CN111321204B - Aptamer sensor-based high-sensitivity method for detecting PD-L1 in serum - Google Patents
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Abstract
A method for detecting PD-L1 in serum with high sensitivity based on an aptamer sensor belongs to the field of medication auxiliary diagnosis. At present, the detection in the serum exosome PD-L1 is an enzyme-linked immunosorbent assay based on antigen-antibody hybridization, and has the defects of high detection cost, high operation difficulty and the like. The invention takes PD-L1 aptamer as an example, realizes multiple amplification of detection signals through enzyme digestion auxiliary target circulation and Q-PCR, and establishes a PD-L1 hypersensitive detection sensor. The method can provide a detection method with lower cost, low operation difficulty and high sensitivity for the serum PD-L1.
Description
Technical Field
The invention belongs to the field of medication auxiliary diagnosis, and particularly relates to a method for detecting serum PD-L1 with high sensitivity by using an aptamer. At present, the detection in the serum exosome PD-L1 is an enzyme-linked immunosorbent assay based on antigen-antibody hybridization, and has the defects of high detection cost, high operation difficulty and the like. The method can provide a detection method with lower cost, low operation difficulty and high sensitivity for the serum PD-L1.
Background
Biomarkers in serum can well reflect the current physical condition of a person, and doctors often use the information for diagnosis, development state, medication assistance and the like of diseases. However, the complex components in serum, which have not been fully studied until now, have made detection of the present extremely minute amounts of biomarkers extremely difficult.
The enzyme-linked immunosorbent assay based on the monoclonal antibody is the most widely applied biomarker detection means at present, but the development of the enzyme-linked immunosorbent assay is hindered by high production and storage cost, difficulty in guaranteeing consistency among batches and the like. Therefore, we propose a method for highly sensitive detection of serum biomarkers (taking programmed cell death ligand 1 as an example) based on aptamer sensors to achieve highly sensitive detection of biomarkers in serum samples.
The aptamer is a good substitute of an antibody, is known as a chemical antibody, refers to a single-chain DNA or RNA micromolecule generated by a ligand systematic evolution by exponential enrichment (SELEX) technology, and has the advantages of low production cost, strong stress resistance, good batch consistency, easy production and the like.
Meanwhile, the method also has great significance for detecting programmed cell death ligand (PD-L1). PD-L1 is a type I transmembrane protein, and is expressed on the surfaces of various tumor cells. Interact with programmed cell death receptor 1(PD-1) on the surface of activated T cells to generate immune checkpoint responses. Based on this interaction, a variety of immune checkpoint inhibitors have been developed for clinical treatment of human cancers. However, clinical benefit is observed in only 20-30% of patients. More seriously, such immunotherapeutic drugs may cause serious side effects in non-benefited patients. Thus, there is an urgent need for prognostic biomarkers to determine which patients will fail early treatment and which patients will achieve a long lasting therapeutic effect.
Currently, a number of biomarkers have been discovered to predict the therapeutic efficacy of PD-1/PD-L1 inhibitors, such as tumor progression-associated PD-L1 expression levels, mismatch repair (MMR) status, Tumor Mutational Burden (TMB), etc., with PD-L1 being the earliest, most abundant one of the studies. At present, PD-L1 expressed on the surface of tumor cells is mainly detected by monoclonal antibody enzyme-linked immunosorbent assay (ELISA). However, the requirement for extraction of tumor tissue from patients and the cost of monoclonal antibody production have made this strategy somewhat unsatisfactory. Recent studies have shown that exocrine PD-L1 in blood can be used as a potential tumor biomarker for prognostic evaluation. Therefore, the technology for detecting PD-L1 in blood or serum has wide application prospect.
Disclosure of Invention
The invention aims to establish a high-sensitivity aptamer detection sensor, reduce the experiment cost and the operation difficulty and realize high-sensitivity detection of biomarkers. The invention takes PD-L1 aptamer as an example, realizes multiple amplification of detection signals through enzyme digestion auxiliary target circulation and Q-PCR, and establishes a PD-L1 hypersensitive detection sensor. By using the sensor, the detection limit of PD-L1 can reach 0.076ng/mL in a buffer system; in a human serum sample, the detection limit of PD-L1 can reach 0.3625 ng/mL.
The above purpose is realized by the following technical scheme:
1. the following oligonucleotide sequences were synthesized:
apt38 of biotin modified at the 5' end, which is marked as Bio-Apt 38:
5’-Biotin-AAG ACG GAC CAG CCT TGC CGC AAG ACG GAC CAG GGA TT-3’
the amplification template strand No. 1, denoted P1, was underlined the base complementary pairing part with Bio-Apt38 (same below):
5’-AAT CCC TGG TCC GTC TTG CGG CAA GGC TGG TCC GTC TTA GCA GCA CAG AGG TCA GAA CGA TGG TCG ACA CAC GAT CAG AAG CTT GGC ATG GAT GAC GAC G-3’
amplification template strand No. 2, denoted P2:
5’-AAT CCC TGG TCC GTC TTG CGG CAA GGC TGG TCC TGA GGA GCA GCA CAG AGG TCA GAA CGA TGG TCG ACA CAC GAT CAG AAG CTT GGC ATG GAT GAC GAC G-3’
amplification template strand No. 3, labeled P3:
5’-AAT CCC TGG TCC GTC TTG CGG CAA GGC TTT GAA TGA GGA GCA GCA CAG AGG TCA GAA CGA TGG TCG ACA CAC GAT CAG AAG CTT GGC ATG GAT GAC GAC G-3’
amplification template strand No. 4, denoted P4:
5’-AAT CCC TGG TCC GTC TTG CGG CAC TTA GTT GAA TGA GGA GCA GCA CAG AGG TCA GAA CGA TGG TCG ACA CAC GAT CAG AAG CTT GGC ATG GAT GAC GAC G-3’
3' -end phosphorylation modified amplification template strand No. 3, labeled P3-Pho:
5’-AAT CCC TGG TCC GTC TTG CGG CAA GGC TTT GAA TGA GGA GCA GCA CAG AGG TCA GAA CGA TGG TCG ACA CAC GAT CAG AAG CTT GGC ATG GAT GAC GAC G-Phosphate-3’
the upstream primer, noted as F1:
5’-AAT CCC TGG TCC GTC TTG CG-3’
the downstream primer, noted as R1:
5’-CGT CGT CAT CCA TGC CAA GC-3’
2. the following buffer solutions were prepared
SELEX binding buffer: 150mM sodium chloride, 5mM potassium chloride, 1mM magnesium chloride, 1mM calcium chloride, 40mM HEPES, pH 7.4.
Streptavidin magnetic bead binding/washing buffer (B)&W buff): 8mM Tris, 1M sodium chloride, 1mM EDTA, 0.02% (V/V)
X-100，pH 7.8。
10 × reaction buffer: 67mM glycine, 6.7mM magnesium chloride, 10mM 2-mercaptoethanol, pH 9.5.
3. Detection principle of signal double-amplification sensor
As shown in FIG. 1, first, Apt38 labeled at the 5' end with biotin, a partially complementary DNA thereof (P3 for example) and a reverse primer R1 were incubated, and a nucleic acid complex was formed by complementary hydrogen bonding hybridization (FIG. 1). 3' terminal phosphorylation and reverse primer hybridization were designed to protect complementary DNA from Exo I digestion. Meanwhile, the 3 'end of Apt38 is hybridized with the 5' end of P3 to form a blunt end, and Apt38 is protected from Exo I digestion. In the absence of PD-L1, P3 was difficult to isolate from Apt38 due to hydrogen bonding between complementary bases. In the presence of PD-L1, binding of PD-L1 to Apt38 promotes the conformational change of the latter, forcing P3 to dissociate from the nucleic acid complex. At the same time, due to the loss of blunt end protection, the Exo I enzyme starts digestion from the 3' end of Apt38 and releases PD-L1 from the aptamer-protein complex. The released PD-L1 can further trigger another specific recognition, release and digestion, allowing more P3 to be released from the nucleic acid complex, thereby achieving a first signal amplification based on the target cycle.
Next, the complementary DNA released simultaneously is amplified and quantified by Q-PCR. The corresponding amount of PD-L1 can be determined by the amount of P3 released as implied by the change in Ct value. Thus, a secondary signal amplification based on PCR amplification is achieved.
4. Signal double amplification sensor construction process
4.1 vortex mixer mix streptavidin magnetic beads, transfer to the micro centrifuge tube, and placed on the magnetic separation frame for 2min, suction and clear supernatant, then with B & W buff washing magnetic beads 2 times, each 200L. Uniformly mixing the Bio-Apt38, the partial complementary strand and the R1, carrying out water bath at 95 ℃ for 5min, carrying out ice water bath at 0 ℃ for 10min, and incubating at room temperature for 60min to enable the three nucleic acid strands to form a nucleic acid compound through base complementary pairing. The treated magnetic beads were added and incubated at 22 ℃ for 30min at 80r/min to form magnetic bead-nucleic acid complexes.
4.2 magnetic separation, and using SELEX combined with buffer washing four times each 200 u L and heavy suspension. 10 Xreaction buffer, Exo I exonuclease and gradient PD-L1 protein solution are added to incubate at 22 ℃ for 60min, and the rotation speed is 80 r/min. Magnetic separating to obtain supernatant, and water bathing at 85 deg.C for 15 min.
4.3 configuring the Q-PCR system in Table 4-1 and setting the Q-PCR program in Table 4-2, the fluorescence threshold was set to 100, the Ct value was measured as a function of the concentration of PD-L1 protein, and data fitting analysis was performed using Origin 8.
TABLE 4-1Q-PCR composition and content
Table 1-6 Composition and content of Q-PCR
TABLE 4-2Q-PCR procedure
Table 4-2 Q-PCR procedure
5. Optimization of experimental parameters
5.1 complementary length optimization: the experimental procedure was as in step 3, with the introduction of strands of different complementary lengths (P1: binding length 38 nt; P2: binding length 33 nt; P3: binding length 28 nt; P4 binding length 23nt) to determine the optimal complementary length. As shown in FIG. 2, the optimal binding length is 28 nt.
5.2 aptamer end protection mode optimization: the experimental method is the same as the step 3, and a plurality of terminal protection modes (1: P3; 2: P3-Pho; 3: P3+ R1; 4: P3-Pho + R1) are introduced to determine the optimal terminal protection mode. As shown in FIG. 3, the best mode of end protection is to use both the end-modified phosphate group and the end-protecting chain, i.e., P3-Pho + R1.
5.3Exo I enzyme dosage optimization: the experimental procedure was the same as in step 3, with different amounts of Exo I enzyme (0, 0.05, 0.1, 0.15, 0.2, 0.5U/. mu.L) being introduced to determine the optimum Exo I enzyme dosage. As shown in FIG. 4, the optimum concentration of Exo I enzyme was 0.05U/. mu.L.
5.4 enzyme digestion reaction time optimization: the experimental method is the same as the step 3, and different enzyme digestion reaction times (20 min, 40 min, 60min, 90 min and 120min) are introduced to determine the optimal enzyme digestion reaction time. As shown in FIG. 5, the optimal enzyme digestion reaction time is 60 min.
6. Linear range and limit of detection in buffer system
6.1 Linear Range: the experimental method is the same as the step 3, and PD-L1 with gradient concentration is added into the buffer solution reaction system for identifying the detection range of the sensor. The PD-L1 concentration was plotted on the abscissa and the Ct value was plotted on the ordinate. As shown in fig. 6, the standard curve of the sensor under the buffer system is 13.647-0.266lnX, the correlation coefficient R2 is 0.991, and the linear detection range is 0.1ng/mL-10000 ng/mL.
6.2 limit of detection (LOD): the experimental method is the same as that in step 3, the negative control group without adding PD-L1 is taken, and the standard deviation (SD value) obtained by subtracting 3 times from the average response value of the negative control is brought into a fitting curve, so that the detection Limit (LOD) of the sensor in a buffer solution system is obtained. The lowest detection limit of the detection sensor under a buffer solution system is 0.076 ng/mL.
7. Sensor specificity study
The experimental procedure was the same as in step 3, with 10ng/mL PD-L1 and 100ng/mL PD-L2, PD-1 and B7-1, respectively, to test the specificity of the sensor. The signal response of the sensor was observed when different proteins were added and the specificity was assessed. As shown in fig. 7, the sensor has good specificity.
8. Linear range and limit of detection in human serum samples
8.1 Linear Range: the experimental method is the same as that in step 3, PD-L1 with gradient concentration is added into a 10% human serum (microvesicle is removed by centrifugation) system for carrying out the detection range identification of the sensor. The PD-L1 concentration was plotted on the abscissa and the Ct value was plotted on the ordinate. As shown in FIG. 8, the standard curve of the sensor under the buffer system is 10.872-0.211lnX, and the correlation coefficient R2Linear detection ranged from 0.1ng/mL to 10000ng/mL, 0.999 ng/mL.
8.2 limit of detection (LOD): the experimental method is the same as that in step 3, the negative control group without adding PD-L1 is taken, and the standard deviation (SD value) obtained by subtracting 3 times from the average response value of the negative control is brought into a fitting curve, so that the detection Limit (LOD) of the sensor in a buffer solution system is obtained. The lowest detection limit of the detection sensor under a human serum sample was obtained to be 0.3625 ng/mL.
9. Research on recovery rate by adding standard
The experimental method is the same as the step 3, PD-L1 protein with different concentrations is added into processed 10% human serum, the final concentrations are respectively 0.1, 1.0, 10 and 100ng/mL, Ct values obtained by detecting through a signal double amplification sensor are substituted into a standard curve of a serum sample to obtain corresponding detected concentrations, and the recovery rate of adding standard is calculated after each group is performed in triplicate. The result is shown in Table 9-1, the recovery rate of the added standard is 88.21% -105.80%, and the sensor has good detection performance.
Table 9-1 the recovery of PD-L1 added to human serum samples was determined using the developed sensor (n ═ 3)
Table 9-1 Recovery rates for added PD-L1 detection in human serum samples using the developed aptasensor(n＝3)
The invention has the advantages that:
(1) the invention uses the aptamer as a key detection element, thereby reducing the detection cost and the detection difficulty.
(2) The invention realizes high-sensitivity detection of the target through double amplification of the signal.
(3) The invention still has better detection performance in complex serum samples.
(4) The invention has great application potential in the field of medication auxiliary diagnosis.
Drawings
FIG. 1: novel PD-L1 detection sensor principle schematic diagram. a synthesis of nucleic acid magnetic bead compound, b signal double amplification aptamer sensor production schematic diagram
FIG. 2: complementary length optimization
FIG. 3: aptamer end protection mode optimization
FIG. 4: exo I enzyme dosage optimization
FIG. 5: optimization of enzyme digestion reaction time
FIG. 6: buffer solution system sensor detection standard curve
FIG. 7: sensor detection specificity
FIG. 8: standard curve of human serum detection system
Detailed Description
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1: the following oligonucleotide sequences were synthesized:
apt38 of biotin modified at the 5' end, which is marked as Bio-Apt 38:
5’-Biotin-AAG ACG GAC CAG CCT TGC CGC AAG ACG GAC CAG GGA TT-3’
the amplification template strand No. 1, denoted P1, was underlined the base complementary pairing part with Bio-Apt38 (same below):
5’-AAT CCC TGG TCC GTC TTG CGG CAA GGC TGG TCC GTC TTA GCA GCA CAG AGG TCA GAA CGA TGG TCG ACA CAC GAT CAG AAG CTT GGC ATG GAT GAC GAC G-3’
amplification template strand No. 2, denoted P2:
5’-AAT CCC TGG TCC GTC TTG CGG CAA GGC TGG TCC TGA GGA GCA GCA CAG AGG TCA GAA CGA TGG TCG ACA CAC GAT CAG AAG CTT GGC ATG GAT GAC GAC G-3’
amplification template strand No. 3, labeled P3:
5’-AAT CCC TGG TCC GTC TTG CGG CAA GGC TTT GAA TGA GGA GCA GCA CAG AGG TCA GAA CGA TGG TCG ACA CAC GAT CAG AAG CTT GGC ATG GAT GAC GAC G-3’
amplification template strand No. 4, denoted P4:
5’-AAT CCC TGG TCC GTC TTG CGG CAC TTA GTT GAA TGA GGA GCA GCA CAG AGG TCA GAA CGA TGG TCG ACA CAC GAT CAG AAG CTT GGC ATG GAT GAC GAC G-3’
3' -end phosphorylation modified amplification template strand No. 3, labeled P3-Pho:
5’-AAT CCC TGG TCC GTC TTG CGG CAA GGC TTT GAA TGA GGA GCA GCA CAG AGG TCA GAA CGA TGG TCG ACA CAC GAT CAG AAG CTT GGC ATG GAT GAC GAC G-Phosphate-3’
the upstream primer, noted as F1:
5’-AAT CCC TGG TCC GTC TTG CG-3’
the downstream primer, noted as R1:
5’-CGT CGT CAT CCA TGC CAA GC-3’
example 2: the following buffer solutions were prepared
SELEX binding buffer: 150mM sodium chloride, 5mM potassium chloride, 1mM magnesium chloride, 1mM calcium chloride, 40mM HEPES, pH 7.4.
Streptavidin magnetic bead binding/washing buffer (B)&W buff): 8mM Tris, 1M sodium chloride, 1mM EDTA, 0.02% (V/V)
X-100，pH 7.8。
10 × reaction buffer: 67mM glycine, 6.7mM magnesium chloride, 10mM
Example 3: detection principle of signal double-amplification sensor
As shown in FIG. 1, first, a nucleic acid complex is formed by incubating Apt38 labeled at the 5' end with biotin, a partially complementary DNA (P3) thereof, and a reverse primer, and hybridizing them by complementary hydrogen bonds (FIG. 1). 3' terminal phosphorylation and reverse primer hybridization were designed to protect complementary DNA from Exo I digestion. Meanwhile, the 3 'end of Apt38 is hybridized with the 5' end of P3 to form a blunt end, Apt38 is protected from Exo I digestion, and P3 is difficult to separate from Apt38 due to the hydrogen bonding effect between complementary bases under the condition without PD-L1 protein. In the presence of PD-L1, binding of PD-L1 to Apt38 promotes the conformational change of the latter, further forcing P3 to dissociate from the nucleic acid complex. At the same time, due to the loss of blunt end protection, the Exo I enzyme starts digestion from the 3' end of Apt38 and releases PD-L1 protein from the aptamer-protein complex. The released PD-L1 protein can further trigger another specific recognition, release and digestion, allowing more P3 to be released from the nucleic acid complex, thereby achieving the first signal amplification based on the target cycle.
Next, the complementary DNA released simultaneously is amplified and quantified by Q-PCR. The corresponding amount of PD-L1 can be determined by the amount of P3 released as implied by the change in Ct value. Thus, a secondary signal amplification based on PCR amplification is achieved.
Example 4 Signal Dual amplification sensor construction Process
4.1 vortex mixer mix streptavidin magnetic beads, transfer to the micro centrifuge tube, and placed on the magnetic separation frame for 2min, suction and clear supernatant, then with B & W buff washing magnetic beads 2 times, each 200L. Uniformly mixing Bio-Apt38, a part of complementary strand and R1 in a molar ratio of 1.4:1.0:1.4, carrying out water bath at 95 ℃ for 5min, carrying out ice water bath at 0 ℃ for 10min, and incubating at room temperature for 60min to enable three nucleic acid chains to form a nucleic acid complex through base complementary pairing. The treated magnetic beads were added and incubated at 22 ℃ for 30min at 80r/min to form magnetic bead-nucleic acid complexes.
4.2 magnetic separation, and using SELEX combined with buffer washing four times each 200 u L and heavy suspension. 10 Xreaction buffer, Exo I exonuclease and gradient PD-L1 protein solution are added to incubate at 22 ℃ for 60min, and the rotation speed is 80 r/min. Magnetic separating to obtain supernatant, and water bathing at 85 deg.C for 15 min.
4.3 configuring the Q-PCR system in Table 4-1 and setting the Q-PCR program in Table 4-2, the fluorescence threshold was set to 100, the Ct value was measured as a function of the concentration of PD-L1 protein, and data fitting analysis was performed using Origin 8.
TABLE 4-1Q-PCR composition and content
Table 1-6 Composition and content of Q-PCR
TABLE 4-2Q-PCR procedure
Table 4-2 Q-PCR procedure
Example 5 optimization of experimental parameters
5.1 complementary length optimization: the experimental procedure was as in step 3, with the introduction of strands of different complementary lengths (P1: binding length 38 nt; P2: binding length 33 nt; P3: binding length 28 nt; P4 binding length 23nt) to determine the optimal complementary length. As shown in FIG. 2, the optimal binding length was 28nt, and the P3 sequence was selected for subsequent experiments.
5.2 aptamer end protection mode optimization: the experimental method is the same as the step 3, and a plurality of terminal protection modes (1: P3; 2: P3-Pho; 3: P3+ R1; 4: P3-Pho + R1) are introduced to determine the optimal terminal protection mode. As shown in FIG. 3, the best end protection method is to use both the end modified phosphate group and the end protection chain, and select the end protection method of P3-Pho + R1 for subsequent experiments.
5.3Exo I enzyme dosage optimization: the experimental procedure was the same as in step 3, with different amounts of Exo I enzyme (0, 0.05, 0.1, 0.15, 0.2, 0.5U/. mu.L) being introduced to determine the optimum Exo I enzyme dosage. As shown in FIG. 4, the optimal action concentration of Exo I enzyme was 0.05U/. mu.L, and the action concentration was selected for subsequent experiments.
5.4 enzyme digestion reaction time optimization: the experimental method is the same as the step 3, and different enzyme digestion reaction times (20 min, 40 min, 60min, 90 min and 120min) are introduced to determine the optimal enzyme digestion reaction time. As shown in FIG. 5, the optimal enzyme reaction time is 60min, and the enzyme reaction time is selected for subsequent experiments.
Example 6 buffer System detection Linear Range and detection Limit
6.1 Linear Range: the experimental method is the same as the step 3, the experimental parameters are obtained from the optimized results, and PD-L1 with gradient concentration is added into the buffer solution reaction system for identifying the detection range of the sensor. The PD-L1 concentration was plotted on the abscissa and the Ct value was plotted on the ordinate. As shown in FIG. 6, the standard curve of the sensor under the buffer system is 13.647-0.266lnX, and the correlation coefficient R2Linear detection range 0.1ng/mL-10000ng/mL, 0.991.
6.2 limit of detection (LOD): the experimental method is the same as that in step 3, the negative control group without adding PD-L1 is taken, and the standard deviation (SD value) obtained by subtracting 3 times from the average response value of the negative control is brought into a fitting curve, so that the detection Limit (LOD) of the sensor in a buffer solution system is obtained. The lowest detection limit of the detection sensor under a buffer solution system is 0.076 ng/mL.
Example 7 sensor-specific study
The experimental method is the same as step 3, the experimental parameters are optimized, and 10ng/mL PD-L1 and 100ng/mL PD-L2, PD-1 and B7-1 are respectively used for detecting the specificity of the sensor. The signal response of the sensor was observed when different proteins were added and the specificity was assessed. As shown in fig. 7, the sensor has good specificity.
Example 8 human serum sample detection Linear Range and detection Limit
8.1 Linear Range: the experimental method is the same as the step 3, the experimental parameters are optimized, and PD-L1 with gradient concentration is added into a 10% human serum (microvesicle is removed by centrifugation) system for the detection range identification of the sensor. The PD-L1 concentration was plotted on the abscissa and the Ct value was plotted on the ordinate. As shown in FIG. 8, the standard curve of the sensor under the buffer system is 10.872-0.211lnX, and the correlation coefficient R2Linear detection ranged from 0.1ng/mL to 10000ng/mL, 0.999 ng/mL.
8.2 limit of detection (LOD): the experimental method is the same as the step 3, the experimental parameters are the optimized results, the negative control group without adding PD-L1 is taken, the standard deviation (SD value) obtained by subtracting 3 times from the average response value of the negative control is brought into a fitting curve, and the detection Limit (LOD) of the sensor under a buffer solution system is obtained. The lowest detection limit of the detection sensor under the buffer system is 0.3625 ng/mL.
Example 9 spiking recovery study
The experimental method is the same as the step 3, the experimental parameters are the optimized results, PD-L1 protein with different concentrations is added into treated 10% human serum, the final concentrations are respectively 0.1, 1.0, 10 and 100ng/mL, Ct values obtained by detecting through a signal double-amplification sensor are substituted into a standard curve of a serum sample to obtain corresponding detected concentrations, and the recovery rate of the added standard is calculated after each group is divided into three groups. The result is shown in Table 9-1, the recovery rate of the added standard is 88.21% -105.80%, and the sensor has good detection performance.
Table 9-1 the recovery of PD-L1 added to human serum samples was determined using the developed sensor (n ═ 3)
Table 9-1 Recovery rates for added PD-L1 detection in human serum samples using the developed aptasensor(n＝3)
Sequence listing
<110> Beijing university of chemical industry
<120> high-sensitivity serum PD-L1 detection method based on aptamer sensor
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 38
<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 1
aagacggacc agccttgccg caagacggac cagggatt 38
<210> 3
<211> 100
<212> DNA
<213> amplification template Strand No. 1(2 Ambystoma laterale x Ambystoma jeffersonanum)
<400> 3
aatccctggt ccgtcttgcg gcaaggctgg tccgtcttag cagcacagag gtcagaacga 60
tggtcgacac acgatcagaa gcttggcatg gatgacgacg 100
<210> 3
<211> 100
<212> DNA
<213> amplification template Strand No. 2 (2 Ambystoma laterale x Ambystoma jeffersonanum)
<400> 3
aatccctggt ccgtcttgcg gcaaggctgg tcctgaggag cagcacagag gtcagaacga 60
tggtcgacac acgatcagaa gcttggcatg gatgacgacg 100
<210> 4
<211> 100
<212> DNA
<213> amplification template Strand No. 3 (2 Ambystoma laterale x Ambystoma jeffersonanum)
<400> 4
aatccctggt ccgtcttgcg gcaaggcttt gaatgaggag cagcacagag gtcagaacga 60
tggtcgacac acgatcagaa gcttggcatg gatgacgacg 100
<210> 5
<211> 100
<212> DNA
<213> amplification template Strand No. 4 (2 Ambystoma laterale x Ambystoma jeffersonanum)
<400> 5
aatccctggt ccgtcttgcg gcacttagtt gaatgaggag cagcacagag gtcagaacga 60
tggtcgacac acgatcagaa gcttggcatg gatgacgacg 100
<210> 6
<211> 100
<212> DNA
<213> amplification template Strand No. 3 modified by phosphorylation of 3' -terminus (2 Ambystoma laterale x Ambystoma jeffersonia)
<400> 6
aatccctggt ccgtcttgcg gcaaggcttt gaatgaggag cagcacagag gtcagaacga 60
tggtcgacac acgatcagaa gcttggcatg gatgacgacg 100
<210> 7
<211> 20
<212> DNA
<213> upstream primer, noted as f1(2 Ambystoma laterale x Ambystoma jeffersonanum)
<400> 7
<210> 8
<211> 20
<212> DNA
<213> downstream primer, noted r1(2 Ambystoma laterale x Ambystoma jeffersonia)
<400> 8
Claims (3)
1. The application of the oligonucleotide sequence in preparing the reagent kit for detecting PD-L1 in serum with high sensitivity based on an aptamer sensor,
the oligonucleotide sequences are shown below:
apt38 of biotin modified at the 5' end, which is marked as Bio-Apt 38:
5’-Biotin-AAG ACG GAC CAG CCT TGC CGC AAG ACG GAC CAG GGA TT-3’
the amplification template strand No. 3, denoted P3, is underlined the base complementary pairing part with Bio-Apt 38:
5’-AATCCCTGGTCCGTCTTGCGGCAAGGCTTT GAA TGA GGA GCA GCA CAG AGG TCA GAA CGA TGG TCG ACA CAC GAT CAG AAG CTT GGC ATG GAT GAC GAC G-3’
the upstream primer, noted as F1:
5’-AAT CCC TGG TCC GTC TTG CG-3’
the downstream primer, noted as R1:
5’-CGT CGT CAT CCA TGC CAA GC-3’
the method is characterized in that the aptamer sensor-based high-sensitivity detection of PD-L1 in serum comprises the following steps:
1) the following buffer solutions were prepared
SELEX binding buffer: 150mM sodium chloride, 5mM potassium chloride, 1mM magnesium chloride, 1mM calcium chloride, 40mM HEPES, pH 7.4;
streptavidin magnetic bead binding/washing buffer B&W buff: 8mM Tris, 1M sodium chloride, 1mM EDTA, 0.02% (V/V)
X-100，pH 7.8；
10 × reaction buffer: 67mM glycine, 6.7mM magnesium chloride, 10mM 2-mercaptoethanol, pH 9.5;
2) firstly, the 5' end Apt38 and P3 labeled by biotin and a reverse primer R1 are used for incubation, and a nucleic acid complex is formed by complementary hydrogen bond hybridization; phosphorylation of the 3' end of P3 and reverse primer hybridization were designed to protect complementary DNA from Exo I digestion; meanwhile, the 3 'end of Apt38 is hybridized with the 5' end of P3 to form a blunt end, so that Apt38 is protected from being digested by Exo I;
secondly, amplifying and quantifying the complementary DNA released simultaneously by using a Q-PCR method;
3) signal double amplification sensor construction process
3.1 mixing streptavidin magnetic beads uniformly by a vortex mixer, transferring the streptavidin magnetic beads to a microcentrifuge tube, placing the microcentrifuge tube on a magnetic separation rack for 2min, sucking and removing supernatant, and then washing the magnetic beads for 2 times by using B & W buff, wherein 200 mu L of the streptavidin magnetic beads is obtained each time; uniformly mixing Bio-Apt38, P3 and R1, carrying out water bath at 95 ℃ for 5min, carrying out ice water bath at 0 ℃ for 10min, and incubating at room temperature for 60min to enable three nucleic acid chains to form a nucleic acid compound through base complementary pairing; adding the processed magnetic beads, and incubating at 22 ℃ for 30min at the rotation speed of 80r/min to form magnetic bead-nucleic acid complexes;
3.2 magnetic separation, and using SELEX combined with buffer washing four times each 200 u L and heavy suspension; adding 10 × reaction buffer solution, Exo I exonuclease and gradient PD-L1 protein solution, and incubating at 22 deg.C for 60min at 80 r/min; magnetically separating to obtain supernatant, and water bathing at 85 deg.C for 15 min;
3.3 configuring a Q-PCR system according to the table 3-1 and setting a Q-PCR program according to the table 3-2, setting a fluorescence threshold value as 100, detecting the relation of the Ct value with the change of the concentration of the PD-L1 protein, and performing data fitting analysis by using Origin 8;
TABLE 3-1Q-PCR composition and content
TABLE 3-2Q-PCR procedure
2. Use according to claim 1, characterized in that:
the action concentration of Exo I enzyme is 0.05U/. mu.L-0.5U/. mu.L.
3. Use according to claim 1, characterized in that:
the enzyme digestion reaction time is 20-120 min.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105063200A (en) * | 2015-08-06 | 2015-11-18 | 常熟理工学院 | Method for determining kanamycin concentration of sample |
| CN110029110A (en) * | 2019-03-22 | 2019-07-19 | 安徽省昂普拓迈生物科技有限责任公司 | A kind of kit and its use, detection method for aptamer screening |
| CN110511935A (en) * | 2019-01-28 | 2019-11-29 | 北京化工大学 | A kind of aptamers truncation optimization method cut based on S1 digestion |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105063200A (en) * | 2015-08-06 | 2015-11-18 | 常熟理工学院 | Method for determining kanamycin concentration of sample |
| CN110511935A (en) * | 2019-01-28 | 2019-11-29 | 北京化工大学 | A kind of aptamers truncation optimization method cut based on S1 digestion |
| CN110029110A (en) * | 2019-03-22 | 2019-07-19 | 安徽省昂普拓迈生物科技有限责任公司 | A kind of kit and its use, detection method for aptamer screening |
Non-Patent Citations (2)
| Title |
|---|
| Ultrasensitive detection of protein using an aptamer-based exonuclease protection assay;Wang Xiao-Li等;《Analytical chemistry》;20040824;第76卷(第19期);第5605-5610页 * |
| 联合检测血清VEGF、Ki67、PD-L1表达水平评估宫颈癌患者预后的价值;赵方等;《中国妇幼保健》;20190715;第34卷(第14期);第3206-3209页 * |
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