Method and kit for simultaneous detection of multi target molecules using magnetic bead-aptamer conjugate

Abstract

This application relates to molecular biology, and more specifically to a method which uses the molecular recognition between a target molecule ligand and an aptamer, magnetic separation and MS qualitative and quantitative analysis to enable the association between the detection of multi molecules and information of multiple functional groups, and effectively determine the correlation between molecules and functional groups of the body or tissue. This application can easily purify the target molecules by magnetic separation and can effectively obtain the target molecule group based on the high specificity and affinity of the aptamer. In addition, based on the MS detection, this application can effectively perform the qualification and quantification of the multi molecules, achieving the secondary molecular detection and improving the detection accuracy. The simultaneous qualification and quantification of multi molecules can not only accurately reflect the relationship among molecules, but also reveal the interrelationship among body functions, playing a significant role in the proteomics and genomics research and clinical molecular detection.

Claims

1. A method for simultaneous detection of multi target molecules using a magnetic bead-aptamer complex, comprising: (1) incubation incubating the magnetic bead-aptamer complex with a target molecule-containing sample to form a magnetic bead-aptamer-target molecule complex, wherein the aptamer are a group of aptamers screened by SELEX and specific to the target molecule-containing sample; and further comprising the step of: preparing the magnetic bead-aptamer-target molecule complex by incubating 100 μL of the target molecule-containing sample with 50 μL of the magnetic bead-aptamer complex at 37° C. for 30 min, washing the reaction mixture once with 3×SSC; and washing the reaction mixture three times with 0.4 mM binding buffer each for a volume of 20 times the volume of the reaction mixture followed by magnetic separation to obtain the magnetic bead-aptamer-target molecule complex: (2) acid elution adding an elution buffer to the magnetic bead-aptamer-target molecule complex under shaking to collect an eluate to which Tris-HCl containing a standard peptide is added to neutralize the eluate for detection of the target molecules, wherein the acid elution comprises the steps of: adding 0.5 mL of the elution buffer to the magnetic bead-aptamer-target molecule complex obtained in step (1) followed by shaking for 1 min to collect the eluate; and immediately neutralizing the eluate with 250 μL of 1 M Tris-HCl (pH 8.0): repeating the elution and neutralization twice; and combining the neutralized eluates to obtain a target molecule solution for mass spectrometry; (3) subjecting the neutralized eluate to mass spectrometry for qualitative and quantitative detection of the target molecules; and (4) analyzing data from the mass spectrometry on a data analytics platform to obtain the detection results.

2. The method according to claim 1, wherein that in the step (1), the target molecule-containing sample comprises at least one of serum, urine, body fluid and cell and tissue suspensions.

3. The method according to claim 1, wherein that in the step (2), an efficiency of the acid elution for separating the target molecules from the aptamer is 98% or more.

4. The method according to claim 1, wherein that in the step (2), the standard peptide is a reference standard peptide that is introduced in a mass spectrometric sample for accurate qualification and quantification of a specified peptide.

5. The method according to claim 1, wherein that in the step (3), the target molecules which are obtained by screening have a specific marker.

6. The method according to claim 1, wherein that in the step (4), a functional mechanism of a specified protein is speculated based on a dose-effect relationship, and an analysis report is made.

7. The method according to claim 1, wherein that in the step (1), the target molecule-containing sample is prepared by the following steps: collecting the required amount of blood by venipuncture; immediately removing a needle and injecting the blood into a test tube containing an anticoagulant; immediately shaking the test tube gently to mix the blood and the anticoagulant uniformly to avoid coagulation; and centrifuging the test tube at 3,000-6,000 rpm to obtain a serum sample.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

(2) FIG. 1 schematically shows Embodiment 1 of the invention. It should be noted that in FIG. 1, aptamers A, B, C and D are aptamers of p120 and p24 antigens. CD4 and membrance protein antibody and antigen.

(3) FIG. 2 schematically shows Embodiment 2 of the invention; It should be noted that in FIG. 2, aptamer A is a specific aptamer shared by gastric cancer, liver cancer and lung cancer; aptamer B is a specific aptamer of gastric cancer; aptamer C is a specific aptamer of liver cancer; and D is a specific aptamer of lung cancer.

(4) FIG. 3 schematically shows Embodiment 3 of the invention. It should be noted that in FIG. 3, aptamer A is a specific aptamer of gastric cancer trauma; aptamer B is a specific aptamer of induced gastric cancer stem cells; aptamer C is a specific aptamer of autophagic antibody of gastric cancer; and D is a specific aptamer of gastric cancer.

DETAILED DESCRIPTION OF EMBODIMENTS

(5) In Figures:

(6) 11 Aptamter A; 12 Aptamter B; 13 Aptamter C; 14 Aptamter D;

(7) 2 Magnetic bead carrier;

(8) 31 Protein ligand of aptamer A; 32 Protein ligand of aptamer B; 33 Protein ligand of aptamer C; 34 Protein ligand of aptamer D;

(9) 41 Maker peptide of protein ligand 4; 42 Maker peptide of protein ligand 8; 43 Maker peptide of protein ligand n; 44 Maker peptide of protein ligand x;

(10) 5 HIV virus;

(11) 6 Aptamer of HIV virus;

(12) 7 Viral RNA;

(13) 8 PCR product.

(14) (1) A sample to be detected is degreased, specifically, the sample is cooled to 4° C. and centrifuged at 3,000 rpm for 20 min, and the upper lipid layer is discarded.

(15) (2) The degreased sample is added to a 0.4 mM binding buffer containing magnetic beads carrying a specific aptamer group to allow a target molecule to bind to the magnetic beads.

(16) (3) The target molecule-binding magnetic beads are washed once with 3×SSC buffer (pH 7.4), and then washed three times with the binding buffer each for a volume of 10 times the volume of the magnetic beads.

(17) (4) The specific marker-binding magnetic beads are eluted with a basic buffer, and the resulting eluate is neutralized with an acidic buffer in a volume ratio of 2:1 to form a target molecule extract.

(18) (The above steps (2)-(4) can be performed in one time by a magnetic bead washing-extracting machine or the like).

(19) (5) The target molecule extract is analyzed by laser-resolution TOF mass spectrometry to determine the content of a specific protein in the sample.

(20) (6) The obtained specific protein content is analyzed and processed by a data platform to determine the pathogenesis of a disease and recommend a treatment regimen.

(21) In an embodiment, a kit of this application includes 5-10 mL of a reagent 1, 5-10 mL of a reagent 2 and a reagent 3, where the reagent 1 is a 0.01-0.1 M pH 7.4 phosphate buffer containing 50% of streptavidin agar magnetic beads with a particle size of 5-50 nm; the reagent 2 is a 0.4 mM pH 7.4 1× binding buffer containing 0.003-0.3 μg/L of a specific biotinylated aptamer; and the reagent 3 is an elution buffer.

(22) Specifically, the agar magnetic beads are bound with the streptavidin via a carboxyl group (or an epoxy group), and then bound with a biotinylated aptamer to form a carrier for the target molecule binding (containing 0.01% sodium azide as a preservative).

(23) 0.4 mM pH 7.4 binding buffer containing 0.01% sodium azide as a preservative is used for the binding between the aptamer and the target molecule, where 1 L 1× binding buffer is prepared as follows. A solution containing 138 mmol/L NaCl, 2.7 mmol/L KCl, 8.1 mmol/L Na2HPO4, 1.1 mmol/L KH2PO4 and 1 mmol/L MgCl2 is first prepared, adjusted to pH 7.4 with HCl, dilute to 1 L with water, sterilized by high pressure steam for 20 min and stored at room temperature for use.

(24) The target molecule-binding magnetic beads are washed with 3×SSC buffer (pH 7.4).

(25) The target molecule-binding magnetic beads are eluted with a pH 3.0 basic eluent (containing 0.1 M glycine, 0.5 M sodium chloride and 0.05% Tween 20) in a volume ratio of 1:2 under shaking for 1 min, and the eluate is collected.

(26) The eluate is neutralized with a pH 8.0 acidic buffer (250 μL of 1 M Tris-HCl) containing a standard peptide reagent in a volume ratio of 2:1.

(27) In an embodiment, this application provides a method for simultaneous detection of multi target molecules using a magnetic bead-aptamer complex, where magnetic beads coated with a specific aptamer are used to extract the target molecule group, and then the target molecule group is subjected to qualification and quantification by MS, finally, the obtained results are analyzed by a data platform to give the reference results. The method specifically includes the following steps.

(28) (1) Sample preparation

(29) The required amount of blood is collected by venipuncture, and the needle is immediately removed. The blood is injected into a test tube containing an anticoagulant, and immediately shaken gently to mix the blood and the anticoagulant uniformly to avoid coagulation. Then, the test tube is centrifuged at 3,000-6,000 rpm to obtain a serum sample.

(30) (2) Formation of a magnetic bead-aptamer-target molecule complex

(31) 100 μL of the serum sample is incubated with 50 μL of a magnetic bead-aptamer complex at 37° C. for 30 min. The reaction mixture is washed once with 3×SSC, and then washed three times with 0.4 mM binding buffer each for a volume of 20 times the volume of the reaction mixture. The reaction mixture is magnetically separated to obtain the magnetic bead-aptamer-target molecule complex.

(32) (3) Preparation of a target molecule sample

(33) The magnetic bead-aptamer-target molecule complex is added with 0.5 mL of a pH 3.0 elution buffer containing 0.1 M glycine, 0.5 M sodium chloride and 0.05% Tween 20 and shaken for 1 min. The eluate is collected and immediately neutralized with 250 μL of 1 M Tris-HCl (pH 8.0) containing a standard peptide for qualification and quantification. Such elution and neutralization are repeated three times and the resulting three eluates are combined to produce a target molecule solution for mass spectrometry.

(34) (4) Mass spectrometry

(35) The target molecule solution is qualitatively and quantitatively determined by mass spectrometry to obtain the information about the target molecule.

(36) (5) The obtained data are analyzed by data platform and an analysis report is generated.

(37) The invention is further described below with reference to embodiments.

Example 1 Shortening of Window Period by Simultaneous Detection of Multiple Proteins and Genes

(38) Referring to FIG. 1, the proteins and genes were both qualitatively and quantitatively analyzed to determine the status of HIV infection, such as window period, silent period and outbreak period.

(39) Sample Preparation (Serum)

(40) After the required materials were prepared, labeled and checked, the required amount of blood was collected by venipuncture, and the needle was immediately removed. The blood was injected into a test tube containing an anticoagulant, and the test tube was immediately shaken gently to mix the blood and the anticoagulant uniformly to avoid coagulation. Then the test tube was centrifuged at 3,000-6,000 rpm to obtain a serum sample which was stored at 4° C. for use.

(41) Preparation of aptamers respectively of P24 antigen, P24 antibody, CD4 and viral envelope antibody

(42) The specific aptamers respectively of P24 antigen, P24 antibody, CD4 and virus envelope antibody were obtained by SELEX.

(43) Preparation of magnetic bead-aptamer-multi-protein (including P24 antigen, P24 antibody, CD4 and virus envelope antibody) “sandwich” complex

(44) Streptavidin magnetic beads were selected, added with biotinylated aptamers (including aptamers of P24 antigen, P24 antibody, CD4 and virus envelope antibody) and incubated for 30 min to produce a magnetic bead-aptamer complex. The magnetic bead-aptamer complex is washed 3 times with PBS containing 0.05% Tween 20 each for 3 min. 10-100 μL of the above magnetic bead-aptamer complex was added to 100-1000 μL of the serum sample. The reaction mixture was incubated for 30 min for binding to form a magnetic bead-aptamer-multi-protein complex. The magnetic bead-aptamer-multi-protein complex was adhered by an electromagnetic pole to be isolated from the unbound serum. After the serum was aspirated, the magnetic bead-aptamer-multi-protein complex was washed 3-12 times with 0.01-0.1 M PBS containing 0.01-0.1 M Tween 20 each for 400 μL and 3-6 min.

(45) Preparation of a Solution for Mass Spectrometry

(46) The acid elution was performed as follows. The magnetic bead-aptamer-multi-protein complex was eluted with 0.5 mL of an elution buffer (pH 3.0) containing 0.1 M glycine, 0.5 M sodium chloride and 0.05% Tween 20 under shaking for 1 min. The resulting eluate was collected and immediately neutralized with 250 μL of 1 M Tris-HCl (pH 8.0). Such elution and neutralization were repeated three times, and the resulting three eluates were collected and combined.

(47) MS Detection

(48) The multiple proteins including P24 antigen, P24 antibody, CD4 and virus envelope antibody were qualitatively and quantitatively determined by a mass spectrometer.

(49) Gene Detection

(50) The serum was added to nucleic acid-extracting magnetic beads to produce a nucleic acid-magnetic bead complex. The nucleic acid-magnetic bead complex was lysed by a lysis buffer, washed and eluted, and the eluate was subjected to reverse transcription-PCR amplification.

(51) The data was collected, processed and analyzed.

(52) A detection report was generated and printed.

(53) This method can simultaneously detect and analyze RNA, p120 and p24 antigens, CD4 and a membrane protein antibody and antigen, which facilitates the comprehensive understanding of the patient's condition, providing a reasonable therapeutic regimen.

Example 2 Detection of Multiple Tumor Makers

(54) Referring to FIG. 2, the occurrence of a tumor and the type of a cancer (gastric cancer, lung cancer and liver cancer) were determined by the dose-effect relationship of the corresponding peptides.

(55) Preparation of a Serum Sample

(56) After the required materials were prepared, labeled and checked, the required amount of blood was collected by venipuncture, and the needle was immediately removed. The blood was injected into a test tube containing an anticoagulant, and the test tube was immediately shaken gently to mix the blood and the anticoagulant uniformly to avoid coagulation. Then the test tube was centrifuged at 3,000 rpm to obtain a serum sample which was stored at 4° C. for use.

(57) Preparation of a Tumor Marker Aptamer

(58) Gastric cancer, liver cancer and lung cancer serums were respectively used as a composite target molecule (non-gastric cancer serum as control) to obtain their corresponding aptamers by SELEX, where the apatmer which was shared by the three tumors was used as a tumor marker aptamer.

(59) Preparation of a Magnetic Bead-Tumor Marker Aptamer Complex

(60) Streptavidin magnetic beads were added with a biotinylated tumor marker aptamer and incubated for 30 min to produce a magnetic bead-aptamer complex. The magnetic bead-aptamer complex was washed 3 times with PBS containing 0.05% Tween 20 each for 3 min.

(61) Preparation of a Magnetic Bead-Aptamer-Multi Tumor Marker “Sandwich” Complex

(62) 10-100 μL of the above agar magnetic bead-tumor marker aptamer complex was added to 100-1000 μL of the serum sample. The reaction mixture was incubated for 30 min for binding to produce a magnetic bead-aptamer-multi-protein complex. The magnetic bead-aptamer-multi-protein complex was adhered by an electromagnetic pole to be isolated from the unbound serum. After the serum was aspirated, the magnetic bead-aptamer-multi-protein complex was washed 3-12 times with 0.01-0.1 M PBS containing 0.01-0.1 M Tween 20 each for 400 μL and 3-6 min.

(63) Preparation of Multiple Tumor Markers

(64) The acid elution was performed as follows. The magnetic bead-aptamer-multi-tumor marker complex was eluted with 0.5 mL of an elution buffer (pH 3.0) containing 0.1 M glycine, 0.5 M sodium chloride and 0.05% Tween 20 under shaking for 1 min. The resulting eluate was collected and immediately neutralized with 250 μL of 1 M Tris-HCl (pH 8.0) containing a standard peptide for the detection of a target molecule. Such elution and neutralization were repeated three times, and the resulting three eluates were combined to give a mixture of multiple tumor markers.

(65) MS Detection

(66) The multiple target molecules were qualitatively and quantitatively determined using a mass spectrometer according to the standard peptide in the mixture. The data was collected, processed and analyzed. In the case that the marker molecules extracted by the three types of tumor aptamers were identified by mass spectrometry to be a common marker molecule, it was confirmed that a tumor occurred. The specific marker molecules respectively corresponding to the three types of tumors can be used for the identification of a tumor.

(67) A test report was generated and printed.

Example 3 Detection of Multiple Marker Molecules Associated with Gastric Cancer Pathogenesis

(68) Referring to FIG. 3, the pathogenesis of gastric cancer was determined according to the dose-effect relationship of a peptide.

(69) Preparation of a Serum Sample

(70) After the required materials were prepared, labeled and checked, the required amount of blood was collected by venipuncture, and the needle was immediately removed. The blood was injected into a test tube containing an anticoagulant, and the test tube was immediately shaken gently to mix the blood and the anticoagulant uniformly to avoid coagulation. Then the test tube was centrifuged at 3,000-6,000 rpm to obtain a serum sample which was stored at 4° C. for use.

(71) Preparation of an Aptamer of Multiple Marker Molecules Associated with the Pathogenesis of Gastric Cancer

(72) Gastric cancer serum (non-gastric cancer serum as control) was used as a composite target molecule to obtain a gastric cancer marker aptamer by SELEX.

(73) Preparation of a Magnetic Bead-Gastric Cancer Marker Aptamer Complex

(74) Streptavidin magnetic beads were added with a biotinylated gastric cancer marker aptamer. The reaction mixture was incubated for 30 min to form a magnetic bead-aptamer complex. Then the magnetic bead-aptamer complex was washed 3 times with PBS containing 0.05% Tween 20 each for 3 min.

(75) Preparation of a Magnetic Bead-Aptamer-Gastric Cancer Marker “Sandwich” Complex

(76) 10-100 μL of the above agar magnetic bead-gastric cancer marker aptamer complex was added to 100-1000 μL of the serum sample and incubated for 30 min for binding to produce a magnetic bead-aptamer-gastric cancer marker complex. The magnetic bead-aptamer-gastric cancer marker complex was adhered by an electromagnetic pole to be isolated from the unbound serum. After the serum was aspirated, the magnetic bead-aptamer-gastric cancer marker complex was washed 3-12 times with 0.01-0.1 M PBS containing 0.01-0.1 M Tween 20 each for 400 μL and 3-6 min.

(77) Preparation of Multiple Gastric Cancer Markers

(78) The acid elution was performed as follows. The magnetic bead-aptamer-gastric cancer marker complex was eluted with 0.5 mL of an elution buffer (pH 3.0) containing 0.1 M glycine, 0.5 M sodium chloride and 0.05% Tween 20 under shaking for 1 min. The resulting eluate was collected and immediately neutralized with 250 μL of 1 M Tris-HCl (pH 8.0) containing a standard peptide for the detection of the target molecule. Such elution and neutralization were repeated three times, and the resulting three eluates were collected and combined to give a mixture of multiple tumor markers.

(79) MS Detection

(80) The multiple target molecules were qualitatively and quantitatively determined using a mass spectrometer according to the standard peptide in the mixture.

(81) Data Collection, Processing and Analysis

(82) The marker target molecules associated with the pathogenesis of gastric cancer were extracted by the gastric tumor aptamers and identified by mass spectrometry to summarize the pathogenesis of gastric cancer.

(83) A test report was generated and printed.

INDUSTRIAL APPLICABILITY

(84) The invention relates to molecular biology, and more specifically to a method and a kit for simultaneous detection of multi target molecules using a magnetic bead-aptamer complex. The method includes the steps of screening out a specific aptamer; quantitatively and qualitatively detecting a target molecule group by mass spectrometry; and processing data and providing an analysis report by a data platform, which enables the simultaneous detection of related multiple target molecules. In addition, the condition of the entirety or a certain function can be comprehensively and clearly shown based on the data analysis of multiple target molecules. The invention uses a specific aptamer to capture the related target molecule group, and then the target molecules were qualitatively and quantitatively analyzed by mass spectrometry to relatively comprehensively reflect the change in function of the body or tissue. Moreover, this method can provide a large amount of data, which greatly improves the accuracy of the omic analysis. Therefore, the invention is of great significance for proteomics and genomics research and clinical molecular detection.

(85) It should be understood that various modifications and changes made by those skilled in the art based on the above description should fall within the scope defined by the appended claims.

(86) These embodiments are merely illustrative of the invention, and are not intended to limit the invention. The modifications made without departing from the spirit of the invention, or the direct use of the technical solutions of the invention in other occasions, should fall within the scope of the invention.