Ultra-sensitive diagnosis pathogenic microorganism by taking hemolysin with metal-bound polypeptide label as biological signal marker
Technical Field
The invention relates to a nano-gold sensor assembled by hemolysin of a metal-bound polypeptide label and a DNA detection probe for diagnosing pathogenic microorganisms with ultrasensitiveness.
Background
Infectious diseases remain a significant risk to human health since the 21 st century, especially in third world countries. The WHO announced that over the last 30 years, 29 new pathogens were newly discovered. At present, the types of microorganisms causing infectious diseases are increasingly complex, the threat of common pathogenic microorganisms is not eliminated, and drug-resistant strains, such as staphylococcus, enterococcus, pseudomonas aeruginosa, escherichia coli and the like, appear, and the appearance of new pathogens brings great difficulty to clinical diagnosis and treatment. SARS causes global disasters and panic in 2003, and serious reality such as bird flu outbreak, human infection caused by streptococcus suis, vibrio parahaemolyticus infection and human infection caused by bovine anthrax occur successively in this year, and the occurrence frequency is more and more frequent, and the like, provides higher requirements for the detection and diagnosis of pathogenic microorganisms. Meanwhile, the definite diagnosis of pathogenic bacteria is also the basis of reasonable medication. The food microorganism inspection and diagnosis in daily life is an essential important component part for food quality management, is a thorough 'prevention-oriented' guideline, can effectively prevent or reduce the occurrence of diseases of food, people and livestock, and guarantees the physical health of people. The food microbiological examination is one of the important indexes for measuring the food sanitation quality and is also one of the scientific bases for judging whether the detected food is eaten or not. Through food microbiological examination, the food processing environment and the food sanitation condition can be judged, the degree of the food polluted by bacteria can be correctly evaluated, and a scientific basis is provided for various sanitation management works.
The biological signal marker is a biochemical index which can mark the change or possible change of the structure or function of a target, a system, an organ, a tissue, a cell and a subcellular, and has very wide application. It can be used for disease diagnosis, microbial infection or to evaluate the effectiveness of new therapies.
The signal marker was chosen according to the following principle: firstly, the selected biomarker must have certain specificity; the selected biomarker must have sufficient sensitivity, i.e., the level of the selected marker must have a dose-response relationship with the level of external exposure, which relationship is maintained at levels of exposure that are not deleterious; the repeatability and individual difference of the selected biomarker analysis are within an acceptable range; and fourthly, the selected biomarker has enough stability and is convenient for transportation, storage and analysis of the sample.
The signal label should have the following properties: the sensitivity is higher than general biological detection indexes, can be detected at low dose and can be operated in a micro-scale manner; secondly, the reaction time effect is achieved, the reaction needs to have certain stable time and needs to be rapid; effect of the effect marker on molecular and biochemical level is closely linked with effect (such as growth and reproduction) on high-level biological level, and effect on each level has causal relationship; and fourthly, the method requires to select indexes with little damage to the tested organisms, and the technology is easy to master. 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.
The field of biosensors is developing low-cost portable devices with higher sensitivity, which are more convenient and can be monitored continuously in real time. These are important for bacterial identification and detection, enabling ultra-sensitive detection, and increasing sensitivity to single molecule detection is critical for preventing pathogen infection. In this regard, biosensors based on nanomaterial signal labeling offer the promise of increased sensitivity while simultaneously detecting multiple targets. Nanomaterials, due to their large surface area-to-volume ratio, increase the amount of biomolecules immobilized on the surface and maximize the number of binding sites. Thus, nanomaterials provide tremendous signal enhancement. Furthermore, future developments in the field of biosensors should integrate microfluidic and micro-optical components in order to be able to position the sample processing in a single handheld device in order to meet the requirements of on-site analysis.
According to the technology, hemolysin is used as a signal label, after hemolysin molecules are dissociated from a nano sensor platform, the hemolysin molecules and a phospholipid double-layer membrane act to generate current signals, the generated current signals are further researched to be in direct proportion to the quantity of the hemolysin in a solution, and the microbial nucleic acid can be detected in an ultra-sensitive mode through the measurement of a pA (paper A) ammeter.
Disclosure of Invention
In order to achieve the purpose, the invention adopts the technical scheme that:
1. a hemolysin with gold metal combined with polypeptide label as biological signal marker for diagnosing pathogenic microbe in ultra-sensitive mode features that the metal is combined with polypeptide label and hemolysin protein
2. The metal-binding polypeptide tag of claim 1, which may comprise gold-binding polypeptides, ferroferric oxide-binding polypeptides, platinum-binding polypeptides, cobalt-binding polypeptides, and iron-platinum alloy-binding polypeptides.
3. The gold-binding polypeptide tag of claim 2, wherein the amino acid sequence comprises VSGSSPDS, LKALPPSRLPS, TGTSVLIATPYV.
4. The ferroferric oxide binding polypeptide label based on the label in the claim 2 has an amino acid sequence: LSTVQTISPSNH are provided.
5. The platinum-binding polypeptide tag of claim 2, having the amino acid sequence: CPTSTGQAC, CTLHVSSYC are provided.
6. The cobalt-binding polypeptide tag of claim 2, having the amino acid sequence: HSVRWLLPGAHP, KLHSSPHTLPVQ are provided.
7. The iron-platinum alloy binding polypeptide tag based on claim 2, wherein the amino acid sequence of the tag is as follows: HNKHLPSTQPLA, SVSVGMKPSPRP, VISNHRESSRPL are provided.
8. The hemolysin protein tag of claim 1, which can include alpha-hemolysin, beta-hemolysin, and gamma-hemolysin.
Drawings
FIG. 1A flow chart for diagnosing microbial nucleic acid molecules based on gold-binding polypeptide labeled hemolysin as a signal marker: target capture (1), hemolysin labeling (2), functionalized nanogold assembly (3), gold-binding polypeptide competitive dissociation (4) and nanopore signal measurement (5).
FIG. 2 SDS-PAGE profiles of three different gold-binding polypeptide labeled hemolysins.
FIG. 3 is a standard curve for diagnosing different concentrations of nucleic acid molecules based on gold-binding polypeptide labeled hemolysin as a signal marker.
FIG. 4 is a standard curve for diagnosing Staphylococcus aureus microorganisms at different concentrations based on gold-binding polypeptide labeled hemolysin as a signal marker.
Detailed Description
The invention is further illustrated by the following examples.
Example 1
Mu.l of streptavidin-functionalized magnetic beads (2 mg/mL) was mixed with 40. mu.l of buffer (0.2M NaCl, 10 mMPBS, 0.1% Tween 20, pH 7.4) and reacted with 20. mu.l of artificially synthesized Staphylococcus aureus target DNA (0.1 pM) for 30 min. The reaction system is washed 10 times by buffer solution, 40 mul of functionalized gold nanoprobe (2.0nM) is added for reaction for 30min, and the reaction system is washed 6 times by buffer solution. 0.1ml of gold-binding polypeptide (2. mu.M) was added thereto and reacted for 20 min. The supernatant was taken and added to a nanopore test system (eONE, Elements SRL, Italy) for testing, and the relevant current signal was collected.
Example 2
Mu.l of streptavidin-functionalized magnetic beads (2 mg/mL) was mixed with 40. mu.l of buffer (0.2M NaCl, 10 mMPBS, 0.1% Tween 20, pH 7.4) and reacted with 20. mu.l of artificially synthesized Staphylococcus aureus target DNA (1 pM) for 30 min. The reaction system is washed 10 times by buffer solution, 40 mul of functionalized gold nanoprobe (2.0nM) is added for reaction for 30min, and the reaction system is washed 6 times by buffer solution. 0.1ml of gold-binding polypeptide (2. mu.M) was added thereto and reacted for 20 min. The supernatant was taken and added to a nanopore test system (eONE, Elements SRL, Italy) for testing, and the relevant current signal was collected.
Example 3
Mu.l of streptavidin-functionalized magnetic beads (2 mg/mL) was mixed with 40. mu.l of buffer (0.2M NaCl, 10 mMPBS, 0.1% Tween 20, pH 7.4) and reacted with 20. mu.l of artificially synthesized Staphylococcus aureus target DNA (10 pM) for 30 min. The reaction system is washed 10 times by buffer solution, 40 mul of functionalized gold nanoprobe (2.0nM) is added for reaction for 30min, and the reaction system is washed 6 times by buffer solution. 0.1ml of gold-binding polypeptide (2. mu.M) was added thereto and reacted for 20 min. The supernatant was taken and added to a nanopore test system (eONE, Elements SRL, Italy) for testing, and the relevant current signal was collected.
Example 4
Mu.l of streptavidin-functionalized magnetic beads (2 mg/mL) was mixed with 40. mu.l of buffer (0.2M NaCl, 10 mMPBS, 0.1% Tween 20, pH 7.4) and reacted with 20. mu.l of artificially synthesized Staphylococcus aureus target DNA (100 pM) for 30 min. The reaction system is washed 10 times by buffer solution, 40 mul of functionalized gold nanoprobe (2.0nM) is added for reaction for 30min, and the reaction system is washed 6 times by buffer solution. 0.1ml of gold-binding polypeptide (2. mu.M) was added thereto and reacted for 20 min. The supernatant was taken and added to a nanopore test system (eONE, Elements SRL, Italy) for testing, and the relevant current signal was collected.
Example 5
Mu.l of streptavidin-functionalized magnetic beads (2 mg/mL) was mixed with 40. mu.l of buffer (0.2M NaCl, 10 mMPBS, 0.1% Tween 20, pH 7.4) and reacted with 20. mu.l of artificially synthesized Staphylococcus aureus target DNA (1 nM) for 30 min. The reaction system is washed 10 times by buffer solution, 40 mul of functionalized gold nanoprobe (2.0nM) is added for reaction for 30min, and the reaction system is washed 6 times by buffer solution. 0.1ml of gold-binding polypeptide (2. mu.M) was added thereto and reacted for 20 min. The supernatant was taken and added to a nanopore test system (eONE, Elements SRL, Italy) for testing, and the relevant current signal was collected.
Example 6
Mu.l of streptavidin-functionalized magnetic beads (2 mg/mL) was mixed with 40. mu.l of buffer (0.2M NaCl, 10 mMPBS, 0.1% Tween 20, pH 7.4) and reacted with 20. mu.l of artificially synthesized Staphylococcus aureus target DNA (10 nM) for 30 min. The reaction system is washed 10 times by buffer solution, 40 mul of functionalized gold nanoprobe (2.0nM) is added for reaction for 30min, and the reaction system is washed 6 times by buffer solution. 0.1ml of gold-binding polypeptide (2. mu.M) was added thereto and reacted for 20 min. The supernatant was taken and added to a nanopore test system (eONE, Elements SRL, Italy) for testing, and the relevant current signal was collected.
Example 7
Mu.l of streptavidin-functionalized magnetic beads (2 mg/mL) was mixed with 40. mu.l of buffer (0.2M NaCl, 10 mMPBS, 0.1% Tween 20, pH 7.4) and reacted with 20. mu.l of artificially synthesized Staphylococcus aureus target DNA (100 nM) for 30 min. The reaction system is washed 10 times by buffer solution, 40 mul of functionalized gold nanoprobe (2.0nM) is added for reaction for 30min, and the reaction system is washed 6 times by buffer solution. 0.1ml of gold-binding polypeptide (2. mu.M) was added thereto and reacted for 20 min. The supernatant was taken and added to a nanopore test system (eONE, Elements SRL, Italy) for testing, and the relevant current signal was collected.
Example 8
Mu.l of streptavidin-functionalized magnetic beads (2 mg/mL) was mixed with 40. mu.l of buffer (0.2M NaCl, 10 mMPBS, 0.1% Tween 20, pH 7.4) and reacted with 20. mu.l of artificially synthesized Staphylococcus aureus target DNA (1000 nM) for 30 min. The reaction system is washed 10 times by buffer solution, 40 mul of functionalized gold nanoprobe (2.0nM) is added for reaction for 30min, and the reaction system is washed 6 times by buffer solution. 0.1ml of gold-binding polypeptide (2. mu.M) was added thereto and reacted for 20 min. The supernatant was taken and added to a nanopore test system (eONE, Elements SRL, Italy) for testing, and the relevant current signal was collected.
Example 9
Mu.l of streptavidin-functionalized magnetic beads (2 mg/mL) was mixed with 40. mu.l of buffer (0.2M NaCl, 10 mMPBS, 0.1% Tween 20, pH 7.4) and reacted with 20. mu.l of Staphylococcus aureus target (1 cfu/mL) for 30 min. The reaction system is washed 10 times by buffer solution, 40 mul of functionalized gold nanoprobe (2.0nM) is added for reaction for 30min, and the reaction system is washed 6 times by buffer solution. 0.1ml of gold-binding polypeptide (2. mu.M) was added thereto and reacted for 20 min. The supernatant was taken and added to a nanopore test system (eONE, Elements SRL, Italy) for testing, and the relevant current signal was collected.
Example 10
Mu.l of streptavidin-functionalized magnetic beads (2 mg/mL) was mixed with 40. mu.l of buffer (0.2M NaCl, 10 mMPBS, 0.1% Tween 20, pH 7.4) and reacted with 20. mu.l of Staphylococcus aureus target (10 cfu/mL) for 30 min. The reaction system is washed 10 times by buffer solution, 40 mul of functionalized gold nanoprobe (2.0nM) is added for reaction for 30min, and the reaction system is washed 6 times by buffer solution. 0.1ml of gold-binding polypeptide (2. mu.M) was added thereto and reacted for 20 min. The supernatant was taken and added to a nanopore test system (eONE, Elements SRL, Italy) for testing, and the relevant current signal was collected.
Example 11
Mu.l of streptavidin-functionalized magnetic beads (2 mg/mL) was mixed with 40. mu.l of buffer (0.2M NaCl, 10 mMPBS, 0.1% Tween 20, pH 7.4) and reacted with 20. mu.l of Staphylococcus aureus target (100 cfu/mL) for 30 min. The reaction system is washed 10 times by buffer solution, 40 mul of functionalized gold nanoprobe (2.0nM) is added for reaction for 30min, and the reaction system is washed 6 times by buffer solution. 0.1ml of gold-binding polypeptide (2. mu.M) was added thereto and reacted for 20 min. The supernatant was taken and added to a nanopore test system (eONE, Elements SRL, Italy) for testing, and the relevant current signal was collected.
Example 12
Mu.l of streptavidin-functionalized magnetic beads (2 mg/mL) was mixed with 40. mu.l of buffer (0.2M NaCl, 10 mMPBS, 0.1% Tween 20, pH 7.4) and reacted with 20. mu.l of Staphylococcus aureus target (103 cfu/mL) for 30 min. The reaction system is washed 10 times by buffer solution, 40 mul of functionalized gold nanoprobe (2.0nM) is added for reaction for 30min, and the reaction system is washed 6 times by buffer solution. 0.1ml of gold-binding polypeptide (2. mu.M) was added thereto and reacted for 20 min. The supernatant was taken and added to a nanopore test system (eONE, Elements SRL, Italy) for testing, and the relevant current signal was collected.
Example 13
Mu.l of streptavidin-functionalized magnetic beads (2 mg/mL) was mixed with 40. mu.l of buffer (0.2M NaCl, 10 mMPBS, 0.1% Tween 20, pH 7.4) and reacted with 20. mu.l of Staphylococcus aureus target (104 cfu/mL) for 30 min. The reaction system is washed 10 times by buffer solution, 40 mul of functionalized gold nanoprobe (2.0nM) is added for reaction for 30min, and the reaction system is washed 6 times by buffer solution. 0.1ml of gold-binding polypeptide (2. mu.M) was added thereto and reacted for 20 min. The supernatant was taken and added to a nanopore test system (eONE, Elements SRL, Italy) for testing, and the relevant current signal was collected.
Example 14
Mu.l of streptavidin-functionalized magnetic beads (2 mg/mL) was mixed with 40. mu.l of buffer (0.2M NaCl, 10 mMPBS, 0.1% Tween 20, pH 7.4) and reacted with 20. mu.l of Staphylococcus aureus target (104 cfu/mL) for 30 min. The reaction system is washed 10 times by buffer solution, 40 mul of functionalized gold nanoprobe (2.0nM) is added for reaction for 30min, and the reaction system is washed 6 times by buffer solution. 0.1ml of gold-binding polypeptide (2. mu.M) was added thereto and reacted for 20 min. The supernatant was taken and added to a nanopore test system (eONE, Elements SRL, Italy) for testing, and the relevant current signal was collected.
Example 15
Mu.l of streptavidin-functionalized magnetic beads (2 mg/mL) was mixed with 40. mu.l of buffer (0.2M NaCl, 10 mMPBS, 0.1% Tween 20, pH 7.4) and reacted with 20. mu.l of Staphylococcus aureus target (105 cfu/mL) for 30 min. The reaction system is washed 10 times by buffer solution, 40 mul of functionalized gold nanoprobe (2.0nM) is added for reaction for 30min, and the reaction system is washed 6 times by buffer solution. 0.1ml of gold-binding polypeptide (2. mu.M) was added thereto and reacted for 20 min. The supernatant was taken and added to a nanopore test system (eONE, Elements SRL, Italy) for testing, and the relevant current signal was collected.
Example 16
Mu.l of streptavidin-functionalized magnetic beads (2 mg/mL) was mixed with 40. mu.l of buffer (0.2M NaCl, 10 mMPBS, 0.1% Tween 20, pH 7.4) and reacted with 20. mu.l of Staphylococcus aureus target (106 cfu/mL) for 30 min. The reaction system is washed 10 times by buffer solution, 40 mul of functionalized gold nanoprobe (2.0nM) is added for reaction for 30min, and the reaction system is washed 6 times by buffer solution. 0.1ml of gold-binding polypeptide (2. mu.M) was added thereto and reacted for 20 min. The supernatant was taken and added to a nanopore test system (eONE, Elements SRL, Italy) for testing, and the relevant current signal was collected.
Example 17
Mu.l of streptavidin-functionalized magnetic beads (2 mg/mL) was mixed with 40. mu.l of buffer (0.2M NaCl, 10 mMPBS, 0.1% Tween 20, pH 7.4) and reacted with 20. mu.l of Staphylococcus aureus target (107 cfu/mL) for 30 min. The reaction system is washed 10 times by buffer solution, 40 mul of functionalized gold nanoprobe (2.0nM) is added for reaction for 30min, and the reaction system is washed 6 times by buffer solution. 0.1ml of gold-binding polypeptide (2. mu.M) was added thereto and reacted for 20 min. The supernatant was taken and added to a nanopore test system (eONE, Elements SRL, Italy) for testing, and the relevant current signal was collected.