 20). mu.L of the successfully constructed sandwich model was mixed with 1. mu.L of SNAPtide (FITC/DABCYL) (final concentration 5. mu.M), the mixture was added to an ELISA plate, and the plate was placed in a BioTek H1 ELISA reader to collect fluorescence signals at 37 ℃ and λ ex: 490 nm and λ em: 523 nm over a period of 180 min. Meanwhile, the color change of the reaction system after the magnetic adsorption separation is observed under the condition of visible light, and the absorbance of the supernatant at 520 nm is detected by using a microplate reader.
Pseudomonas aeruginosa target DNA (10) artificially synthesized by 30 muL (2 mg/mL) staphylococcus aureus capture probe streptavidin functionalized magnetic beads and 40 muL4aM) is used as a template to carry out double-stranded product reaction of unequal length PCR for 30 min. Washing the reaction system with PB buffer solution (10 mM PB pH 7.4) for 3 times, adding 70 muL of staphylococcus aureus functionalized gold nanoprobe (2.0 nM) to react for 30 timesmin, wash the reaction 2 times with buffer (50 mM HEPES, pH 7.4, 500 mM NaCl, 0.05% TWEEN20) and resuspend with 36 μ L buffer (50 mM HEPES, pH 7.4, 2.3M TMAO, 0.05% TWEEN 20). mu.L of the successfully constructed sandwich model was mixed with 1. mu.L of SNAPtide (FITC/DABCYL) (final concentration 5. mu.M), the mixture was added to an ELISA plate, and the plate was placed in a BioTek H1 ELISA reader to collect fluorescence signals at 37 ℃ and λ ex: 490 nm and λ em: 523 nm over a period of 180 min. Meanwhile, the color change of the reaction system after the magnetic adsorption separation is observed under the condition of visible light, and the absorbance of the supernatant at 520 nm is detected by using a microplate reader.
Pseudomonas aeruginosa target DNA (10) artificially synthesized by 30 muL (2 mg/mL) staphylococcus aureus capture probe streptavidin functionalized magnetic beads and 40 muL3aM) is used as a template to carry out double-stranded product reaction of unequal length PCR for 30 min. The reaction system was washed 3 times with PB buffer (10 mM PB pH 7.4), reacted for 30 min with 70 μ L Staphylococcus aureus functionalized gold nanoprobe (2.0 nM), washed 2 times with buffer (50 mM HEPES, pH 7.4, 500 mM NaCl, 0.05% TWEEN20) and resuspended in 36 μ L buffer (50 mM HEPES, pH 7.4, 2.3M TMAO, 0.05% TWEEN 20). mu.L of the successfully constructed sandwich model was mixed with 1. mu.L of SNAPtide (FITC/DABCYL) (final concentration 5. mu.M), the mixture was added to an ELISA plate, and the plate was placed in a BioTek H1 ELISA reader to collect fluorescence signals at 37 ℃ and λ ex: 490 nm and λ em: 523 nm over a period of 180 min. Meanwhile, the color change of the reaction system after the magnetic adsorption separation is observed under the condition of visible light, and the absorbance of the supernatant at 520 nm is detected by using a microplate reader.
Pseudomonas aeruginosa target DNA (10) artificially synthesized by 30 muL (2 mg/mL) staphylococcus aureus capture probe streptavidin functionalized magnetic beads and 40 muL2aM) is used as a template to carry out double-stranded product reaction of unequal length PCR for 30 min. The reaction system was washed 3 times with PB buffer (10 mM PB pH 7.4), reacted for 30 min with 70. mu.L Staphylococcus aureus functionalized gold nanoprobe (2.0 nM), washed 2 times with buffer (50 mM HEPES, pH 7.4, 500 mM NaCl, 0.05% TWEEN20) and 36. mu.L buffer (50 mM HEPES, pH 7.4, 2.3)M TMAO, 0.05% TWEEN 20). mu.L of the successfully constructed sandwich model was mixed with 1. mu.L of SNAPtide (FITC/DABCYL) (final concentration 5. mu.M), the mixture was added to an ELISA plate, and the plate was placed in a BioTek H1 ELISA reader to collect fluorescence signals at 37 ℃ and λ ex: 490 nm and λ em: 523 nm over a period of 180 min. Meanwhile, the color change of the reaction system after the magnetic adsorption separation is observed under the condition of visible light, and the absorbance of the supernatant at 520 nm is detected by using a microplate reader.
Pseudomonas aeruginosa target DNA (10) artificially synthesized by 30 muL (2 mg/mL) staphylococcus aureus capture probe streptavidin functionalized magnetic beads and 40 muL1aM) is used as a template to carry out double-stranded product reaction of unequal length PCR for 30 min. The reaction system was washed 3 times with PB buffer (10 mM PB pH 7.4), reacted for 30 min with 70 μ L Staphylococcus aureus functionalized gold nanoprobe (2.0 nM), washed 2 times with buffer (50 mM HEPES, pH 7.4, 500 mM NaCl, 0.05% TWEEN20) and resuspended in 36 μ L buffer (50 mM HEPES, pH 7.4, 2.3M TMAO, 0.05% TWEEN 20). mu.L of the successfully constructed sandwich model was mixed with 1. mu.L of SNAPtide (FITC/DABCYL) (final concentration 5. mu.M), the mixture was added to an ELISA plate, and the plate was placed in a BioTek H1 ELISA reader to collect fluorescence signals at 37 ℃ and λ ex: 490 nm and λ em: 523 nm over a period of 180 min. Meanwhile, the color change of the reaction system after the magnetic adsorption separation is observed under the condition of visible light, and the absorbance of the supernatant at 520 nm is detected by using a microplate reader.
Pseudomonas aeruginosa target DNA (10) artificially synthesized by 30 muL (2 mg/mL) staphylococcus aureus capture probe streptavidin functionalized magnetic beads and 40 muL0aM) is used as a template to carry out double-stranded product reaction of unequal length PCR for 30 min. The reaction system was washed 3 times with PB buffer (10 mM PB pH 7.4), reacted for 30 min with 70 μ L Staphylococcus aureus functionalized gold nanoprobe (2.0 nM), washed 2 times with buffer (50 mM HEPES, pH 7.4, 500 mM NaCl, 0.05% TWEEN20) and resuspended in 36 μ L buffer (50 mM HEPES, pH 7.4, 2.3M TMAO, 0.05% TWEEN 20). mu.L of the successfully constructed "sandwich" model was mixed with 1. mu.L of SNAPtide (FITC/DABCYL) (final concentration 5. mu.M) and addedAnd (3) placing the enzyme label plate into a BioTek H1 enzyme label instrument to collect fluorescence signals within 180 min at 37 ℃, lambda ex 490 nm and lambda em 523 nm. Meanwhile, the color change of the reaction system after the magnetic adsorption separation is observed under the condition of visible light, and the absorbance of the supernatant at 520 nm is detected by using a microplate reader.
30 mu L of staphylococcus aureus capture probe streptavidin functionalized magnetic beads (2 mg/mL) and 40 mu L of staphylococcus aureus capture probe streptavidin functionalized magnetic beads take artificially synthesized pseudomonas aeruginosa target DNA (0 aM) as a template to carry out double-strand product reaction of unequal length PCR for 30 min. The reaction system was washed 3 times with PB buffer (10 mM PB pH 7.4), reacted for 30 min with 70 μ L Staphylococcus aureus functionalized gold nanoprobe (2.0 nM), washed 2 times with buffer (50 mM HEPES, pH 7.4, 500 mM NaCl, 0.05% TWEEN20) and resuspended in 36 μ L buffer (50 mM HEPES, pH 7.4, 2.3M TMAO, 0.05% TWEEN 20). mu.L of the successfully constructed sandwich model was mixed with 1. mu.L of SNAPtide (FITC/DABCYL) (final concentration 5. mu.M), the mixture was added to an ELISA plate, and the plate was placed in a BioTek H1 ELISA reader to collect fluorescence signals at 37 ℃ and λ ex: 490 nm and λ em: 523 nm over a period of 180 min. Meanwhile, the color change of the reaction system after the magnetic adsorption separation is observed under the condition of visible light, and the absorbance of the supernatant at 520 nm is detected by using a microplate reader.
As can be seen from FIG. 3, the fluorescence and colorimetric methods of the sensor of the present invention can detect synthetic targets of 1 aM and 100 aM Pseudomonas aeruginosa, respectively.
Detection of sensitivity of microbial concentration:
30 muL staphylococcus aureus capture probe streptavidin functionalized magnetic bead (2 mg/mL) and 40 muL pseudomonas aeruginosa (10 muL)6 cfu•ml-1) Taking cDNA as a template to perform double-stranded product reaction of unequal length PCR for 30 min. The reaction system was washed 3 times with PB buffer (10 mM PB pH 7.4), reacted for 30 min with 70 μ L Staphylococcus aureus functionalized gold nanoprobe (2.0 nM), washed 2 times with buffer (50 mM HEPES, pH 7.4, 500 mM NaCl, 0.05% TWEEN20) and resuspended in 36 μ L buffer (50 mM HEPES, pH 7.4, 2.3M TMAO, 0.05% TWEEN 20). 9 μ L of a successfully constructed "The sandwich model was mixed with 1. mu.L of SNAPtide (FITC/DABCYL) (final concentration 5. mu.M) and added to an ELISA plate, and the plate was placed in a BioTek H1 ELISA reader to collect fluorescence signals at 37 ℃ and λ ex: 490 nm and λ em: 523 nm over a period of 180 min. Meanwhile, the color change of the reaction system after the magnetic adsorption separation is observed under the condition of visible light, and the absorbance of the supernatant at 520 nm is detected by using a microplate reader.
30 muL staphylococcus aureus capture probe streptavidin functionalized magnetic bead (2 mg/mL) and 40 muL pseudomonas aeruginosa (10 muL)5 cfu•ml-1) Taking cDNA as a template to perform double-stranded product reaction of unequal length PCR for 30 min. The reaction system was washed 3 times with PB buffer (10 mM PB pH 7.4), reacted for 30 min with 70 μ L Staphylococcus aureus functionalized gold nanoprobe (2.0 nM), washed 2 times with buffer (50 mM HEPES, pH 7.4, 500 mM NaCl, 0.05% TWEEN20) and resuspended in 36 μ L buffer (50 mM HEPES, pH 7.4, 2.3M TMAO, 0.05% TWEEN 20). mu.L of the successfully constructed sandwich model was mixed with 1. mu.L of SNAPtide (FITC/DABCYL) (final concentration 5. mu.M), the mixture was added to an ELISA plate, and the plate was placed in a BioTek H1 ELISA reader to collect fluorescence signals at 37 ℃ and λ ex: 490 nm and λ em: 523 nm over a period of 180 min. Meanwhile, the color change of the reaction system after the magnetic adsorption separation is observed under the condition of visible light, and the absorbance of the supernatant at 520 nm is detected by using a microplate reader.
30 muL staphylococcus aureus capture probe streptavidin functionalized magnetic bead (2 mg/mL) and 40 muL pseudomonas aeruginosa (10 muL)4 cfu•ml-1) Taking cDNA as a template to perform double-stranded product reaction of unequal length PCR for 30 min. The reaction system was washed 3 times with PB buffer (10 mM PB pH 7.4), reacted for 30 min with 70 μ L Staphylococcus aureus functionalized gold nanoprobe (2.0 nM), washed 2 times with buffer (50 mM HEPES, pH 7.4, 500 mM NaCl, 0.05% TWEEN20) and resuspended in 36 μ L buffer (50 mM HEPES, pH 7.4, 2.3M TMAO, 0.05% TWEEN 20). Mixing 9 μ L of successfully constructed sandwich model with 1 μ L of SNAPtide (FITC/DABCYL) (final concentration 5 μ M), adding into the ELISA plate, placing the ELISA plate into BioTek H1 ELISA reader, and collectingFluorescence signals at 37 ℃ and λ ex 490 nm and λ em 523 nm over a period of 180 min. Meanwhile, the color change of the reaction system after the magnetic adsorption separation is observed under the condition of visible light, and the absorbance of the supernatant at 520 nm is detected by using a microplate reader.
30 muL staphylococcus aureus capture probe streptavidin functionalized magnetic bead (2 mg/mL) and 40 muL pseudomonas aeruginosa (10 muL)3 cfu•ml-1) Taking cDNA as a template to perform double-stranded product reaction of unequal length PCR for 30 min. The reaction system was washed 3 times with PB buffer (10 mM PB pH 7.4), reacted for 30 min with 70 μ L Staphylococcus aureus functionalized gold nanoprobe (2.0 nM), washed 2 times with buffer (50 mM HEPES, pH 7.4, 500 mM NaCl, 0.05% TWEEN20) and resuspended in 36 μ L buffer (50 mM HEPES, pH 7.4, 2.3M TMAO, 0.05% TWEEN 20). mu.L of the successfully constructed sandwich model was mixed with 1. mu.L of SNAPtide (FITC/DABCYL) (final concentration 5. mu.M), the mixture was added to an ELISA plate, and the plate was placed in a BioTek H1 ELISA reader to collect fluorescence signals at 37 ℃ and λ ex: 490 nm and λ em: 523 nm over a period of 180 min. Meanwhile, the color change of the reaction system after the magnetic adsorption separation is observed under the condition of visible light, and the absorbance of the supernatant at 520 nm is detected by using a microplate reader.
30 muL staphylococcus aureus capture probe streptavidin functionalized magnetic bead (2 mg/mL) and 40 muL pseudomonas aeruginosa (10 muL)2 cfu•ml-1) Taking cDNA as a template to perform double-stranded product reaction of unequal length PCR for 30 min. The reaction system was washed 3 times with PB buffer (10 mM PB pH 7.4), reacted for 30 min with 70 μ L Staphylococcus aureus functionalized gold nanoprobe (2.0 nM), washed 2 times with buffer (50 mM HEPES, pH 7.4, 500 mM NaCl, 0.05% TWEEN20) and resuspended in 36 μ L buffer (50 mM HEPES, pH 7.4, 2.3M TMAO, 0.05% TWEEN 20). mu.L of the successfully constructed sandwich model was mixed with 1. mu.L of SNAPtide (FITC/DABCYL) (final concentration 5. mu.M), the mixture was added to an ELISA plate, and the plate was placed in a BioTek H1 ELISA reader to collect fluorescence signals at 37 ℃ and λ ex: 490 nm and λ em: 523 nm over a period of 180 min. Meanwhile, observing the reaction system under the condition of visible light through magnet adsorptionThe color after separation changed and the absorbance of the supernatant at 520 nm was measured with a microplate reader.
30 muL staphylococcus aureus capture probe streptavidin functionalized magnetic bead (2 mg/mL) and 40 muL pseudomonas aeruginosa (10 muL)1 cfu•ml-1) Taking cDNA as a template to perform double-stranded product reaction of unequal length PCR for 30 min. The reaction system was washed 3 times with PB buffer (10 mM PB pH 7.4), reacted for 30 min with 70 μ L Staphylococcus aureus functionalized gold nanoprobe (2.0 nM), washed 2 times with buffer (50 mM HEPES, pH 7.4, 500 mM NaCl, 0.05% TWEEN20) and resuspended in 36 μ L buffer (50 mM HEPES, pH 7.4, 2.3M TMAO, 0.05% TWEEN 20). mu.L of the successfully constructed sandwich model was mixed with 1. mu.L of SNAPtide (FITC/DABCYL) (final concentration 5. mu.M), the mixture was added to an ELISA plate, and the plate was placed in a BioTek H1 ELISA reader to collect fluorescence signals at 37 ℃ and λ ex: 490 nm and λ em: 523 nm over a period of 180 min. Meanwhile, the color change of the reaction system after the magnetic adsorption separation is observed under the condition of visible light, and the absorbance of the supernatant at 520 nm is detected by using a microplate reader.
30 muL staphylococcus aureus capture probe streptavidin functionalized magnetic bead (2 mg/mL) and 40 muL pseudomonas aeruginosa (10 muL)0 cfu•ml-1) Taking cDNA as a template to perform double-stranded product reaction of unequal length PCR for 30 min. The reaction system was washed 3 times with PB buffer (10 mM PB pH 7.4), reacted for 30 min with 70 μ L Staphylococcus aureus functionalized gold nanoprobe (2.0 nM), washed 2 times with buffer (50 mM HEPES, pH 7.4, 500 mM NaCl, 0.05% TWEEN20) and resuspended in 36 μ L buffer (50 mM HEPES, pH 7.4, 2.3M TMAO, 0.05% TWEEN 20). mu.L of the successfully constructed sandwich model was mixed with 1. mu.L of SNAPtide (FITC/DABCYL) (final concentration 5. mu.M), the mixture was added to an ELISA plate, and the plate was placed in a BioTek H1 ELISA reader to collect fluorescence signals at 37 ℃ and λ ex: 490 nm and λ em: 523 nm over a period of 180 min. Meanwhile, the color change of the reaction system after the magnetic adsorption separation is observed under the condition of visible light, and the absorbance of the supernatant at 520 nm is detected by using a microplate reader.
30 mu L golden grapeStaphylococcus capture probe streptavidin functionalized magnetic bead (2 mg/mL) and 40 muL pseudomonas aeruginosa (0 cfu.ml)-1) Taking cDNA as a template to perform double-stranded product reaction of unequal length PCR for 30 min. The reaction system was washed 3 times with PB buffer (10 mM PB pH 7.4), reacted for 30 min with 70 μ L Staphylococcus aureus functionalized gold nanoprobe (2.0 nM), washed 2 times with buffer (50 mM HEPES, pH 7.4, 500 mM NaCl, 0.05% TWEEN20) and resuspended in 36 μ L buffer (50 mM HEPES, pH 7.4, 2.3M TMAO, 0.05% TWEEN 20). mu.L of the successfully constructed sandwich model was mixed with 1. mu.L of SNAPtide (FITC/DABCYL) (final concentration 5. mu.M), the mixture was added to an ELISA plate, and the plate was placed in a BioTek H1 ELISA reader to collect fluorescence signals at 37 ℃ and λ ex: 490 nm and λ em: 523 nm over a period of 180 min. Meanwhile, the color change of the reaction system after the magnetic adsorption separation is observed under the condition of visible light, and the absorbance of the supernatant at 520 nm is detected by using a microplate reader.
As can be seen from FIG. 4, the fluorescence method and the colorimetric method of the sensor of the present invention can detect 1 cfu.ml, respectively-1And 10 cfu.ml-1Pseudomonas aeruginosa.
Example 3 microbial diagnostic sensor based on gold-binding polypeptide tag functionalized botulinum toxin detects 6 different bacterial microorganisms
30 mu L of staphylococcus aureus capture probe streptavidin functionalized magnetic beads (2 mg/mL) and 40 mu L of staphylococcus aureus capture probe streptavidin functionalized magnetic beads are subjected to double-strand product reaction of unequal length PCR for 30 min by using enterococcus faecalis, streptococcus pyogenes, staphylococcus aureus, pseudomonas aeruginosa, escherichia coli and salmonella typhi cDNA as templates. The reaction system was washed 3 times with PB buffer (10 mM PB pH 7.4), reacted for 30 min with 70 μ L Staphylococcus aureus functionalized gold nanoprobe (2.0 nM), washed 2 times with buffer (50 mM HEPES, pH 7.4, 500 mM NaCl, 0.05% TWEEN20) and resuspended in 36 μ L buffer (50 mM HEPES, pH 7.4, 2.3M TMAO, 0.05% TWEEN 20). mu.L of the successfully constructed sandwich model was mixed with 1. mu.L of SNAPtide (FITC/DABCYL) (final concentration 5. mu.M), the mixture was added to an ELISA plate, and the plate was placed in a BioTek H1 ELISA reader to collect fluorescence signals at 37 ℃ and λ ex: 490 nm and λ em: 523 nm over a period of 180 min. Meanwhile, the color change of the reaction system after the magnetic adsorption separation is observed under the condition of visible light, and the absorbance of the supernatant at 520 nm is detected by using a microplate reader.
As can be seen from FIG. 5, the sensor of the present invention can specifically detect enterococcus faecalis, Streptococcus pyogenes, Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and Salmonella typhi by fluorescence or colorimetry.

Claims (8)

1. A microbial diagnostic sensor based on gold-binding polypeptide tag functionalized botulinum toxin.
2. The microbial diagnostic sensor of claim 1, wherein detection of the microbes incorporates unequal length PCR nucleic acid amplification and botulinum toxin functionalized nanogold nucleic acid probes.
3. The microbial diagnostic sensor of claim 1, wherein the detection of the microbe employs a quenching probe pair of a botulinum toxin action substrate.
4. The microbial diagnostic sensor of claim 1, wherein said gold-binding polypeptide tag functionalized botulinum toxin comprises botulinum toxin type A, botulinum toxin type B, botulinum toxin type C, botulinum toxin type D, botulinum toxin type E, botulinum toxin type F, botulinum toxin type G, botulinum toxin type H.
5. The microbial diagnostic sensor of claim 1, wherein the gold-binding polypeptide tag functionalized botulinum toxin comprises the light chain domain of botulinum toxin types a-H.
6. The microbial diagnostic sensor of claim 2, wherein the primer pair for unequal length PCR is a forward primer containing spacer modified PCR and a reverse primer containing biotin modification.
7. The microbial diagnostic sensor of claim 3, wherein the pair of quenching probes comprises SNAPtide (FITC/DABCYL), SNAPtide (o-Abz/Dnp), VAMPtide (FITC/DABCYL), VAMPtide (Pya/Nop), SNAP tide (o-Abz/Dnp), and SYNTAXtide (o-Abz/Dnp).
8. The microbial diagnostic sensor of claim 6, wherein said spacer comprises one or more C18 s.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114392354A (en) * 2022-01-14 2022-04-26 国家纳米科学中心 DNA nano system for tumor targeting and preparation method and application thereof

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010054159A2 (en) * 2008-11-06 2010-05-14 University Of Florida Research Foundation, Inc. Materials and methods for detecting toxins, pathogens and other biological materials
US20130065259A1 (en) * 2007-06-05 2013-03-14 City Of Hope Methods for detection of botulinum neurotoxin
CN105316398A (en) * 2014-07-30 2016-02-10 益善生物技术股份有限公司 Amplification primer for detecting food-borne pathogenic microorganisms and liquid chip kit
CN108562742A (en) * 2018-04-10 2018-09-21 海南大学 A kind of hemolysin of metal-binding polypeptides label diagnoses pathogenic microorganism as bio signal marker hypersensitive
CN110408676A (en) * 2019-07-19 2019-11-05 海南大学 A kind of microbe diagnosis sensor combining Polypeptide tags functionalization trypsase based on gold

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130065259A1 (en) * 2007-06-05 2013-03-14 City Of Hope Methods for detection of botulinum neurotoxin
WO2010054159A2 (en) * 2008-11-06 2010-05-14 University Of Florida Research Foundation, Inc. Materials and methods for detecting toxins, pathogens and other biological materials
CN105316398A (en) * 2014-07-30 2016-02-10 益善生物技术股份有限公司 Amplification primer for detecting food-borne pathogenic microorganisms and liquid chip kit
CN108562742A (en) * 2018-04-10 2018-09-21 海南大学 A kind of hemolysin of metal-binding polypeptides label diagnoses pathogenic microorganism as bio signal marker hypersensitive
CN110408676A (en) * 2019-07-19 2019-11-05 海南大学 A kind of microbe diagnosis sensor combining Polypeptide tags functionalization trypsase based on gold

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
REZA EIVAZZADEH-KEIHAN等: "Recent progress in optical and electrochemical biosensors for sensing of Clostridium botulinum neurotoxin", 《TRAC TRENDS IN ANALYTICAL CHEMISTRY》 *
YI WANG等: "Time-resolved botulinum neurotoxin A activity monitored using peptide-functionalized Au nanoparticle energy transfer sensors", 《CHEM. SCI.》 *
YI WAN等: "Electrical Signal Reporter, Pore-Forming Protein, for Rapid, Miniaturized, and Universal Identification of Microorganisms", 《ANAL CHEM》 *
邵侃凯等: "酿酒酵母中表达神经肉毒素A及其功能研究", 《食品与生物技术学报》 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114392354A (en) * 2022-01-14 2022-04-26 国家纳米科学中心 DNA nano system for tumor targeting and preparation method and application thereof
CN114392354B (en) * 2022-01-14 2023-11-07 国家纳米科学中心 DNA nano system for tumor targeting and preparation method and application thereof

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