CN111912892B - Application of aerolysin nanopore channel in biological phosphorylation and related enzyme analysis - Google Patents
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Abstract
The invention discloses an application of an aerolysin nanopore channel in biological phosphorylation and related enzyme analysis. Specifically, an aerolysin nanopore channel is constructed, voltage is applied to two ends of the constructed aerolysin nanopore channel, an object to be detected or a probe molecule related to the object to be detected is added to one end of a detection pool, the object to be detected passes through the aerolysin nanopore under the driving of the voltage to generate a blocking current signal and blocking current time, and the blocking current signal and the blocking current time are compared and analyzed to obtain corresponding detection information of the molecule to be detected. The invention discloses a new application of an aerolysin nanopore channel, which can be used for phosphorylation detection analysis of nucleic acid, polypeptide and protein, does not need DNA motor protein, has high sensitivity and convenient detection, can further realize activity analysis and quantitative analysis of a plurality of enzymes such as kinase, phosphatase, enzyme inhibitor and the like, and can realize real-time monitoring of enzyme activity.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to application of aerolysin nanopores in biological phosphorylation and related enzyme analysis.
Background
Phosphorylation and dephosphorylation of DNA or proteins are two processes of great importance in the metabolism of nucleic acids. The abnormal phosphorylation of the 3 'end or the dephosphorylation of the 5' end of the DNA is related to diseases such as Alzheimer disease, cancer and the like. Accurate analysis of DNA or protein phosphorylation and dephosphorylation is therefore of great importance. Wherein DNA phosphorylation and dephosphorylation mainly comprises four different states: 5 'terminal phosphorylation, 5' terminal dephosphorylation, 3 'terminal phosphorylation and 3' terminal dephosphorylation. At present, methods for detecting DNA or protein phosphorylation and dephosphorylation mainly comprise methods such as high-resolution mass spectrometry, fluorescence and radioisotope labeling, however, the ionization efficiency of the mass spectrometry method is low when the phosphorylation site on the DNA is determined, and errors are introduced into the final mass spectrometry result without pretreatment of a Polymerase Chain Reaction (PCR) method sample; in addition, the labeling method is limited by the complicated labeling process and the low specificity of the phosphorylated antibody, and only reflects the single phosphorylation state of the 3 'or 5' end of the DNA. Therefore, the direct recognition of multiple phosphorylation states of single DNA molecules, protein molecules and polypeptide molecules still faces huge challenges, and the ideal method is a means of single molecule analysis at present.
In the prior art, α -hemolysin nanopores have been used to resolve a single DNA molecule, but the sensitivity of recognition for multiple phosphorylated states of a DNA molecule is insufficient; and for the CsgG nanopore, due to the requirement of the CsgG nanopore on DNA motor protein, the dual requirements of simultaneously detecting the DNA phosphorylation state and DNA sequencing cannot be met. Therefore, the current research direction is mainly to explore an effective nanopore sensing interface, so that the nanopore sensing interface has a stable interaction with a charged group of DNA, and direct measurement of various phosphorylation states is realized.
The process of reversible phosphorylation plays a crucial role in the regulation of almost all biological functions, the level of DNA/protein phosphorylation in vivo is regulated mainly by kinases and phosphatases, and the process of signal transduction requires the co-action of multiple kinases and phosphatases. Although long-term studies have shown the importance of kinases and phosphatases, the process of dynamic phosphorylation, the study of the co-action of multiple kinases and phosphatases remains a significant challenge. To fully understand this dynamic complex process, there is a need to simultaneously identify and characterize the processes by which kinases and phosphatases synchronously mediate cellular phosphorylation. The current methods for evaluating kinase and phosphatase activities mainly include methods such as radioactive determination, fluorescence, electrochemistry, surface plasmon resonance, mass spectrometry, etc., but these methods are effective only for one enzyme (kinase or phosphatase) or one substrate (DNA or protein), and in addition, the above methods have the following limitations: a harmful radioactive label; complex and expensive fluorescent labeled peptide, multiple detection steps, insufficient reactant contact and expensive specific recognition protein, so that the above method is difficult to develop into a universal method for simultaneously evaluating the efficacy of kinase and phosphatase without substrate limitation, and further fails to realize the screening of kinase/phosphatase signal pathway inhibitors and the real-time monitoring of catalytic phosphorylation process on a single molecule level. Therefore, it remains a great challenge to develop assays suitable for both kinases and phosphatases to catalyze the process of DNA, polypeptide and protein phosphorylation.
Some nanopores have been reported to be useful for studying the effect of different inhibitors and substrates on kinase efficacy, but require that kinase substrate peptides be encoded onto biological nanopores by genetic engineering techniques or that corresponding DNA sequences be attached to substrate models by chemical modification. In addition, previous reports utilized α -hemolysin as a nanopore, but the nanopore has a weak interaction with DNA molecules that are highly accessible through the nanopore and are not sensitive enough for detection of phosphorylation states. Therefore, there is a need for an effective nanopore sensing interface that has a strong interaction with a phosphorylated group, thereby enabling direct detection of phosphorylation/dephosphorylation of DNA or protein substrates, and further enabling application in related enzyme assays.
The invention patent of publication No. CN 104651500B discloses an aerolysin nanopore channel, which can be self-assembled in an aqueous solution to form a heptameric structure and inserted into a phospholipid membrane through conformational change to form a nano-sized channel, and can be applied to DNA sequencing, DNA damage and Micro-RNA detection.
Disclosure of Invention
The purpose of the invention is as follows: in order to solve the problems in the prior art, the invention provides the application of the aerolysin nanopore channel in the analysis and detection of biological phosphorylation or/and related enzymes, and provides a new application of the aerolysin nanopore channel.
The technical scheme is as follows: the invention provides an application of an aerolysin nanopore channel in analysis and detection of biological phosphorylation or/and related enzymes.
Wherein, the analysis and detection of the biological phosphorylation comprises one or more of nucleic acid phosphorylation, nucleic acid dephosphorylation, polypeptide phosphorylation, polypeptide dephosphorylation, protein phosphorylation and analysis and detection (such as phosphorylation position, phosphorylation degree and the like) of protein dephosphorylation; the related enzyme is related to biological phosphorylation, and the analysis and detection of the related enzyme comprises one or more of enzyme activity analysis, real-time enzyme activity monitoring and enzyme quantitative analysis.
The nucleic acid phosphorylation comprises 5 'terminal phosphorylation or/and 3' terminal phosphorylation, and the nucleic acid dephosphorylation comprises 5 'terminal dephosphorylation or/and 3' terminal dephosphorylation.
The nucleic acid is DNA or RNA of 1-10 kbp such as 1-20bp, 5-50bp, 10-100 bp and 1 k-3 kbp; the nucleic acid is DNA or RNA of 1-10 kbp; the length of the polypeptide is 2-100 amino acids such as 2-20, 10-50 and 50-80; the molecular weight of the protein is 1-500 kDa; the related enzyme is hydrolase, kinase, phosphatase, enzyme inhibitor, phosphorylase, phosphatase or glycosyltransferase.
The process of phosphorylation of a nucleic acid, e.g., the 3 'or 5' end of DNA, significantly reduces the blocking time of the blocking current signal generated by the first entry of that end (i.e., the phosphorylated end) into the aerolysin nanopore, and thus, the phosphorylation or dephosphorylation process at each end of the nucleotide can be read directly from the duration of PI/PII. Mainly, the phosphorylation of the nucleic acid terminal increases two negative charges at the corresponding terminal, so that the nucleic acid terminal is subjected to larger electric field force under a detection system, the speed of the perforation event that the terminal firstly enters the nanopore is accelerated, the blocking time of the current blocking signal is greatly reduced, and the influence on the speed of the perforation event that the other non-phosphorylated terminal firstly enters the nanopore is small, so that the blocking time of the current blocking signal does not show obvious change.
The unphosphorylated polypeptide hardly generates a blocking current signal under the condition of applying a positive potential, so that the unphosphorylated polypeptide is less distributed on a scatter diagram of blocking time-blocking current, when the polypeptide is phosphorylated, the electrophoretic force of the polypeptide entering an aerolysin nanopore is enhanced, and more blocking current signals are generated, so that the phosphorylation degree of the polypeptide can be evaluated through the event frequency of perforation before and after the phosphorylation of the polypeptide.
Phosphatase such as alkaline phosphatase can catalyze nucleic acid molecules to remove phosphate groups, so that the phosphate groups at the ends of DNA or RNA are converted into hydroxyl groups, kinase can transfer the phosphate groups onto a substrate, and researches show that before and after reaction, blocking current signals, blocking current time and the like passing through aerolysin nanopores are obviously changed, and the frequency of the blocking signals is positively correlated with the concentration of the phosphatase or the kinase, so that qualitative and quantitative analysis can be carried out on the phosphatase or the kinase by using the aerolysin nanopores, and further real-time detection can be carried out.
The analysis and detection method comprises the following steps: under the action of voltage, the substance to be detected or the related probe molecule of the substance to be detected passes through the nanopore channel constructed by aerolysin, and the generated blocking current signal and blocking current time are analyzed to obtain the corresponding detection information of the substance to be detected. Further, the method specifically comprises the following steps: the method comprises the following steps:
(1) constructing an aerolysin nanopore channel: assembling a detection pool, adding electrolyte solution at two ends of the detection pool, and constructing and forming an aerolysin nanopore channel in the detection pool;
(2) applying voltage to two ends of the constructed aerolysin nanopore channel, adding an object to be detected or a probe molecule related to the object to be detected into one end (Cis end) of the detection cell, and enabling the object to be detected to pass through the aerolysin nanopore under the driving of the voltage to generate a blocking current signal and blocking current time;
(3) and comparing and analyzing the blocking current signal and the blocking current time to obtain corresponding detection information of the object to be detected.
In the step (1), the aerolysin is one of wild type aerolysin and an aerolysin mutant.
The electrolyte solution is selected from one or more of buffer solution, cell lysate, blood and intracellular fluid.
The buffer solution is a Tris buffer solution with the concentration of 0.1-3 mol/L; the pH range of the buffer solution is 4-11, and further 7-9; in polypeptide phosphorylation, proteinIn the analysis and detection of phosphorylation and related enzymes, 10-30 mmol/L Mg is added into electrolyte liquid2+、10～30mmol/L Ca2+Or 10 to 30mmol/L Zn2+。
The voltage in the step (2) is-300 mV to +300mV, except 0V.
The aerolysin nanopore channel is generally obtained by activating aerolysin and then embedding the activated aerolysin into a phospholipid bilayer, wherein the phospholipid bilayer can be prepared by a pulling method, when the activated aerolysin is embedded into the phospholipid bilayer, the aerolysin is added to the forward end of a detection pool, then a voltage of 100-300 mV is applied, and when the aerolysin forms a stable nanopore on a phospholipid bilayer membrane, the ion flow jumps.
Has the advantages that:
(1) the aerolysin nanopore channel can realize direct recognition of multiple phosphorylation states of nucleic acid molecules, does not need DNA motor protein, has high sensitivity, can detect ultralow-concentration nucleic acid (100fmol/L) in a solution, and recognizes different phosphorylation degrees (non-phosphorylation, one-end phosphorylation and two-end phosphorylation) and different phosphorylation sites (5 'phosphorylation or 3' phosphorylation), which is difficult to be effectively recognized by the existing alpha-hemolysin nanopore technology. Direct determination of nucleic acid phosphorylation status and nucleic acid sequence.
(2) The aerolysin nanopore channel is used for detecting the phosphorylation of the polypeptide, the polypeptide is not required to be modified by a genetic engineering or chemical modification method, the sensitivity is high, and the detection is convenient.
(3) The activity analysis and quantitative analysis of a plurality of enzymes such as kinase, phosphatase, enzyme inhibitor and the like can be realized by using the aerolysin nanopore, and the real-time monitoring of the enzymes can be realized.
(4) The aerolysin nanopore can be used for detecting the nucleic acid phosphorylation/dephosphorylation process, and realizing the research of an enzyme signal path and the real-time monitoring of the catalytic phosphorylation process.
Drawings
FIG. 1 is a graph of the analysis of the blocking current signal in different states of 5'-dA14-3' dephosphorylation/phosphorylation;
FIG. 2 is a graph showing the distribution of blocking signals before and after the action of P-5'-dA14-3' -P with alkaline phosphatase;
FIG. 3 is a graph of the current signal blocked by the polypeptide LRRASLG (S-peptide) and the phosphorylated polypeptide LRRASLG (P-peptide);
FIG. 4 is a graph of the frequency of blocking signal as a function of kinase A (PKA) concentration.
Detailed Description
The invention will be further elucidated with reference to the following specific examples.
In the following examples 1 to 4, the method disclosed in the patent with the title of the invention of the aerolysin nanopore channel and its application, for example, the method of example 1 in the patent can be specifically used, and specifically the following steps are included, for example, in the step (1) of constructing the aerolysin nanopore channel with reference to publication No. CN 104651500B:
(1) pretreatment of aerolysin
Mixing the trypsin-EDTA solution and aerolysin at a ratio of 1:100, culturing at room temperature for 10min, preparing the activated aerolysin by using PBS buffer solution, and storing in a refrigerator at the temperature of-20 ℃ at a storage concentration of 0.1 mg/ml.
(2) Preparation of phospholipid bilayers by Czochralski method
Preparing a phospholipid bilayer using a polyacetal resin detection cell as a carrier, the polyacetal resin detection cell comprising a detection cell I (namely, a cis chamber 1) and a detection cell II (namely, a trans chamber), the detection cell II being embedded in the detection cell I; the polyacetal resin detection cell was divided into two regions after the formation of the phospholipid bilayer: because the aerolysin nanopore is unidirectional when being inserted into the phospholipid membrane, a cis chamber (namely a detection pool I) corresponding to the large-opening end and a reverse chamber (namely a detection pool II) corresponding to the small-opening end are defined after the aerolysin nanopore is embedded into the phospholipid membrane; a small hole with the diameter of 50 mu m is formed in the detection cell II and is used for forming a phospholipid bilayer; a lifting hole communicated with the tank body is formed in the side edge of the detection tank I and is used for inserting an injector to lift the internal solution; the 1, 2-diphytanoyl phospholipid used for forming the phospholipid bilayer membrane is stored in a chloroform solution and stored in a refrigerator at-20 ℃. The specific process is as follows:
firstly, coating phospholipid n-decane solution, extracting chloroform from 1, 2-diphytanoyl phospholipid chloroform solution before preparing the phospholipid bilayer membrane, and then adding 90 mu l of n-decane into the extracted 1, 2-diphytanoyl phospholipid to prepare the phospholipid n-decane solution.
Coating phospholipid N-decane solution, uniformly coating the phospholipid N-decane solution on the inner side and the outer side of the small hole 4 of the 1mL detection pool II by using a mink hair painting brush, and drying by using N2.
And thirdly, applying voltage, respectively adding 1mL of electrolyte solution after assembling the detection pool I and the detection pool II, immersing a pair of Ag/AgCl electrodes into the electrolyte solution, applying 100mV voltage to two ends of the phospholipid bilayer membrane through a current amplifier probe, and designating a cis chamber as a virtual grounding end.
Repeatedly pulling the solution, forming a phospholipid bilayer membrane at the small hole of the reverse chamber, monitoring the formation quality of the phospholipid bilayer membrane through capacitance in the phospholipid bilayer membrane forming process, observing the mechanical strength of the phospholipid bilayer membrane by using voltage, and applying 400mV voltage to the obtained phospholipid membrane; re-pulling if the phospholipid bilayer membrane is broken, wherein the capacitance of the re-pulled phospholipid bilayer membrane is the same as or larger than that of the broken phospholipid bilayer membrane, and the phospholipid bilayer membrane can be used for forming a nanopore; if the capacitance decreases, the 400mV voltage should continue to be applied.
And fifthly, detecting and repeating, if the phospholipid bilayer membrane cannot be broken under the voltage of 400mV, brushing the phospholipid bilayer membrane with a mink hair painting brush, and then pulling the phospholipid bilayer membrane again to form a membrane, and repeating the steps III and IV until the phospholipid bilayer membrane capable of forming the nanopore is obtained.
(3) Formation of aerolysin nanopore channels
After the stable phospholipid bilayer membrane is formed, 10 mu l of aerolysin is added into the cis chamber, 100mV voltage is applied to enable the aerolysin to be embedded into the phospholipid bilayer membrane, and when the aerolysin forms a stable nano-channel on the phospholipid bilayer membrane, the ion flow can jump; single aerolysin nanopore channels with currents of 50. + -.5 pA were obtained at 100mV voltage.
Example 1: nucleic acid phosphorylation/dephosphorylation assays
(1) Constructing an aerolysin nanopore channel: assembling a detection pool, adding electrolyte solution (Tris buffer solution, 1mol/L) with the pH value of 8 at two ends of the detection pool, constructing a phospholipid bilayer at a micropore with the diameter of 50 mu m in the detection pool, adding aerolysin at one end of the detection pool, and forming an aerolysin nanopore on the phospholipid bilayer;
(2) applying 300mV voltage on two ends of an aerolysin nanopore channel, adding 10uL of an object to be detected (the addition amount is determined according to the detection requirement and is generally 1-100uL) into one end (Cis end) of a detection cell, and enabling the object to be detected to pass through the aerolysin nanopore to generate a blocking current signal and blocking current time under the drive of potential;
(3) and comparing and analyzing the blocking current signal and the blocking current time to obtain corresponding detection information of the object to be detected.
The DNA of the substances to be detected, which are respectively 5'-dA14-3' without phosphorylation, 5'-dA14-3' with 5'-dA 3526-3' with 5 '-end phosphorylation, 5' -dA14-3 'with 5' -end phosphorylation and 5'-dA14-3' with 5 '-end phosphorylation and 3' -end phosphorylation, are respectively detected, the current blocking signal and the current blocking time are analyzed and compared to obtain the corresponding detection information of the molecules to be detected, and the detection result graph is shown in figure 1.
In FIG. 1, I0Defined as the opening current of the nanopore of aerolysin, I is defined as the current value of a single blocking current signal, I/I0The blocking Current degree caused by the single molecule to be detected passing through the nanopore in the pore is represented by duration, namely the blocking time of a single blocking Current signal, and Current is a Current value. By taking the blocking time as an ordinate and the blocking current degree as an abscissa, 5'-dA14-3' in different phosphorylation states all generate a large number of blocking current signals, and the blocking current signals are distributed obviously differently on the coordinate axis. The unphosphorylated 5' -dA14-3' was detected using aerolysin nanopores, which resulted in two different blocking current states, one with a lesser degree of blocking current and a longer blocking time defined as PI, and the other with a greater degree of blocking current, as PII, from 5' -dA14-3' the act of perforation occurs as a result of the different directions in the perforation process, i.e. the 3' end (PI) or the 5' end (PII) first enters the nanopore. Then, 5'-dA14-3' which is commonly phosphorylated at any end and two ends of 5'-dA14-3' is detected by using a monalysin nanopore, and each current blocking signal is reduced by using data analysis processing software to make a scatter diagram, so that the phosphorylation of 5'-dA14-3' can greatly shorten the blocking time of the perforation process. For 5' -end phosphorylated 5' -dA14-3', the blocking time of PII distribution is shortened by nearly 30 times, while the blocking time of PI distribution is almost unchanged; in contrast, for the 3' end phosphorylated 5' -dA14-3', the blocking time for PI distribution was greatly reduced, approximately 52-fold, while the blocking time for PII distribution was almost unchanged; p-5'-dA14-3' -P is co-phosphorylated at the 5 'end and the 3' end, and the blocking time of PI and PII distribution is greatly reduced. The above data indicate that the phosphorylation process at the 3 'or 5' end significantly reduces the blocking time of the blocking current signal generated by the first entry of the end into the nanopore, and thus it is known that the phosphorylation or dephosphorylation process at each end of the oligonucleotide can be directly read from the duration of PI/PII.
In addition, the method is also suitable for the phosphorylation/dephosphorylation detection of RNA, and the detection method and the analysis of the detection result are the same as those of the phosphorylation/dephosphorylation detection of DNA.
Example 2: detection of alkaline phosphatase
(1) Constructing an aerolysin nanopore channel: assembling the detection cell and adding MgCl at two ends2Electrolyte solution (Tris buffer, 1mol/L) of pH 7.5, MgCl2The concentration of the phospholipid is 20mmol/L, a phospholipid bilayer is constructed at a micropore with the diameter of 50 mu m on the detection pool, aerolysin is added at one end of the detection pool, and an aerolysin nanopore is formed on the phospholipid bilayer;
(2) applying 100mV voltage to two ends of an aerolysin nanopore channel, adding 10ul of an object to be detected into one end (Cis end) of a detection cell, and enabling the object to be detected to pass through the aerolysin nanopore to generate a blocking current signal under the drive of potential;
(3) and comparing and analyzing the blocking current signal and the blocking current time to obtain corresponding detection information of the object to be detected.
The substances to be detected are respectively phosphorylated DNA chains (P-5' -dA)14-3' -P) and the products of the reaction with alkaline phosphatase are respectively detected, and the corresponding detection information of the molecules to be detected is obtained by analyzing and comparing the blocking current signals and the blocking current time. The detection result is shown in FIG. 2.
In FIG. 2, I0Defined as the opening current of the nanopore of aerolysin, I is defined as the current value of a single blocking current signal, I/I0The Duration is the blocking time of a single blocking current signal, which is the degree of blocking current in the pore caused by a single molecule to be detected passing through the nanopore. Phosphorylated DNA strand (P-5' -dA) with blocking time as ordinate and degree of blocking current as abscissa14-3' -P) has a distinctly different distribution on the axis of the coordinate axis than the product after the alkaline phosphatase has been added. Phosphorylated DNA strand (P-5' -dA)14-3' -P) has a distinctly different distribution on the axis of the coordinate axis than the product after the alkaline phosphatase has been added. Phosphorylated DNA strand (P-5' -dA)14-3 '-P) significantly increased signal intensity and increased blocking time after alkaline phosphatase addition, mainly due to the catalysis of P-5' -dA by alkaline phosphatase14Removing phosphate groups at the 3 'end and the 5' end of the-3 '-P, and sequentially generating P-5' -dA14-3 'and 5' -dA14-3'. By comparing the blocking current signals before and after the reaction, the activity information of the alkaline phosphatase can be obtained, the frequency of the blocking signal is positively correlated with the concentration of the alkaline phosphatase, and then the alkaline phosphatase can be quantitatively analyzed, and the catalytic activity of the phosphatase on dephosphorylation of different sites of nucleic acid can be compared.
The method is also suitable for the analysis of the catalytic activity of the alkaline phosphatase on RNA and the quantitative analysis of the alkaline phosphatase.
Example 3: detection of phosphorylation of polypeptides
(1) Constructing an aerolysin nanopore channel: assembling the detection cell and adding MgCl at two ends2Electrolyte solution (Tris buffer, 1mol/L) of pH 7.5, MgCl2At a concentration of 20mmol/L, phosphorus was formed at 50 μm micropores on the inside of the detection cellThe lipid bilayer, adding aerolysin at one end of the detection pool, and forming an aerolysin nanopore on the phospholipid bilayer;
(2) 300mV voltage is applied to two ends of the nano channel, 10ul of the object to be detected is added to one end (Cis end) of the detection cell, and the object to be detected passes through the aerolysin nano hole under the driving of the potential to generate a current blocking signal and current blocking time.
(3) And comparing and analyzing the blocking current signal and the blocking current time to obtain corresponding detection information of the object to be detected.
The test substance takes LRRASLG as model polypeptide, respectively detects unphosphorylated polypeptide LRRASLG (S-peptide) and phosphorylated polypeptide LRRASLG (P-peptide), obtains corresponding detection information of the test substance by analyzing and comparing the time of blocking current signal and blocking current, and the detection result chart is shown in figure 3.
In FIG. 3, I0Defined as the opening current of the nanopore of aerolysin, I is defined as the current value of a single blocking current signal, I/I0The blocking current degree caused by the single molecule to be detected passing through the nanopore in the pore is represented by duration, which is the blocking time of a single blocking current signal. The blocking time is used as an ordinate, the blocking current degree is used as an abscissa, and the S-peptide and the phosphorylated P-peptide generate different blocking current signals and have obviously different distributions on the coordinate axis. First, the S-peptide is detected by using an aerolysin nanopore, the polypeptide has a net charge of +2 at pH 7.5, because a blocking current signal is difficult to generate under the condition of positive potential application, so that the distribution is less on a scatter diagram of blocking time-blocking current, after Ser-5 is phosphorylated, the net charge of the P-peptide is reduced to 0, the electrophoretic force of the P-peptide entering the aerolysin nanopore is enhanced, and more blocking current signals are generated, so that the phosphorylation degree of the polypeptide can be evaluated through the event frequency of perforation of the S-peptide and the P-peptide.
Example 4: phosphorylation Activity of kinase A
(1) Constructing an aerolysin nanopore channel: assembling the detection cell and adding MgCl at two ends2Electrolyte solution (Tris buffer, 1 m) of pH 7.5ol/L)，MgCl2The concentration of the phospholipid is 20mmol/L, a phospholipid bilayer is constructed at a micropore with the diameter of 50 mu m on the detection pool, aerolysin is added at one end of the detection pool, and an aerolysin nanopore is formed on the phospholipid bilayer;
(2) the voltage of +100mV is applied to two ends of the nano channel, 10ul of substance to be detected is added to one end (Cis end) of the detection cell, and the substance to be detected passes through the aerolysin nano hole under the drive of the potential to generate a current blocking signal and current blocking time.
(3) And comparing and analyzing the blocking current signal and the blocking current time to obtain corresponding detection information of the object to be detected.
Using unphosphorylated polypeptide LRRASLG (S-peptide) as substrate, and respectively using S-peptide and products (in ATP and Mg) acted with kinase A (PKA) with different concentrations as test substances2+Fully reacting S-peptide with PKA in the presence of the ATP, heating and quenching to perform reaction and inactivate enzyme, transferring a gamma-phosphate group of ATP to a serine residue of the S-peptide by the PKA, adding a solution after the reaction into an aerolysin nanopore detection cell for analysis), and comparing a blocking current signal with a blocking current time to obtain corresponding detection information of a molecule to be detected, wherein a detection result graph is shown in figure 4.
In fig. 4, the concentration of PKA is used as an abscissa, the blocking current frequency (reciprocal of the period of occurrence of the blocking signal) is used as an ordinate, and at a potential of +100mV, S-peptide hardly generates a blocking current signal, but generates an obvious blocking current signal after the PKA acts, and the frequency of the blocking current signal is positively correlated with the concentration of PKA, so that qualitative and quantitative analysis of PKA can be performed.
The method can also be used for real-time detection of PKA, the specific steps of the experiment are as above, the reactant reacts with PKA, the solution after the reaction is collected at different reaction moments (0-48h) is added into the aerolysin nanopore detection cell for analysis, the concentration of the reaction product is gradually increased along with the reaction time, the frequency of the characteristic blocking current is positively correlated with the reaction time, the signal frequency and the reaction time are plotted, the information of the PKA catalytic reaction at different moments can be obtained, and the real-time analysis of PKA is realized.
The invention is not only suitable for the activity analysis, real-time detection and quantitative analysis of alkaline phosphatase and kinase A, but also suitable for other kinases, phosphatases and enzyme inhibitors.
Claims (6)
1. The application of the aerolysin nanopore channel in the analysis and detection of biological phosphorylation or related enzymes is characterized in that the analysis and detection of biological phosphorylation comprises one or more of the analysis and detection of nucleic acid phosphorylation, nucleic acid dephosphorylation, polypeptide phosphorylation, polypeptide dephosphorylation, protein phosphorylation and protein dephosphorylation, the analysis and detection of related enzymes comprises one or more of enzyme activity analysis, enzyme activity real-time monitoring and enzyme quantitative analysis, and under the action of voltage, a substance to be detected or a related probe molecule of the substance to be detected passes through the nanopore channel constructed by the aerolysin to analyze a generated blocking current signal and blocking current time so as to obtain the corresponding detection information of the substance to be detected; the application specifically comprises the following steps:
(1) constructing an aerolysin nanopore channel: assembling a detection pool, adding electrolyte solution at two ends of the detection pool, and constructing and forming an aerolysin nanopore channel in the detection pool;
(2) applying voltage to two ends of the constructed aerolysin nanopore channel, adding an object to be detected or a probe molecule related to the object to be detected into one end of a detection pool, and enabling the object to be detected to penetrate through the aerolysin nanopore under the driving of the voltage to generate a blocking current signal and blocking current time;
(3) and comparing and analyzing the blocking current signal and the blocking current time to obtain corresponding detection information of the object to be detected.
2. Use of an aerolysin nanopore channel according to claim 1 in the analytical detection of biological phosphorylation or related enzymes, wherein nucleic acid phosphorylation comprises 5 'end phosphorylation or/and 3' end phosphorylation and nucleic acid dephosphorylation comprises 5 'end dephosphorylation or/and 3' end dephosphorylation.
3. The use of an aerolysin nanopore channel according to claim 1 in the analytical detection of biological phosphorylation or related enzymes, wherein said nucleic acid is 1-10 kbp DNA or RNA; the length of the polypeptide is 2-100 amino acids; the molecular weight of the protein is 1-500 kDa; the related enzyme is hydrolase, kinase, phosphatase, enzyme inhibitor, phosphorylase, phosphatase or glycosyltransferase.
4. The use of an aerolysin nanopore channel according to claim 1 in the analytical detection of biological phosphorylation or related enzymes, wherein said aerolysin is one of wild-type aerolysin and an aerolysin mutant.
5. The use of the aerolysin nanopore channel of claim 1 in the analytical detection of biological phosphorylation or related enzymes, wherein said electrolyte solution of step (1) is selected from the group consisting of buffer, cell lysate, blood, and intracellular fluid; the voltage in the step (2) is-300 mV to +300mV, except 0V.
6. The use of the aerolysin nanopore channel according to claim 5 in the analytical detection of biological phosphorylation or related enzymes, wherein said buffer is Tris buffer at a concentration of 0.1-3 mol/L; the pH range of the buffer solution is 4-11; in the analysis and detection of polypeptide phosphorylation, protein phosphorylation and related enzymes, 10-30 mmol/L Mg is added into an electrolyte solution2+、10~30 mmol/L Ca2+Or 10 to 30mmol/L Zn2+。
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