CN115725685A - Method for detecting drug molecules based on biological ehmitis pores - Google Patents

Method for detecting drug molecules based on biological ehmitis pores Download PDF

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CN115725685A
CN115725685A CN202210757743.8A CN202210757743A CN115725685A CN 115725685 A CN115725685 A CN 115725685A CN 202210757743 A CN202210757743 A CN 202210757743A CN 115725685 A CN115725685 A CN 115725685A
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pore
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drug
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CN115725685B (en
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耿佳
包锐
柯博文
魏于全
陈路
赵长健
王裕
朱益波
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Sichuan University
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Abstract

The invention belongs to the field of nanopore detection, and particularly relates to a method for detecting a drug molecule based on a biological angstrom pore. The method provided by the invention comprises the following steps: s1 adding the sample to a rice bore system, the rice bore system comprising: the dielectric layer comprises a Hermitian pore, an insulating film, a first dielectric and a second dielectric, wherein the Hermitian pore is an MscS Hermitian pore, and has a heptamer structure which is radially symmetrical and is shaped like a cylinder, and the heptamer structure comprises 7 side openings and 1 bottom opening; the sample is added to the first medium; s2, applying a driving force to the first medium and the second medium, wherein the drug molecules in the sample interact with the pores and generate an electric signal; s3, analyzing the electric signal, and further identifying the drug molecules in the sample. The method provided by the invention can detect the drug molecules and the concentration thereof (quantitative analysis), and can also directly detect the drug molecules in the whole blood sample.

Description

Method for detecting drug molecules based on biological ehmitis pores
Chinese patent application No. CN2021110062606, entitled "biomacropore system for dNTPs and neocoronavirus detection based on PaMscS, filed on 30.08.2021, and chinese patent application No. CN2021110042496, entitled" biomacropore system for small molecule drug detection and whole blood detection based on PaMscS, filed on 30.2021, both of which are incorporated by reference in their entireties.
Technical Field
The invention belongs to the field of nanopore detection, and particularly relates to a method for detecting drug molecules based on biological ehmitic pores.
Background
Blood concentration monitoring plays an important role in rational and effective use of drugs to reduce side effects. The current methods for clinical monitoring of blood drug concentrations are mainly HPLC, LS/MS2 and ELISA, among others. However, these existing blood monitoring devices tend to require relatively high costs and complex operations. First, these devices often complicate and expensive blood drug monitoring, severely limiting the popularity of blood drug monitoring. Due to the lack of adequate methods, some medical treatments are performed based on the clinical experience of the physician rather than on accurate Therapeutic Drug Monitoring (TDM) equipment, resulting in an increased incidence of medical malpractice. Secondly, most of the currently clinically applied blood drug monitoring is discrete and cannot accurately display the continuous process of drug metabolism, however, a continuous drug monitoring method has an urgent clinical need. At present, the method can realize accurate measurement of the concentration of the drug in blood, but mainly depends on chemical reaction, aptamer or antibody, and although the method has higher specificity, the detection range of the drug is limited.
Nanopore sensing is a single molecule sensing technology with detection principles similar to the coulter counter (Kurt counter). This technique has the characteristic of real-time and direct monitoring at the single molecule level, and generally does not require labeling or modification of the analyte. These advantages make nanopores an emerging technology for biosensing and biodetection. Most biological nanopores have diameters between about 1 nm and about 4nm (e.g., mspA, α -HL20, and phi 29DNA packaging motors), and are suitable for sensing of single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA). However, for sensing of smaller molecules, it is often necessary to label or adapt, such as site-directed mutagenesis or to modify a particular adapter. Taking alpha-HL as an example, the limited pore diameter is about 1.4nm, so the application range is only limited in the analysis of ssDNA, RNA or other molecules, and the dNMPs can be directly detected by using cyclodextrin (cyclodextrin) modification without fluorescent labeling. However, the modification of the pore size of the biological nanopore by a modification means requires a great deal of assistance of bioengineering technology, and in addition, the continuous detection of drug molecules by the existing method and technology combined with appropriate ligands is difficult, and only standard products can be detected. Therefore, continuous detection of drugs in whole blood samples through nanopores remains a challenge.
Based on this, the invention develops a drug molecule monitoring strategy based on the hermite sensing in order to alleviate one or more of the existing problems.
Disclosure of Invention
In view of this, the invention provides a method for detecting drug molecules based on an Hermite well, and the specific technical scheme is as follows.
A method of detecting a drug molecule in a sample, comprising the steps of:
s1 adding the sample to a rice bore system, the rice bore system comprising: an angstrom pore, an insulating film, a first dielectric, a second dielectric, wherein said angstrom pore is embedded in said insulating film, said insulating film separates said first dielectric from said second dielectric, said angstrom pore provides a channel that connects said first dielectric with said second dielectric, said angstrom pore is an MscS angstrom pore, said angstrom pore has a radially symmetric and cylindrically shaped heptamer structure, said heptamer structure comprises 7 side openings and 1 bottom opening; the sample is added to the first medium;
s2, applying a driving force to the first medium and the second medium, wherein the drug molecules in the sample interact with the pores and generate an electric signal;
s3, analyzing the electric signal, and further identifying the drug molecules in the sample.
Further, the charge properties and/or pore size of the openings are adjustable.
Further, the adjustment of the opening includes subjecting the insulating film to a mechanical force and/or changing the physical state of the insulating film.
Further, the mechanical force stimulus includes one or more of a change in osmotic pressure difference of a medium on both sides of the insulating film, a direct physical stimulus of a micro needle to the insulating film, and a stimulus of a negative pressure of air to the insulating film.
Further, the aperture of the opening may be adjusted according to the following:
(1) Selecting a type of the first medium and the second medium; and/or
(2) A difference in osmotic pressure between the first medium and the second medium.
Further, the hermite pore is an MscS variant hermite pore.
Further, the MscS variants comprise a side-hole volume variant and/or a side-hole charge variant.
Further, the E.oryzae is derived from a bacillus.
Further, the hermite pores include one or more of pseudomonas aeruginosa, escherichia coli, thermophilic anaerobes tengchongensis, and helicobacter pylori.
Further, the hermite pore is a pamsccs variant hermite pore.
Further, the hermite comprises one or more of the following variants: 130A, 130H, 180R, 271I, 130S and 130P.
Further, the molecular weight of the drug molecule is less than 1000g/mol.
Furthermore, the molecular weight of the drug molecule is 177.98-712.72 g/mol.
Further, the concentration of the drug molecule is greater than 10nM.
Further, the sample is a body fluid sample.
Further, the body fluid sample comprises one or more of urine, blood, serum, plasma, lymph, cyst fluid, pleural fluid, ascites fluid, peritoneal fluid, amniotic fluid, epididymis fluid, cerebrospinal fluid, bronchoalveolar lavage fluid, breast milk, tears, saliva, and sputum.
Further, the sample size of the body fluid sample is more than 10 μ L.
Further, the concentration of drug molecules in the body fluid sample is greater than 10nM.
Further, the method further comprises S4: communicating a dialysis device with said first medium via a conduit such that said blood sample enters said emmetro-pore system via said dialysis device, wherein S4 precedes S1.
Further, the insulating film includes a phospholipid film and/or a polymer film.
Compared with the prior art, the invention has the beneficial effects that:
the invention provides a method for detecting a drug molecule in a sample using a hermite system, wherein the hermite system comprises an MscS hermite. The invention creatively utilizes the characteristic of a small conductance mechanical force sensitivity channel (MscS) to detect the drug molecules in the sample, which is specifically embodied as follows:
1) The MscS Hermitian pore is narrow in diameter. It is estimated that MscS angstrom pores have a pore size in the range of 6-16 angstrom meters, much smaller than nanopores commonly used in the prior art (e.g., alpha-hemolysin nanopores have a pore size of about 1.4-2.4nm, i.e., 14-24 angstrom meters).
2) The MscS Hermitian pore size is tunable (also can be understood as structurally flexible). The MscS emmetropore can convert a mechanical stimulus to an electrical or biochemical signal within milliseconds, thereby initiating modulation of the tunnel configuration. By utilizing the sensitivity of the MscS angstrom pores to the mechanical force stimulation on the insulating film and/or the change of the physical state of the insulating film, the adjustment of the MscS angstrom pores can be realized by influencing the insulating film without complicated chemical modification. For example, the concentration of the first medium and the second medium (i.e., 30mM NaCl/300mM NaCl, 100mM NaCl/300mM NaCl, and 300mM NaCl/300mM NaCl) can be adjusted to adjust the osmotic pressure difference across the insulating film and thus the pore size, to optimize the selectivity for the analyte and to improve the discrimination of the analyte. Protein nanopores in the prior art usually have a fixed channel structure, and additional protein engineering modification or chemical modification and the like are required to realize channel structure adjustment. The MscS angstrom pore diameter related by the invention can realize reversible in-situ adjustment only by changing external conditions, and is suitable for direct detection of various types and sizes of drug molecules. Specifically, drug molecules such as aminoglycoside antibiotics and glutamate all cause a corresponding blocking current signal in the MscS hermite, which enables detection of drug molecules at the single molecule level.
3) The MscS emmetropore can realize the quantitative analysis of the drug molecules. The gradient concentration measurement of the drug molecules presents a good linear relationship between the signal frequency and the drug concentration, so that the MscS hermite can not only detect the drug molecules but also realize the detection (quantitative analysis) of the concentration of the drug molecules.
4) The MscS emmer hole has strong anti-interference capability. The MscS cytoplasmic end is a sieve-like structure with 1 bottom opening at the bottom and 7 side openings at the side, and the tunnel of each opening (pore) is narrow, which is favorable for passing ions and small molecules, but can block macromolecular substances (such as proteins) outside the tunnel, so that the biological macromolecules can not enter the tunnel and can not cause obstruction to the tunnel. Thus, mscS exhibits a strong interference rejection and enables direct detection of a bodily fluid sample (e.g., a whole blood sample). More specifically, the method can be used together with devices such as a dialysis device and the like to realize real-time and continuous monitoring of the blood concentration.
As used herein, the term "derived from" refers not only to proteins produced by the strain of organism in question, but also to proteins encoded by DNA sequences isolated from such strains and produced in a host organism containing such DNA sequences.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. It is obvious that the drawings in the following description are some embodiments of the invention, and that for a person skilled in the art, other drawings can be derived from them without inventive exercise.
FIG. 1 shows electrophysiological measurements of PaMscS-Amicron pores;
FIG. 2 shows drug unimolecular biosensing based on PaMscS2 Ammi pore;
FIG. 3 shows drug concentration measurements of whole blood samples;
FIG. 4 shows a proof of concept for continuous monitoring of drug concentration in vivo in living rats via Ammi well;
FIG. 5 shows the SDS-PAGE results of PaMscS proteins;
fig. 6 shows the overall structure of PaMscS;
FIG. 7 shows a continuous current trace for a PaMscS2 (V271I) Amyloric pore with gentamicin sulfate;
FIG. 8 shows a continuous current trace for a PaMscS2 (V271I) Amyloric pore with neomycin sulfate;
FIG. 9 shows that high concentrations of gentamicin sulfate and neomycin sulfate can block PaMscS2 (V271I) epothilone pores for extended periods of time;
FIG. 10 shows that MspA-2NNN E-Mi pores can be frequently blocked by a whole blood sample (10 μ L of whole blood sample to add 1mL end);
FIG. 11 shows a continuous current trace for a PaMscS2 (V271I) Ammi pore in a blood sample;
FIG. 12 shows the current trace for direct measurement of a rat whole blood sample through a PaMscS2 (V271I) Ammi pore;
FIG. 13 shows the current signal for gentamicin sulfate through a PaMscS2 (V271I) epothilone pore from-50 mV to-80 mV;
FIG. 14 shows the current signal for sisomicin through a PaMscS2 (V271I) angstrom pore from-50 mV to-80 mV;
FIG. 15 shows the transport frequency of dNTPs through a PaMscS3 (W130A) pore under different osmotic pressure conditions;
FIG. 16 shows dNTP detection based on a wild-type PaMscS emittor;
fig. 17 shows the current trace for PaMscS1 detection of glutamate;
FIG. 18 shows a PaMscS-Ammi pore-based amino acid detection scheme and different amino acid blocking current profiles;
FIG. 19 shows a single channel embedded current trace for wild-type EcMscS (voltage +100mV, conductivity 30mM;
FIG. 20 shows the channel scan voltage (-100 mV to 100 mV) for wild-type EcMscS;
figure 21 shows conductance profiles of wild-type EcMscS;
figure 22 shows the sequence alignment of PaMscS with MscS of other bacteria.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention. It is to be understood that the embodiments described are only a few embodiments of the present invention, and not all embodiments. All other embodiments, which can be obtained by a person skilled in the art without inventive step based on the embodiments of the present invention, are within the scope of protection of the present invention.
It should be noted that, in this document, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising one of 8230, and" comprising 8230does not exclude the presence of additional like elements in a process, method, article, or apparatus comprising the element.
As used in this specification, the term "about" typically means +/-5% of the stated value, more typically +/-4% of the stated value, more typically +/-3% of the stated value, more typically +/-2% of the stated value, even more typically +/-1% of the stated value, and even more typically +/-0.5% of the stated value.
In this specification, certain embodiments may be disclosed in a range of formats. It should be understood that this description of "within a certain range" is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, the description of the range 1 to 6 should be read as having specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within this range, such as 1,2,3,4, 5, and 6. The above rules apply regardless of the breadth of the range.
Detailed description of the drawings
FIG. 1: A. schematic diagram of an electrophysiological measurement tip. PaMscS1 (K180R) angstrom pores and PaMscS2 (V271I) single pore insertion at +50mV voltage, both PaMscS1 and PaMscS2 angstrom pores maintain stable tunnel current under these conditions. I-V relationship (N =4, respectively) for PaMscS1 and PaMscS2 emm pores at a voltage range of-50 mV to +50 mV. Conductance profile of pamscs s2 angstrom pores at +50mV voltage (N = 108). The electrolyte conditions were-cis terminal: 300mM NaCl, -trans end: 30mM NaCl,10mM HEPES, pH 7.0.
FIG. 2: A. results for gentamicin sulfate, including representative current traces and occlusion signals, were detected through the PaMscS2 (V271I) emmetron pore. B. Results for neomycin sulfate detection by PaMscS2 emmer pores. C. Quantitative standard curve for gentamicin sulfate (N = 3) and heat map of occlusion signal (right, 878 occlusion events). D. Quantitative standard curve for neomycin sulfate (middle, N = 4) and heat map of occlusion signal (right, 883 occlusion events). The electrolyte conditions were-cis terminal: 300mM NaCl, -trans end: 30mM NaCl,10mM HEPES, pH7.0, and the voltage for drug detection was-50 mV. E. The results of the detection of 1.5. Mu.M gentamicin sulfate between LC-MS and PaMscS2 Ammi wells were compared. The electrolyte conditions were-cis terminal: 130mM NaCl, -trans end: 130mM NaCl,10mM HEPES, pH7.0, the detection voltage of the drug was-50 mV.
FIG. 3: A. the whole blood sample was assayed directly through a PaMscS2 (V271I) Amylor pore, which remained open after the addition of the whole blood sample. The electrolyte conditions were-cis terminal: 130mM NaCl, -trans end: 130mM NaCl,10mM HEPES, pH7.0, voltage-50 mV. B. After addition of 20. Mu.L rat blood, the-cis terminal conductance buffer turned red. C. Percentage of the channel of the whole blood sample open. D. The quantitative standard curve range of gentamicin sulfate is 0 to 3 μ M. E. Drug concentrations were measured by PaMscS2 emmetropium in rats at various time intervals after gentamicin injection. The electrolyte conditions for whole blood emittor assay were-cis: 130mM NaCl, -trans end: 130mM NaCl,10mM HEPES, pH7.0, voltage of-50 mV, N.gtoreq.3.
FIG. 4 is a schematic view of: A. rat drug concentration monitoring system. B. The quantitative standard curve for gentamicin sulfate ranged from 0 to 30 μ M. C. Typical current traces for PaMscS2 (V271I) Am pores during drug concentration monitoring with 4mg/kg and 20mg/kg gentamicin sulfate injections. D. Results of continuous drug blood concentration monitoring of rats through PaMscS s2 angstrom wells at different doses of gentamicin sulfate (N = 1). Grey data points indicate that the drug blocking signal frequency is higher than the highest signal frequency within the standard curve range, and that the double step signal occurs frequently and makes the quantification inaccurate. The electrolyte conditions were-cis terminal: 130mM NaCl, -trans end: 130mM NaCl,10mM HEPES, pH7.0, at a voltage of-50 mV.
FIG. 5: the strip includes: a label; a PaMscS1 (K180R) mutant; a PaMscS2 (V271I) mutant.
FIG. 6: alignment of sequences of the MscS family. Residues highlighted in red are identical in the 4 sequences. The cylinders above the sequence are designated as alpha helix and beta strand.
FIG. 7 is a schematic view of: current trace against the background of the ehmitic well and the trace after addition of gentamicin sulfate. The electrolyte conditions were-cis terminal: 300mM NaCl, -trans end: 30mM NaCl,10mM HEPES, pH7.0, voltage-50mV, N =3.
FIG. 8: background current traces from the epothilone wells and traces after addition of neomycin sulfate. The electrolyte conditions were-cis terminal: 300mM NaCl, -trans end: 30mM NaCl,10mM HEPES, pH7.0, voltage-50mV, N =3.
FIG. 9: at high concentrations of drug, the blocking signal of the drug is difficult to count and the PaMscS2 emmetrope can be blocked for long periods of time, leading to an inability to calculate quantitatively. The electrolyte conditions were-cis terminal: 300mM NaCl, -trans end: 30mM NaCl,10mM HEPES, pH7.0, and the voltage for drug detection was-50 mV.
FIG. 10: the electrolyte conditions were-cis terminal: 300mM NaCl, -trans end: 300mM NaCl,10mM HEPES, pH7.0, voltage +100mV, N =3.
FIG. 11: current trace against the ehmi well background and the trace after addition of the whole blood sample.
FIG. 12: mu.L of rat whole blood was added to the-cis end (1 mL) and the PaMscS 2A-rice well worked well in the presence of the whole blood sample.
FIG. 13: the electrolyte conditions were-cis terminal: 130mM NaCl, -trans end: 130mMNaCl,10mM HEPES, pH 7.0. It is worth noting that two peaks occur in the gradient voltage.
FIG. 14: the electrolyte conditions were-cis terminal: 130mM NaCl, -trans end: 130mMNaCl,10mM HEPES, pH 7.0. The structure of sisomicin is close to the C1a component of gentamicin sulfate. At the gradient voltage, only one blocking current peak was observed.
FIG. 15 is a schematic view of: dCTP (expressed in orange) and dGTP (expressed in blue) were tested for translocation frequency under different differential osmotic pressures, symmetric (a, 300mM NaCl. Four sets of dNTPs concentrations, 0.5mM, 1.0mM, 1.5mM and 2.0mM, were tested for dCTP and dGTP translocation. D. Relationship between translocation frequency and dCTP/dGTP concentration under symmetric, low and high differential osmotic pressure conditions (each data point n = 3). E. Under 3 different osmotic pressure conditions, f dCTP And f dGTP The rate of increase of (c).
Hermite eye
The emittor pore used in the invention is a small conductance mechanical force sensitive channel (MscS), preferably a PaMscS (pseudomonas aeruginosa small conductance mechanical force sensitive channel) or a variant thereof. The variant (which may also be understood as a "mutant") may be a naturally occurring variant expressed by an organism, such as Pseudomonas aeruginosa. Variants also include non-naturally occurring variants produced by recombinant techniques. In the present invention, "pamsccs variant", "mutant pamsccs", "pamsccs mutant" mean the same unless otherwise specified.
In one embodiment of the invention, the emmetropic pore may be an MscS variant. Amino acid substitutions, for example single or multiple amino acid substitutions, may be made to the amino acid sequences of SEQ ID NO 1 or 2 or 3 or 4. Substitutions may be conservative or non-conservative. Preferably, one or more positions of the amino acid sequence of SEQ ID NO 1 or 2 or 3 or 4 are non-conservatively substituted, wherein the amino acid residue to be substituted is replaced by an amino acid of significantly different chemical and/or physical size. Further, the MscS variants can be divided into side hole volume variants and side hole charge variants. A side hole volume variant refers to a variant in which the mutation site is located at the lateral opening (also understood as "side hole") of the cytoplasmic end and the side hole volume is changed by changing the amino acid at that site. The side-hole charge variant refers to a variant in which the mutation site is located at the side opening of the cytoplasmic end and the side-hole charge thereof is changed by changing the amino acid at the site. For example, the side-hole volume variant can be a substitution of a larger volume of an amino acid (e.g., tryptophan (W)) for a smaller volume of an amino acid (e.g., alanine (a), serine (S), or proline (P)), or vice versa. The side-hole charge variants may be obtained by substituting an amino acid with a certain charge for an amino acid with an opposite charge or neutral charge, or by substituting an amino acid with a neutral charge for an amino acid with a charge. In general, non-limiting examples of positively charged amino acids include histidine, arginine, and lysine; non-limiting examples of negatively charged include aspartic acid and glutamic acid; neutral, non-limiting examples include glycine, alanine, phenylalanine, valine, leucine, isoleucine, cysteine, asparagine, glutamine, serine, threonine, tyrosine, methionine, proline, and tryptophan. Conservative or non-conservative substitutions of amino acids, as well as many different types of modifications (deletions, substitutions, additions) of amino acids, etc., are well known in the art, and one skilled in the art can modify MscS as appropriate to obtain the corresponding MscS variant. Means for modification include modification of the corresponding DNA sequence (e.g., direct synthesis of the corresponding protein after modification of DNA sequence information or site-directed mutagenesis of the DNA sequence by PCR) to obtain the corresponding variant (and its corresponding DNA sequence).
In a particular embodiment, the MscS variant can be a PaMscS variant. The PaMscS variants include, for example, one or more of 130A, 130H, 180R, 271I, 130S, and 130P. Side-hole volume mutants of PaMscS include, for example, 130A, 130S, 130P, side-hole charge variants of PaMscS include, for example, 130H, 180R, 271I. Such modification can change the pore size (also understood as "pore size") of the modified side pore, thereby improving the detection capability for analytes of a particular molecular volume; the local charge characteristics of the modified side-hole channel can also be changed, so that the detection capability of a specific charged analyte is improved; the stability of the protein channel currents of the PaMscS variants can also be enhanced.
In one embodiment of the invention, the hermite pore may be a wild-type pamsccs that has a high background noise but still has the ability to detect an analyte.
In one embodiment of the invention, the hermite pore may be a wild-type EcMscS (e.coli small conductance mechanical force sensitive channel) or a variant thereof. The EcMscS has a highly similar structure to pammscs and also forms a stable tunnel current, enabling the detection of analytes. Sequence similarity of PaMscS to EcMscS was 60%. Conservative substitutions or non-conservative substitutions for amino acids, as well as many different types of modifications (deletions, substitutions, additions) to amino acids, are well known in the art, and one skilled in the art can modify EcMscS to obtain corresponding EcMscS variants, as appropriate.
In another embodiment of the invention, the herminium pores may be derived from other bacilli, such as anaerobe tengcnatus (Thermoanaerobacter tengconsis) and helicobacter pylori (helicobacter pylori), in addition to Escherichia coli (Escherichia coli) and Pseudomonas aeruginosa. PaMscS is highly similar in structure to TtMscS and HpMscS, and the sequence similarity is 55% and 44%, respectively. In combination with the results of actual electrophysiological measurements of PaMscS and EcMscS, it can be seen that MscS is able to detect analytes as hermite pores because of its highly similar structure and similar function. Conservative or non-conservative substitutions for amino acids, as well as many different types of modifications (deletions, substitutions, additions) to amino acids, are known in the art, and one skilled in the art can modify MscS to obtain corresponding MscS variants, as the case may be.
Analyte
The analyte is a charged species. An analyte is charged if it carries a net charge. The analyte may be negatively or positively charged. An analyte is negatively charged if it has a net negative charge. An analyte is positively charged if it has a net positive charge. Suitable analytes should be substances with a size smaller than or equal to the pore size of the hermite pores, preferably nucleotides, amino acids, peptides, drug molecules.
In one embodiment of the invention, the analyte may be a nucleotide. "nucleotide" refers to a monomeric unit consisting of a heterocyclic base, a sugar, and a phosphate group. It is understood that heterocyclic bases include naturally occurring bases (guanine (G), adenine (A), cytosine (C), thymine (T), and uracil (U)) as well as non-naturally occurring base analogs. Sugars include naturally occurring sugars (deoxyribose and ribose) as well as non-naturally occurring sugar analogs. The nucleotides include deoxyribonucleotides and ribonucleotides such as ATP, dATP, CTP, dCTP, GTP, dGTP, UTP, TTP, dUTP, GMP, UMP, TMP, CMP, dGMP, dAMP, dTMP, dCMP, dUMP, ADP, GDP, TDP, UDP, CDP, dADP, dGDP, dTDDP, dCDP. The nucleotides include naturally occurring nucleotides and non-naturally occurring nucleotide analogs that hybridize to nucleic acids in a manner similar to naturally occurring nucleotides. The nucleotides are free (or, alternatively, can be understood as "single"). Preferably, the nucleotide is ATP, dATP, CTP, dCTP, GTP, dGTP, UTP, TTP, dUTP.
In one embodiment of the invention, the analyte may be an amino acid. "amino acid" refers to any of the 20 naturally occurring amino acids found in proteins, the D-stereoisomers of naturally occurring amino acids (e.g., D-threonine), unnatural amino acids, and chemically modified amino acids. Each of these amino acid types is not mutually exclusive. The following abbreviations are used for the 20 naturally occurring amino acids: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine (Cys; C), glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y) and valine (Val; V). The specific properties (e.g., polarity, charge, hydrophilicity, average volume) of an amino acid are known to those of skill in the art. In another embodiment of the invention, the analyte may be a short peptide, such as a dipeptide.
In one embodiment of the invention, the analyte may be a drug molecule. The drug molecule may be a compound. More specifically, a "drug molecule" can be a drug having a molecular weight of 1000g/mol or less (e.g., less than 800, 700, 600, 500, 400, 300, or 200 g/mol). Preferably, the drug molecule may be an aminoglycoside antibiotic. In another embodiment of the invention, the drug molecules include amino acids and salts thereof (including non-druggable amino acids) and peptides.
Angstrom pore system
The "angstrom pore system" includes pores having angstrom-scale dimensions (simply referred to as "angstrom pores"), an insulating film, a first dielectric, and a second dielectric. In one embodiment of the invention, the pores having angstrom-scale dimensions are small conductance mechanical force sensitive channel (MscS) angstrom pores. The pores having angstrom-scale dimensions are preferably heptameric structures having radial symmetry and a cylinder-like shape, comprising 7 side openings and 1 bottom opening. In one embodiment of the invention, the pores with angstrom-scale dimensions have a typical heptameric structure radially symmetric and shaped like a cylinder, comprising 8 openings, with 7 equal openings distributed on the side, the 8 th opening distributed on the bottom and formed by 7 subunits; the aperture sizes of the 8 openings can be adjusted. The pores having a size on the order of angstroms allow the analyte to translocate from one side of the insulating film to the other.
In one embodiment of the present invention, the pores with angstrom scale size are embedded in the insulating film, the insulating film (which may also be understood as a composite of the angstrom scale size pores and the insulating film) separates the first medium from the second medium, and the pore passages with the angstrom scale size pores provide a passage for communicating the first medium with the second medium; upon application of a driving force between the first medium and the second medium, an analyte located in the first medium interacts with the MscS Eimeria pores to form an electrical current (i.e., an electrical signal). In the present invention, "first medium" refers to the medium in which the analyte is located when added to the hermite system; the "second dielectric" refers to the other side of the "first dielectric" in the two parts of the dielectric separated by the insulating film. In the present invention, the driving force refers to the force that drives the analyte to interact with the pores by means of electrical potential, electroosmotic flow, concentration gradient, and the like.
The first medium and the second medium may be the same or different, and the first medium and the second medium may comprise an electrically conductive liquid. The electric conduction liquid is an alkali metal halide aqueous solution, and specifically is sodium chloride (NaCl), lithium chloride (LiCl), cesium chloride (CsCl), potassium chloride (KCl) and sodium bromide (NaCl). In one embodiment of the present invention, the first medium and the second medium contain different concentrations of the electrically conductive liquid, in other words, there is a difference in the concentrations of the electrically conductive liquid in the first medium and the second medium, so that there is a difference in osmotic pressure across the insulating film. The first medium and/or the second medium may also include a buffer, such as HEPES. The concentration of the first medium and/or the second medium may range from 30mM to 3M.
The insulating film refers to a film having the ability to carry angstrom pores (or nanopores) and block an ion current passing through non-angstrom pores (or nanopores). The insulating film may include a phospholipid film and/or a polymer film. Exemplary phospholipid membranes include DPHPC, DOPC, e.
The present angstrom pore system may comprise any of the small conductance mechanical force sensitive channels described herein, such as wild-type PaMscS (SEQ ID NO: 1), wild-type EcMscS (SEQ ID NO: 2), wild-type TtMscS (SEQ ID NO: 3), and wild-type HpMscS (SEQ ID NO: 4), and their corresponding variants, with specific sequence information for the four MscS described above as shown in Table 1. For example, the small conductance mechanical force-sensitive channel can be a mutant PaMscS1 (K180R), a mutant PaMscS2 (V271I), a mutant PaMscS3 (W130A).
In one embodiment of the invention, the angstrom pore system includes two electrolyte chambers separated by an insulating film to form a trans (-trans) compartment and a cis (-cis) compartment, the pores of the angstrom pore being embedded in the insulating film, there being only small conductance mechanical force sensitive channels on the insulating film to communicate the two electrolyte chambers. When an electric potential is applied to the above two electrolyte chambers, electrolyte ions in the solution in the electrolyte chamber move through the angstrom pore by electrophoresis.
In one embodiment of the invention, the small conductance mechanical force sensitive tunnel (MscS) angstrom pores may be embedded in an insulating film, but retain the ability to change the structure of proteins in response to mechanical stimuli to which the insulating film is subjected and changes in the physical state of the insulating film. Specifically, the mechanical force stimulation includes osmotic pressure change on both sides of the insulating film, direct physical stimulation of the insulating film by micro-needles, stimulation of the insulating film by air pressure negative pressure, and the like. The physical changes of the insulating film include changes in thickness of the insulating film, changes in composition of the insulating film, and changes in curvature of the surface of the insulating film. The altering the protein structure comprises altering a charge property and/or pore size of an opening of the MscS. Further, the charge properties and/or pore size of the openings altered by the MscS-angstrom pores can be used to detect different analytes. The adjustable range of the aperture of the hermite hole related by the invention can be 5-15 hermite.
Interaction between the ehmitic pore and an analyte
The analyte may be in contact with the eminence pore on either side of the insulating film. The analyte may be in contact with either side of the insulating film such that the analyte passes through the passage of the hermite to the other side of the insulating film. In this case, the analyte interacts with the angstrom pore as it passes through the insulating film via the passage of the pore. Alternatively, the analyte may be in contact with a side of the insulating film that allows the analyte to interact with the hermite such that it is separated from the hermite and resides on the same side of the insulating film. The analyte may interact with the pores in any manner and at any site. The analyte may also impinge on the angstrom pore, interacting with the angstrom pore, causing it to separate from the angstrom pore and reside on the same side of the insulating film.
During interaction of the analyte with the emittor pore, the analyte affects the current flowing through the emittor pore in a manner specific to the analyte, i.e., the current flowing through the emittor pore is characteristic of a particular analyte. Control experiments can be performed to determine the effect of a particular analyte on the current flowing through the hermite pores, and then to identify the particular analyte in the sample or to determine whether the particular analyte is present in the sample. More specifically, the presence, absence, concentration, or the like of an analyte can be identified based on a comparison of a current pattern obtained by detecting the analyte with a known current pattern obtained using a known analyte under the same conditions.
The hermite system of the present invention may further comprise one or more measuring devices, such as a patch clamp amplifier or a data acquisition device, that measure the current flowing through the hermite.
Sample(s)
The analyte may be present in any suitable sample. The invention is typically performed on samples known to contain or suspected of containing the analyte. The invention may be performed on samples containing one or more species of unknown analyte. Alternatively, the invention may identify the identity of the one or more analytes known to be present or expected to be present in the sample.
The sample may be a biological sample. The invention may be carried out in vitro on a sample obtained or extracted from any organism or microorganism. The invention may also be carried out in vitro on a sample obtained or extracted from any virus. Preferably, the sample is a fluid sample. The sample typically comprises a bodily fluid. The sample may be a bodily fluid sample, such as urine, blood, serum, plasma, lymph, cyst fluid, pleural fluid, ascites fluid, peritoneal fluid, amniotic fluid, epididymal fluid, cerebrospinal fluid, bronchoalveolar lavage fluid, breast milk, tears, saliva, sputum, or a combination thereof. The sample may be derived from a human or from another mammal. The sample may be a non-biological sample. The non-biological sample is preferably a fluid sample, such as drinking water, sea water, river water, and reagents for laboratory testing.
The sample may be untreated prior to analysis, for example by detecting the analyte directly in whole blood. The sample may also be processed prior to analysis, for example by centrifugation, filtration, dilution, precipitation or other physical or chemical means known in the art.
In one embodiment of the invention, the sample is a whole blood sample.
Method for detecting drug molecules in a sample
The present invention also provides a method of detecting a drug molecule in a sample, the method comprising: s1 adding said sample to a hermite system, said hermite system comprising: an angstrom pore, an insulating film, a first dielectric, a second dielectric, wherein said angstrom pore is embedded in said insulating film, said insulating film separates said first dielectric from said second dielectric, said angstrom pore provides a channel that connects said first dielectric with said second dielectric, said angstrom pore is an MscS angstrom pore, said angstrom pore has a radially symmetric and cylindrically shaped heptamer structure, said heptamer structure comprises 7 side openings and 1 bottom opening; the sample is added to the first medium; s2, applying a driving force to the first medium and the second medium, wherein the drug molecules in the sample interact with the pores and generate an electric signal; and S3, analyzing the electric signal so as to identify the drug molecules in the sample.
In one embodiment of the invention, the sample is a body fluid sample. The body fluid sample may be urine, blood, serum, plasma, lymph, cyst fluid, pleural fluid, ascites fluid, peritoneal fluid, amniotic fluid, epididymal fluid, cerebrospinal fluid, bronchoalveolar lavage fluid, breast milk, tears, saliva, sputum, or a combination thereof. The sample may be untreated prior to analysis, for example by detecting the analyte directly in whole blood (blood). Of course, the sample may also be processed prior to analysis, for example by centrifugation, filtration, dilution, precipitation or other physical or chemical means known in the art. The samples referred to in the present invention include unprocessed samples and processed samples.
In one embodiment of the invention, the detectable range of the drug molecule may be greater than 10nM (which may also be understood as a lower limit of detection of 10 nM). Preferably, the detectable range of the drug molecule may be 10nM to 1mM. If the concentration of the drug molecule is much greater than 10nM (e.g., 10 mM), it can be diluted to a concentration of 10nM to 1mM.
In one embodiment of the invention, the drug molecule is a compound. More specifically, a "drug molecule" can be a drug having a molecular weight of 1000g/mol or less (e.g., less than 800, 700, 600, 500, 400, 300, or 200 g/mol). Preferably, the drug molecule may be an aminoglycoside antibiotic. In another embodiment of the invention, the drug molecules include amino acids and salts thereof (including non-druggable amino acids) and peptides.
In one embodiment of the present invention, the present invention detects a drug molecule in a sample of body fluid, wherein the detection limit of the drug molecule is 10nM. In other words, the detectable range of the drug molecule in the body fluid sample may be greater than 10nM.
In one embodiment of the present invention, the present invention detects drug molecules in a whole blood sample (also understood as "blood sample"). After the whole blood sample is added to the hermite system provided by the invention, cells (such as red blood cells, white blood cells and blood platelets) and macromolecular substances (such as proteins) in the whole blood sample cannot pass through the hermite, that is, the cells and the macromolecular substances in the whole blood sample cannot block the hermite system provided by the invention. The drug molecules existing in the whole blood sample can pass through the hermite hole and generate specific current, and the hermite hole provided by the invention can sensitively identify the drug molecules with lower concentration, so that the existence and the concentration of the drug molecules in the whole blood sample can be judged.
In one embodiment of the invention, the methods provided herein can be used for continuous monitoring of blood drug levels of drug molecules in a subject.
In one embodiment of the invention, paMscS1 (K180R) and PaMscS2 (V271I) Amylor pores are used to detect drug molecules in whole blood samples, but other MscS and their corresponding variants are also within the scope of the invention, based on the ability of the variants to perform a sensory detection of drug molecules.
Example one
Materials and methods
Chemical product: sodium chloride (NaCl, >99.0%, CAS # 7647-14-5), yeast extract (CAS # 8013-01-2), trypsin (CAS # 73049-73-7), ampicillin sodium salt (≧ 98.5%, CAS # 69-52-3), tris (≧ 99.9%, CAS # 77-86-1), imidazole (≧ 99%, CAS # 288-32-4), n-dodecyl- β -D-maltoside (DDM) (≧ 99%, CAS # 69227-93-6), isopropyl- β -D-thiogalactoside (IPTG) (≧ 99%, CAS # 367-93-1), phenylmethylsulfonyl fluoride (PMSF) (≧ 99%, CAS # 329-98-6), 4- (2-hydroxyethyl) piperazine-1-ethanesulfonic acid (HEPES, >99.5%, CAS # 65-7345-9) were purchased from Sigma-rich. Coli polar lipid extract (100600P) was purchased from Avanti.
Expression and purification of mutant PaMscS:
the gene for PaMscS was amplified from pseudomonas aeruginosa genomic DNA by Polymerase Chain Reaction (PCR) using gene-specific primers. The gene was inserted into a plasmid using the Clonexpress II One Step Cloning Kit (Vazyme). Coli BL21 (DE 3) cells containing a plasmid containing the PaMscS gene were cultured in Luria-Bertani (LB) medium in the presence of 50. Mu.g/mL ampicillin at 37 ℃ and purified for expression. The peak was determined by SDS-PAGE analysis. In particular, the steps of purification and expression of the wild-type and mutant proteins in the present invention are identical, but there is a difference in the plasmid synthesis stage due to the difference in the sequence of the wild-type protein and the mutant protein.
Drug measurements by LC-MS:
LC-MS and LC-MS/MS analyses were performed on a Shimadzu ultra fast liquid chromatography system (UFLC, shimadzu) and an AB SCIEX Qtrap 5500 mass spectrometer equipped with a Turbo Spray ion source (Turbo Spray ion source). Collection and analysis of chromatographic and mass spectral data was done by Analyst 1.6.2 software (AB SCIEX, USA).
The chromatographic separation was carried out on a WatersACQUITY UPLC BEH C18 column (2.1 mm. Times.100mm I.D.,1.7 μm). The mobile phase consisted of water (a) and acetonitrile (B), eluting with a gradient as follows: 0-1.0 min, 10-90% by weight B;1-2.0 minutes, 90% B. The flow rate was 0.5mL/min. The column temperature and the autosampler temperature were maintained at 35 ℃ and 15 ℃ respectively. The injection volume was 1. Mu.L.
In MS/MS analysis, positive ionization mode was used for sample detection, with the following optimized mass spectrometry parameters: ion spray voltage, 5500V; declustering voltage, 100V; temperature, 500 ℃. The MRM (multiple reaction monitoring) mode was chosen for quantification of gentamicin sulfate and IS (internal standard), the ion pairs were 450.2-160.1, 464.2-160.1, 478.2-157.1 and 265.2-232.2, respectively.
Gentamicin sulfate was continuously monitored from a dialysis device connected to a blood vessel of a living rat:
male Sprague-Dawley rats (250-300 g) were provided by Wydowski laboratories, inc., and all animal experiments in the present invention were approved by the ethical Committee of the Wayne Hospital, sichuan university (approval No. 2021885A).
Rats (n = 1) were anesthetized with 2% pentobarbital and the left femoral vein was isolated and catheterized to provide one intravenous injection port. Next, an initial 0.4mL heparin solution (250U/mL) was injected through the catheter, followed by 0.1mL per 40 minute cycle to prevent clot formation during monitoring. The left femoral artery was then isolated, catheterized, and immediately connected to the device through a pre-designed tube with dialysis membrane. After the air in the device is expelled by the blood stream, the tube is connected to an intravenous catheter to form a stable circulation. For continuous monitoring, a baseline signal was first recorded in the absence of the target drug, followed by a slow infusion of a specific concentration of gentamicin sulfate through an intravenous catheter. For the interval measurement of the blood concentration, a similar cycle is established, with neither equipment nor dialysis membrane. Blood samples were collected from the arterial catheters at 0, 15, 30, 45 and 60 minutes and drug concentrations were measured using PaMscS2 (V271I) emmer pores. After each experiment, the rats were sacrificed by cervical dislocation.
And (3) whole blood detection:
collecting 10 microliter of rat blood, adding the rat blood into a sample groove fused with an Hermite hole (-cis end), applying bias voltage, recording the signal frequency in a certain time after sample addition, subtracting the blank blood control frequency from the signal frequency, and calculating to obtain the corresponding concentration of the molecule according to the standard curve of the detected molecule.
Example two
Detection system comprising an emmetropore
This example provides a detection system comprising an icometer well of the present invention
The system includes two electrolyte chambers separated by an insulating membrane to form cis (-cis) and trans (-trans) compartments, with only a small conductance mechanical force sensitive channel in the membrane communicating the chambers, with the pores of the pores being embedded in the insulating membrane, and electrolyte ions in solution move through the pores by electrophoresis when a voltage is applied to the electrolyte chambers. The insulating film includes a phospholipid film, DPHPC, DOPC, e.
EXAMPLE III
Structure of PaMscS angstrom hole
Two PaMscS mutants, including PaMscS1 (K180R) and PaMscS2 (V271I), are described in detail in this example (fig. 5). Researchers analyzed the structure of PaMscS 1. Modeling has shown that the functional channels of the hermite pore form a classical homo-heptamer, which is radially symmetric, resembling a cylinder. It contains 8 openings, 7 on the side and 1 on the bottom. Topologically, paMscS can be divided into two parts, a transmembrane region and a large cytoplasmic portion. Each monomer contributes 3N-terminal transmembrane helices, including TM1 (residues 17 to 52), TM2 (residues 58 to 83), and TM3 (residues 90 to 22). And the C-terminal cytoplasmic domain can be divided into an intermediate β domain and a COOH-terminal domain. TM1 and TM2 in each subunit are aligned together in an anti-parallel orientation, with TM1 crossing the bilayers outside the channel and TM2 forming the central layer, so that they form a permeation pathway around the axis of the channel. The TM3 helix can be described as 2 helix segments, TM3a and TM3b, at Gly108There is a significant kink at 53 °, a conserved residue on the homology. TM3a passes through the layers with different deflections like TM1, while TM3b returns to the cytoplasm, interacting with the cytoplasmic region. Furthermore, the 7 subunits form a radius of
Figure BDA0003720097560000101
And a central bore that senses the tension and is associated with the conformational change. In the cytoplasmic region, the middle beta domain (residues 123 to 172) contains 5 beta strands, assembled with other strands of different subunits. While the C-terminal domain (residues 177 to 273) forms 5 beta strands and 2 alpha helices, which is a mixed structure. Between the domains of these two adjacent monomers there are 7 clearly visible equal openings with a radius of approximately
Figure BDA0003720097560000111
This is proposed to be the reason for ion penetration in EcMscS. In addition to these entries, the 8 th opening is present at the bottom of the protein, which consists of 7 beta strands with the narrowest radius
Figure BDA0003720097560000112
In all dimensions, extend
Figure BDA0003720097560000113
Parallel to the seven-fold axis, and extending in width in the vertical direction
Figure BDA0003720097560000114
The structure of PaMscS is similar to that of EcMscS in the off state (PDB: 2 OAU), with TM domains exceeding 101C α Atom has
Figure BDA0003720097560000115
However, in the open state (PDB: 2VV 5), the difference in the TM domain is large, and there is
Figure BDA0003720097560000116
Rmsd of (1). These results indicate that the conformation of PaMscS in the structure reflects the off-state. PaMscS processThe unique and elegant structure of the variant hermite also shows great potential for detection.
Example four
Electrophysiological detection of PaMscS emmetropic pores:
single-channel electrophysiological studies of PaMscS hermite pores were performed in planar lipid bilayer membrane structures (fig. 1A). PaMscS1 (K180R) angstrom pores and PaMscS2 (V271I) angstrom pores can form stable tunnel currents at +50mV at 34.9. + -. 7.0pA (mean. + -. SD from 18 independent insertion events) and 37.6. + -. 3.8pA (mean. + -. SD from 108 independent insertion events), respectively (FIG. 1B, electrolyte conditions-cis: 300mM NaCl, -trans: 30mM NaCl,10mM HEPES, pH 7.0). The IV curve from-50 mV to +50mV indicates that PaMscS1 and PaMscS2 do not have voltage gating in this voltage range (fig. 1C), and were selected for further testing due to the high efficiency of PaMscS2 emma pores in lipid bilayer membrane fusion. According to the conductance profile, the peak conductivity of the PaMscS2 angstrom pore at +50mV was 0.78 nS (FIG. 1D).
Drug monomolecular biosensing based on PaMscS emmer pores:
according to the channel structure characteristic analysis of PaMscS angstrom pores, small molecule drugs (molar mass less than 1000 g/mol) are selected for detection. Quantitative determination was carried out by taking gentamicin sulfate (molecular weight: MW 561.65) and neomycin sulfate (molecular weight: MW 712.72) as examples, and since the two drugs are clinically used in large scale, convenient TDM is urgently needed. The test for gentamicin sulfate and neomycin sulfate was carried out in the presence of 300mM NaCl (-cis terminal), 30mM NaCl (-trans terminal), 10mM HEPES, pH7.0 electrolyte, and the voltage for drug detection was-50 mV. Significant blocking current signals appeared after drug addition to the detection system (fig. 2A, 2B, fig. 7, fig. 8). Standard curves for gentamicin sulfate showed linear detection ranging from 10nM to 10 μ M (N = 3), and 2D density plots of typical gentamicin sulfate signals showed peak occlusion currents of 11.57 ± 0.02pA and peak residence times of 1.33 ± 0.02ms (fig. 2C, peak of gaussian fit, 878 occlusion events). The standard curve for neomycin sulfate showed a linear detection range of 100nM to 100 μ M (N = 3), and the 2D density plot of a typical neomycin sulfate signal showed a peak occlusion current of 9.44 ± 0.02pA and a peak residence time of 1.06 ± 0.01ms (fig. 2D, gaussian-fitted peak, 883 occlusion events). Higher drug concentrations may cause severe obstruction of the channels and make drug concentration calculation difficult, so the system is more suitable for detecting trace concentrations of drug molecules (gentamicin sulfate: 10nM to 10. Mu.M; neomycin sulfate: 100nM to 100. Mu.M) (FIGS. 9A-B).
Besides gentamicin sulfate and neomycin sulfate, the PaMscS E.oryzae pores can also sense other drugs, such as sisomicin (MW: 447.53), pyrophosphate (MW: 177.975), in a single molecule.
To assess the accuracy of detection of the drug by the PaMscS2 emm-pores, LC-MS was used to measure the concentration of gentamicin sulfate. The PaMscS2 Ames pore and LC-MS exhibited similar detection results for the 1.5 μ M gentamicin sulfate sample, indicating that the detection of the PaMscS2 Ames pore has good accuracy (FIG. 2E).
Measurement of drug concentration in whole blood sample:
blood is the main medium of TDM, but the whole blood components are complex, which can cause serious interference to the detection of the hermite, i.e. the blockage in the whole blood detection means that the hermite is blocked by substances other than detected molecules and influences the detection effect of target molecules. The commonly used Aminous MspA-2NN was blocked after addition of 10. Mu.L of whole blood sample to the-cis end (1 mL volume at the-cis and trans ends), and had a probability of opening channels of only 5% + -4.7% at +100mV within 300s (FIG. 10, electrolyte conditions-cis end: 300mM NaCl, -trans end: 300mM NaCl,10mM HEPES, pH7.0, N = 3). In contrast, a PaMscS2 Amicro well can maintain the open channel with a 99% + -1% probability of opening the channel at +100mV within 300s even when 20. Mu.L of whole blood is added to the-cis end (FIGS. 3A, 3B, 3C, electrolyte conditions of-cis end: 130mM NaCl, -trans end: 130mM NaCl,10mM HEPES, pH7.0, N = 3). In view of the stable detection capability of the PaMscS s2 ehmitum wells, gentamicin sulfate plasma concentrations in rats were measured directly at various time intervals (0 min, 15 min, 30 min, 45 min, and 60 min, fig. 11, fig. 12) after injection of 4mg/mL gentamicin sulfate into rats. The standard curve of gentamicin sulfate ranges from 0 to 3 μ M and is used for calculating the drug concentration (N is more than or equal to 3 in figure 3D). Of the 5 monitoring points, the drug concentration was highest at 15 minutes and the concentration decreased to the pre-injection level (0 minutes) at 60 minutes (FIG. 3E, N.gtoreq.3). The drug concentration trend measured by the PaMscS2 angstrom hole accords with the pharmacokinetic rule, and shows that the PaMscS angstrom hole can accurately measure the change of the drug concentration in the living rat body. In addition, the occlusion current profile of gentamicin sulfate showed two peaks at higher negative voltages in the buffer of 130mM NaCl (fig. 13), since gentamicin drug is a mixture of multiple component drugs, whose components have different molecular structures, and thus, multiple occlusion current peaks may be associated with their components. Whereas the single-component sisomicin (sisomicin) showed only one peak of the blocking current under the same conditions (fig. 14). Continuous monitoring of the drug by dialysis devices connected to the blood vessels of living rats:
due to the wide variation in drug metabolism rates among patients, particularly critically ill patients, it is difficult to meet the needs of precision medicine with the spaced TDM technique. Therefore, continuous monitoring of drug concentration is highly desirable. To achieve long-term monitoring of drug concentrations in living animals, experimenters have introduced dialysis membranes to avoid blood consumption. In TDM processes, a small amount of blood flows through the tubing to the dialysis unit and then back into the body. Drug molecules in the blood can permeate into the conducting fluid at the-cis end through the dialysis device without substantial depletion of the blood (fig. 4A). The standard curve for gentamicin sulfate ranged from 0 to 30 μ M for continuous monitoring drug concentration calculations (fig. 4B). In the feasibility verification experiments in live rats, a clear signal of gentamicin sulfate was continuously observed up to 3 hours after injection, based on a simple dialysis device (fig. 4C). Different doses of gentamicin sulfate, including 4mg/kg and 20mg/kg, in rats were distinguishable by an epothilone monitoring device (FIG. 4D). These results indicate that the system can continuously monitor the drug concentration in live animals with minimal loss.
dNTPs detection of PaMscS3 (W130A) Aminopore:
mechanical forces considering PaMscS3 (W130A) Am poresSensitivity, the experimenter adjusted the selectivity of the PaMscS3 emm pore by applying different osmotic pressure differentials. To maintain constant charge characteristics of dCTP and dGTP at different osmotic pressure differences, the experimenter maintained the conductivity buffer concentration at the-cis end at 300mM and varied the conductivity buffer concentration at the-trans end to vary the osmotic pressure difference. The detection ability of PaMscS3 angstrom pores for large and small molecule dGTP and dCTP was tested under 3 conditions of differential osmotic pressure, including symmetric (symmetric) conditions (FIG. 15A,300mM NaCl/300mM NaCl, +50mV bias), low differential osmotic pressure conditions (FIG. 15B,100mM NaCl/300mM NaCl, +50mV bias), and high differential osmotic pressure conditions (FIG. 15C,30mM NaCl/300mM NaCl, +50mV bias). Under the condition of symmetric osmotic pressure, the translocation frequency of dCTP is from 0.16 +/-0.03 s -1 Increase to 0.22 + -0.07 s -1 And dGTP is from 0.09 +/-0.02 s -1 Change to 0.07. + -. 0.003 s -1 . The translocation frequency of dCTP was from 0.34. + -. 0.1s under low differential permeability (LOD) conditions -1 Increase to 0.67 + -0.14 s -1 And dGTP is from 0.06 +/-0.01 s -1 Increased to 0.3 + -0.04 s -1 . Under high osmotic pressure (HOD) conditions, the translocation frequency of dCTP is from 0.12 + -0.04 s -1 Increase to 0.22 + -0.07 s -1 And dGTP is from 0.37 +/-0.08 s -1 Increased to 1.12 + -0.12 s -1 (fig. 15D, n =3, mean ± s.e.m per experiment). Figure 15E summarizes the detection of dGTP and dCTP, and it is summarized that low differential osmotic pressure conditions showed the highest increase in translocation events for dCTP, while high differential osmotic pressure conditions showed the highest increase in translocation events for dGTP (figure 15E). The low osmotic pressure conditions show balanced capture capability for both dCTP and dGTP, compared to the reduced capture efficiency of dCTP under high osmotic pressure conditions. Given that the size and charge characteristics of dGTP and dCTP remain constant under given experimental conditions, and that the size of the MscS family of channels can vary under different pressure, osmolarity conditions, or membrane potentials, the experimenter can conclude that: the difference in selectivity of PaMscS3 (W130A) Aminopores for dNTPs is caused by the change in channel size under different osmotic pressure conditions. The wild-type PaMscS emmer pore exhibited 2 peaks for the four dNTPs cocktail blocking rates (fig. 16).
EXAMPLE five
Amino acid detection based on PaMscS emmetropores
In this example, amino acid detection was performed using PaMscS3 (W130A) as an example. Glutamic acid (10 mM) was assayed under the conditions of 300mM NaCl (-cis terminal) and 30mM NaCl (-trans terminal), 10mM HEPES, pH7.0 electrolyte, and the voltage for drug detection was-50 mV. The current trace of glutamic acid is shown in fig. 17.
Short peptides (e.g., dipeptides) can be detected by the hermite pores of the present invention in addition to single amino acids. As shown in the left panel of FIG. 18, the amino acid to be tested and the aspartic acid carrier are subjected to dehydration condensation to form a dipeptide, the formed dipeptide is detected under the conditions of 300mM NaCl (-cis terminal), 30mM NaCl (-trans terminal), 10mM HEPES and pH7.0 electrolyte, and a current signal generated by the dipeptide is compared with a specific current signal of the tested amino acid to judge the type of the amino acid to be tested.
EXAMPLE six
Electrophysiological detection based on ecMscS hermite
When the wild-type EcMscS channel is embedded in a Bilayer Lipid Membrane (BLM), stable channel current hopping can be observed at a voltage of +100mV (fig. 19). Wild-type EcMscS channel currents remained stable at voltages ranging from-100 mV to +100mV (fig. 20). The conductance of the wild-type EcMscS E-rice wells was 0.334. + -. 0.028nS (-cis end: 300mM NaCl, -trans end: 30mM NaCl) (FIG. 21). Fig. 22a-c show the structures of EcMscS, ttMscS and HpMscS, respectively, which are highly similar to the structures of pamsccs, i.e. heptameric structures that are both radially symmetric and shaped like cylinders. In addition, fig. 22a-c and fig. 6 further compare the sequences of PaMscS with EcMscS, ttMscS, and HpMscS (sequence information see table 1 below), and show that EcMscS, ttMscS, and HpMscS have some homology to pammscs, but the homology is not highly homologous. EcMscS and pammscs are only 60% similar, but both have the ability to detect analytes. Thus, it will be appreciated by those skilled in the art that the key to determining the ability of bacteria to detect analytes as hermite pores is their radially symmetric and cylindrically shaped heptameric structure and channel pore size, not merely homology.
Table 1: amino acid sequence information for four MscS
Figure BDA0003720097560000131
Summary of the invention
The invention provides a therapeutic drug monitoring method based on a mutant PaMscS emittor (an ultra-narrow tunnel derived from Pseudomonas aeruginosa). Mutant PaMscS hermite pores exhibit robustness and remain sensitive under complex biological conditions (e.g., whole blood). Rat blood samples were tested using the hermite and their ability to measure drug concentration was demonstrated. Proof-of-concept studies demonstrated continuous monitoring of drugs from dialysis devices attached to blood vessels of live rats.
The mutant PaMscS hermite pores have also been shown to play a unique role in the direct detection of small molecules. The feasibility of a PaMscS Ammi pore for TDM was verified. The pore membrane system comprising the hermite pore can directly detect the drug without modification, aptamer or antibody, and has the potential to realize low-cost and convenient TDM in scientific research and clinical treatment.
The present study also shows that the PaMscS omega pore produces a stable tunnel current when a bias voltage is applied and can directly measure the drug concentration in whole blood with nanomolar sensitivity. Experiments on live rats show that the system has the potential to continuously monitor drug concentrations in live animals. This kind of TDM system based on the emittor hole is suitable for the development of micro-sampling, high-throughput and POCT-oriented portable blood drug monitoring equipment and long-term therapeutic drug monitoring for patients and experimental animals.
While the present invention has been described with reference to the particular illustrative embodiments, it is to be understood that the invention is not limited to the disclosed embodiments, but is intended to cover various modifications, equivalent arrangements, and equivalents thereof, which may be made by those skilled in the art without departing from the spirit and scope of the invention as defined by the appended claims.
Reference documents
1.Sanchez-Spitman,A.et al.Tamoxifen Pharmacogenetics and Metabolism:Results From the Prospective CYPTAM Study.J.Clin.Oncol.37,636-646(2019).
2.Seger,C.,Ramsay,S.L.,
Figure BDA0003720097560000141
W.,Tentschert,K.&Griesmacher,A.A rapid HPLC-MS/MS method for the simultaneous quantification ofcyclosporine A,tacrolimus,sirolimus and everolimus in human blood samples.Nat. Protoc.4,526-534(2009).
3.Schmitz,E.M.et al.Therapeutic drug monitoring of infliximab:performance evaluation of three commercial ELISA kits.Clin.Chem.Lab.Med.54,1211-1219(2016).
4.Gao,B.et al.Evidence for Therapeutic Drug Monitoring of Targeted Anticancer Therapies.J.Clin.Oncol.30, 4017-4025(2012).
5.Argollo,M.,Kotze,P.G.,Kakkadasam,P.&D'Haens,G.Optimizing biologic therapy in IBD:how essential is therapeutic drug monitoringNatRev GastroenterolHepatol.17,702-710(2020).
6.WENK,M.,VOZEH,S.&FOLLATH,F.Serum level monitoring of antibacterial drugs.A review.Clin. Pharmacokinet.9,475-492(1984).
7.Rybak,M.J.et al.Therapeutic monitoring ofvancomycin in adults summary of consensus recommendations from the American Society ofHealth-System Pharmacists,the Infectious Diseases Society of America,and the Society ofInfectious Diseases Pharmacists.Pharmacotherapy.29,1275-1279(2009).
8.Griss,R.et al.Bioluminescent sensor proteins for point-of-care therapeutic drug monitoring.Nat.Chem.Biol.10, 598-603(2014).
9.Liu,J.et al.Electrochemical microfluidic chip based on molecular imprinting technique applied for therapeutic drug monitoring.Biosensors andBioelectronics.91,714-720(2017).
10.Tai,L.C.et al.Methylxanthine Drug Monitoring with Wearable Sweat Sensors.Adv.Mater.30,e1707442(2018).
11.Li,H.,Dauphin-Ducharme,P.,Ortega,G.&Plaxco,K.W.Calibration-Free Electrochemical Biosensors Supporting Accurate Molecular Measurements Directly in Undiluted Whole Blood.J.Am.Chem.Soc.139, 11207-11213(2017).
12.Arroyo-Currás,N.et al.Real-time measurement of small molecules directly in awake,ambulatory animals. Proceedings oftheNationalAcademy ofSciences.114,645-650(2017).
13.Ferguson,B.S.et al.Real-Time,Aptamer-Based Tracking of Circulating Therapeutic Agents in Living Animals. Sci.Transl.Med.5,165r-213r(2013).
14.Poudineh,M.et al.A fluorescence sandwich immunoassay for the real-time continuous detection of glucose and insulin in live animals.Nature BiomedicalEngineering.5,53-63(2021).
15.GRAHAM,M.The Coulter principle:foundation of an industry.Journal of the Association for Laboratory Automation.8,72-81(2003).
16.Quick,J.et al.Real-time,portable genome sequencing for Ebola surveillance.Nature.530,228-232(2016).
17.Gilpatrick,T.et al.Targeted nanopore sequencing with Cas9-guided adapter ligation.Nat.Biotechnol.38,433-438 (2020).
18.Gu,W.et al.Rapid pathogen detection by metagenomic next-generation sequencing of infected body fluids.Nat. Med.27,115-124(2021).
19.Laszlo,A.H.et al.Decoding long nanopore sequencing reads of natural DNA.Nat.Biotechnol.32,829-833 (2014).
20.Wang,Y.et al.Nanopore-based detection of circulating microRNAs in lung cancer patients.Nat.Nanotechnol, 6(10),668–674(2011).
21.Wendell,D.et al.Translocation of double-stranded DNA through membrane-adapted phi29 motor protein nanopores.Nat.Nanotechnol,4(11),765–772(2009).
22.Clarke,J.et al.Continuous base identification for single-molecule nanopore DNA sequencing.Nat.Nanotechnol.4, 265-270(2009).
23.Chen,C.et al.Development of a structure-switching aptamer-based nanosensor for salicylic acid detection. Biosensors andBioelectronics.140,111342(2019).
24.Liu,Y.M.,Fang,X.Y.,Fang,F.&Wu,Z.Y.Investigation of hairpin DNA and chelerythrineinteraction by a single bio-nanopore sensing interface.Analyst.144,4081-4085(2019).
25.Feng,S.,Chen,C.,Wang,W.&Que,L.An aptamer nanopore-enabled microsensor for detection oftheophylline. Biosens.Bioelectron.105,36-41(2018).
26.Wang,J.,Bafna,J.A.,Bhamidimarri,S.P.&Winterhalter,M.Small-Molecule Permeation across Membrane Channels:Chemical Modification to Quantify Transport across OmpF.AngewChemInt Ed Engl.58,4737-4741 (2019).
27.
Figure BDA0003720097560000151
U.et al.Tension-activated channels in the mechanism of osmotic fitness in Pseudomonas aeruginosa.J. Gen.Physiol.149,595-609(2017).
28.Zhang,Y.et al.Visualization of the mechanosensitive ion channel MscS under membrane tension.Nature.590, 509-514(2021).
29.Gamini,R.,Sotomayor,M.,Chipot,C.&Schulten,K.Cytoplasmic Domain Filter Function in the Mechanosensitive Channel ofSmall Conductance.Biophys.J.101,80-89(2011).
30.Perozo,E.&Rees,D.C.Structure and mechanism in prokaryotic mechanosensitive channels.CurrOpinStruct Biol.13,432-442(2003).
31.Lopez-Novoa,J.M.,Quiros,Y.,Vicente,L.,Morales,A.I.&Lopez-Hernandez,F.J.New insights into the mechanism ofaminoglycoside nephrotoxicity:an integrative point ofview.Kidney Int.79,33-45(2011).
32.Llanos-Paez,C.C.,Staatz,C.E.,Lawson,R.&Hennig,S.A Population Pharmacokinetic Model ofGentamicin in Pediatric Oncology Patients To Facilitate Personalized Dosing.Antimicrob Agents Chemother.61,(2017).
33.Yu,L.et al.Stable polymer bilayers for protein channel recordings at high guanidinium chloride concentrations. Biophys.J.,(2021).
34.Zheng,S.Q.et al.MotionCor2:anisotropic correction of beam-induced motion for improved cryo-electron microscopy.Nat.Methods.14,331-332(2017).
35.Punjani,A.,Rubinstein,J.L.,Fleet,D.J.&Brubaker,M.A.cryoSPARC:algorithms for rapid unsupervised cryo-EM structure determination.Nat.Methods.14,290-296(2017).
36.Punjani,A.,Zhang,H.&Fleet,D.J.Non-uniform refinement:adaptive regularization improves single-particle cryo-EM reconstruction.Nat.Methods.17,1214-1221(2020).
37.Kefauver,J.M.,Ward,A.B.&Patapoutian,A.Discoveries in structure and physiology of mechanically activated ion channels.Nature 587,567–576(2020).
38.Hurst AC,Petrov E,Kloda A,Nguyen T,Hool L,Martinac B.MscS,the bacterial mechanosensitive channel of small conductance.Int J Biochem Cell Biol.2008；40(4):581-585.
39.Malcolm,H.R.and Maurer,J.A.(2012),The Mechanosensitive Channel of Small Conductance(MscS) Superfamily:Not Just Mechanosensitive Channels Anymore.ChemBioChem,13:2037-2043. 。
SEQUENCE LISTING
<110> Sichuan university
<120> method for detecting drug molecules based on biological ehmitis pores
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Claims (10)

1. A method of detecting a drug molecule in a sample, comprising the steps of:
s1 adding the sample to a rice bore system, the rice bore system comprising: an angstrom pore, an insulating film, a first dielectric, a second dielectric, wherein said angstrom pore is embedded in said insulating film, said insulating film separates said first dielectric from said second dielectric, said angstrom pore provides a passageway that connects said first dielectric with said second dielectric, said angstrom pore is an MscS angstrom pore, said angstrom pore has a radially symmetric and cylindrically shaped heptamer structure, said heptamer structure comprises 7 side openings and 1 bottom opening; the sample is added to the first medium;
s2, applying a driving force to the first medium and the second medium, wherein the drug molecules in the sample interact with the pores and generate an electric signal;
and S3, analyzing the electric signal so as to identify the drug molecules in the sample.
2. The method of claim 1, wherein the charge properties and/or pore size of the opening are adjustable; the manner of adjustment of the opening optionally comprises subjecting the insulating film to a mechanical force stimulus, optionally comprising one or more of a change in the osmotic pressure difference of the medium across the insulating film, a direct physical stimulus of the insulating film by micro-needles and a stimulus of the insulating film by negative pressure of air pressure, and/or a change in the physical state of the insulating film.
3. The method of claim 1, wherein the aperture of the opening is adjustable according to:
(1) Selecting a type of the first medium and the second medium; and/or
(2) A difference in osmotic pressure between the first medium and the second medium.
4. The method of claim 1, wherein the emmer pore is an MscS variant emmer pore, the MscS variant optionally comprising a side hole volume variant and/or a side hole charge variant.
5. The method of claim 1, wherein the E.oryzae is derived from a Bacillus, optionally comprising one or more of Pseudomonas aeruginosa, escherichia coli, thermoanaerobacter tengchongensis, and helicobacter pylori.
6. The method of claim 1, wherein the angstrom pore is a PaMscS variant angstrom pore, optionally including one or more of the following variants: 130A, 130H, 180R, 271I, 130S and 130P.
7. The method of claim 1, wherein the drug molecule has a molecular weight of less than 1000g/mol, optionally 177.98-712.72 g/mol; the concentration of the drug molecule is optionally greater than 10nM.
8. The method of claim 1, wherein the sample is a body fluid sample optionally comprising one or more of urine, blood, serum, plasma, lymph, cyst fluid, pleural fluid, ascites, peritoneal fluid, amniotic fluid, epididymal fluid, cerebrospinal fluid, bronchoalveolar lavage fluid, breast milk, tears, saliva, sputum.
9. The method of claim 8, wherein the sample size of the bodily fluid sample is optionally greater than 10 μ L; the concentration of drug molecules in the body fluid sample is optionally greater than 10nM.
10. The method of claim 9, wherein the method further comprises S4: communicating a dialysis device with said first medium via a conduit such that said blood sample enters said hermite system via said dialysis device, wherein S4 precedes S1.
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101680873A (en) * 2007-04-04 2010-03-24 加利福尼亚大学董事会 Use composition, equipment, the system and method for nano-pore
US20190383720A1 (en) * 2018-06-07 2019-12-19 Nanohmics, Inc. Sensor And Methods For Detecting and Quantifying Ions And Molecules
CN112739712A (en) * 2018-09-11 2021-04-30 格罗宁根大学 Biological nanopores with tunable pore size and their use as analytical tools

Family Cites Families (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2422497A1 (en) * 2000-09-15 2002-03-21 Incyte Genomics, Inc. Transporters and ion channels
US20050063989A1 (en) * 2003-09-22 2005-03-24 Commissariat A L'energie Atomique Ion channel hybrid proteins as electrical sensors and use thereof
EP1722374B1 (en) * 2005-05-13 2013-08-14 nAmbition GmbH Method for determining the state of activation of a protein
WO2008060788A2 (en) * 2006-10-11 2008-05-22 The General Hospital Corporation Compositions, methods, and devices for treating liver disease
US8685755B2 (en) * 2009-07-20 2014-04-01 The Board Of Regents Of The University Of Texas System Combinatorial multidomain mesoporous chips and a method for fractionation, stabilization, and storage of biomolecules
KR102023754B1 (en) * 2011-07-27 2019-09-20 더 보오드 오브 트러스티스 오브 더 유니버시티 오브 일리노이즈 Nanopore sensors for biomolecular characterization
WO2015160952A1 (en) * 2014-04-15 2015-10-22 Pittenger Mark Frings Apparatus or device for cellular therapy
KR20220151011A (en) * 2016-09-23 2022-11-11 알베오 테크놀로지스 인크. Methods and compositions for detecting analytes
CN112041331B (en) * 2018-02-28 2024-05-28 豪夫迈·罗氏有限公司 Alpha-hemolysin variants and uses thereof
KR102106040B1 (en) * 2018-12-05 2020-05-04 한국과학기술원 Method for Detecting Target Nucleic Acid using Personal Glucose Meter
WO2020168286A1 (en) * 2019-02-14 2020-08-20 University Of Washington Systems and methods for improved nanopore-based analysis of nucleic acids
CN111521766B (en) * 2020-04-10 2022-02-15 浙江大学 Artificial synthesized large ring structure molecule nano-pore structure, preparation method and application

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101680873A (en) * 2007-04-04 2010-03-24 加利福尼亚大学董事会 Use composition, equipment, the system and method for nano-pore
US20190383720A1 (en) * 2018-06-07 2019-12-19 Nanohmics, Inc. Sensor And Methods For Detecting and Quantifying Ions And Molecules
CN112739712A (en) * 2018-09-11 2021-04-30 格罗宁根大学 Biological nanopores with tunable pore size and their use as analytical tools

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