A method for detecting C-reactive protein based on aptamers

Abstract

This invention discloses a method for detecting C-reactive protein (CRP) based on aptamers; the sequence of the aptamer is shown in SEQ ID NO.1. A label-free CRP detection method based on the aptamer is established. The required aptamer requires no chemical modification or immobilization, and no antibodies or reagent kits are needed, thus reducing detection costs, batch-to-batch reagent variability, and synthesis time. Furthermore, this method can sensitively and specifically determine the level of high-sensitivity CRP in serum, which can be used to predict the risk of cardiovascular events. Moreover, the required aptamer requires no chemical modification or immobilization, and CRP detection is rapid and time-efficient; the linear range is 0.0125–15 μg / mL, and the detection limit is 4 ng / mL (35 pM). This significantly reduces reagent costs and synthesis time.

Description

Technical Field

[0001] This invention belongs to the field of biochemistry technology, specifically relating to a method for detecting C-reactive protein based on aptamers. Background Technology

[0002] Among methods for measuring C-reactive protein (CRP), diffusion methods have low sensitivity, latex aggregation methods are easily affected by other factors, leading to false positive results, thus requiring high standards for sample pretreatment. Immunoturbidimetric and immunoturbidimetric methods are not sensitive enough to predict the risk of cardiovascular events. Aptamer- and gold nanoparticle-based visualization methods for CRP can produce results within 5 minutes, but this method can only be used for urine samples. Immunoassay methods based on antigen-antibody interactions also have drawbacks such as high antibody acquisition costs, long processing times, immunogenicity, difficulty in modification, and difficulty in preservation. Standardization of CRP clinical testing remains a challenge. Currently, there are significant differences in hs-CRP measurement methods and even between different kits, affecting the comparison between individual patient data and the upper limit of normal values ​​established through epidemiology. Therefore, there is an urgent need for a method that can accurately measure C-reactive protein. Summary of the Invention

[0003] The purpose of this invention is to provide a method for detecting C-reactive protein.

[0004] The technical solution adopted in this invention is:

[0005] This invention provides a method for detecting C-reactive protein, comprising the following steps:

[0006] S1: Mix the aptamer with the sample to be tested to obtain mixture A;

[0007] S2: Add the complementary DNA of the nicking endonuclease and aptamer to mixture A for enzymatic digestion, and then centrifuge to obtain the supernatant;

[0008] S3: Take the supernatant for capillary electrophoresis detection.

[0009] In some embodiments of the present invention, the sequence of the nucleic acid aptamer is shown in SEQ ID NO.1.

[0010] In some embodiments of the present invention, the nucleotide sequence of the complementary DNA is shown in SEQ ID NO.2.

[0011] In some embodiments of the present invention, the aptamer needs to be pretreated, specifically by heating the aptamer at 93-97°C for 8-12 minutes and then cooling it at 13-17°C for 8-12 minutes.

[0012] In some embodiments of the present invention, the mixing conditions in step S1 are 13-17°C and 27-33 min.

[0013] In some embodiments of the present invention, the enzymatic digestion reaction conditions in step S2 are 35–39°C and 55–65 min.

[0014] In some embodiments of the present invention, after the enzymatic digestion reaction is completed, it is necessary to stop the digestion and the treatment method is high-temperature inactivation, specifically heating at 75-85°C for 18-24 minutes.

[0015] In some embodiments of the present invention, the enzyme activity of the endonuclease is 5-15 U.

[0016] In some embodiments of the present invention, the electrophoresis conditions are: voltage 12-18 kV; injection time 3-5 min; UV detection wavelength: 250-300 nm.

[0017] In some embodiments of the present invention, the sample injection during detection is gravity injection, wherein the injection height is 15-16 cm.

[0018] In some embodiments of the present invention, the buffer solution for electrophoresis is composed of 40-50 mM borax and 60-80 mM Tris.

[0019] In some embodiments of the present invention, the buffer solution has a pH of 8 to 10.

[0020] The beneficial effects of this invention are:

[0021] This invention designs a nucleic acid aptamer for detecting C-reactive protein (CRP). By adding three bases to the aptamer, a nicking endonuclease (Nb.BbvCI) recognition site is incorporated, giving the aptamer both CRP-specific recognition capability and the nicking endonuclease recognition site. The required aptamer requires no chemical modification or immobilization, significantly reducing reagent costs and synthesis time.

[0022] This invention also develops a sensitive, low-cost, label-free method for detecting C-reactive protein (CRP) based on nucleic acid aptamers. This method utilizes nicking endonuclease-assisted signal amplification and capillary electrophoresis-UV detector to measure CRP in human serum. The nicking endonuclease can recognize specific sequences in the aptamer and complementary DNA double-stranded structure, and only cleaves the complementary DNA. Furthermore, the signal probe in this invention is single-stranded DNA, requiring no modification or immobilization, greatly saving reagent costs and synthesis time. Capillary electrophoresis also has the advantage of requiring small sample volumes. Therefore, this method requires fewer reagents and samples, is low-cost, highly sensitive, requires no modification of the aptamer or sample derivatization, and rapidly completes CRP detection in a short time; the linear range is 0.0125-15 μg / mL, and the detection limit is 4 ng / mL (35 pM). This method eliminates the need for antibodies and kits, reducing detection costs, batch-to-batch reagent variability, and synthesis time. Moreover, this method can sensitively and specifically measure the level of ultrasensitive CRP in serum, which can be used to predict the risk of cardiovascular events. Attached Figure Description

[0023] Figure 1 This is a schematic diagram of the experimental principle.

[0024] Figure 2 This is to verify the feasibility of the method.

[0025] Figure 3 To optimize the conditions for capillary electrophoresis. Figure 3 A represents the optimization of borax concentration in the electrophoresis buffer; Figure 3 B represents the optimization of the Tris concentration in the electrophoresis buffer; Figure 3 C represents the optimization of the pH in the electrophoresis buffer; Figure 3 D represents the optimization of injection time; Figure 3 E represents voltage optimization; Figure 3 F represents the optimization of DNA signal length.

[0026] Figure 4 Optimize reaction conditions for nicking endonuclease. Figure 4 A represents the effect of enzyme amount on peak height with or without CRP; Figure 4 B represents the effect of enzyme amount on the peak height difference with and without CRP; Figure 4 C represents the effect of enzyme digestion time on the peak height with or without CRP. Figure 4 D represents the effect of enzyme digestion time on the peak height difference with and without CRP.

[0027] Figure 5 For the determination of aptamer and CRP affinity (Kd).

[0028] Figure 6 This demonstrates the specificity of the detection method for CRP detection.

[0029] Figure 7 This is an electrophoresis image of a mouse model of sepsis. Detailed Implementation

[0030] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0031] Example 1: A method for detecting C-reactive protein

[0032] The experimental principle is described below. Figure 1 When the sample does not contain CRP, the aptamer binds to cDNA to form double-stranded DNA. This double-stranded DNA contains the specific recognition sequence 5′-GCTGAGG-3′ of the nicking endonuclease (Nb.BbvCI), which only cleaves the cDNA to produce signal DNA. The digested cDNA forms two fragments. Due to the decrease in Tm value, the two fragments dissociate from the aptamer. The released aptamer can bind to new cDNA, completing the digestion cycle and generating a large amount of signal DNA. This signal DNA shows a high signal value when detected by CE. When the sample contains CRP, the high affinity between the aptamer and CRP leads to specific binding, reducing the aptamer content and thus the signal DNA content after digestion. The signal DNA shows a low signal value when detected by CE. The CRP content can be determined by the change in the peak area of ​​the signal DNA.

[0033] The specific testing steps are as follows:

[0034] 1.1 Sample Preparation:

[0035] The CRP sample contained: 1×NEB buffer, aptamer (20 nm), cDNA (1 μM), and CRP (2 μg / mL). -1 ) and nicking endonuclease (10U);

[0036] CRP-free samples contain: 1×NEB buffer, aptamer (20 nm), cDNA (1 μM), and nicking endonuclease (10 U).

[0037] The sample preparation process was as follows: the aptamer (20 nm) was heated at 95 °C for 10 min and then rapidly cooled at 15 °C for 10 min. Subsequently, the aptamer and CRP were reacted in binding buffer (20 mM HEPES, 120 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2; pH = 7.35) at 15 °C for 30 min to form an aptamer / CRP complex. The nucleic acid sequences are shown in Table 1.

[0038] Table 1 Nucleic Acid Sequences

[0039]

[0040]

[0041] aptamers a The bold text represents the three bases added to the original aptamer.

[0042] The signal DNAs involved, 1, 2, and 3, are used as standards for optimizing the length of signal DNA in capillary electrophoresis and determining the position of signal peaks.

[0043] Add cDNA (1 μM), NEase (10 U), and NEB buffer 2 (1×) to the above solution, bringing the total sample volume to 20 μL. Incubate at 37 °C for 60 minutes. After the reaction, heat the sample at 80 °C for 20 minutes to terminate the reaction. Finally, centrifuge the sample at 5000 rpm for 1 minute to remove protein precipitate and collect the supernatant for later use.

[0044] 1.2 Capillary electrophoresis analysis:

[0045] New capillary tubes were rinsed with ultrapure water for 1 hour, then with 1M NaOH for 1 hour, and finally with ultrapure water for 1 hour. Before each daily use of the capillary tubes, they were rinsed for 5 minutes each with ultrapure water, 0.1M NaOH, ultrapure water, and electrophoresis buffer (45mM borax, 70mM Tris, pH 9.5), while simultaneously preheating the capillary electrophoresis apparatus. Between continuous sample injections, the tubes were rinsed for 2 minutes each with ultrapure water, 0.1M NaOH, and ultrapure water, followed by a 3-minute rinse with electrophoresis buffer.

[0046] The capillary electrophoresis conditions were as follows: sample injection for 4 min, voltage: 14 kV; detection wavelength: 260 nm; gravity injection height: 15.5 cm.

[0047] Example 2 Feasibility Verification Experiment

[0048] 2.1 Sample Preparation: Sample 1: 1×NEB buffer; Sample 2: Aptamer, cDNA, and 1×NEB buffer; Sample 3: 1×NEB buffer, cDNA, and nicking endonuclease; Sample 4: 1×NEB buffer, aptamer, cDNA, CRP, and nicking endonuclease; Sample 5: 1×NEB buffer, aptamer, cDNA, and nicking endonuclease. Other conditions are the same as in 1.1.

[0049] 2.2 Capillary electrophoresis analysis: Same as 1.2.

[0050] See results Figure 2 Sample 1 shows some absorption peaks in the electrophoresis pattern of 1×NEB buffer. Samples 2 and 3 show no signal peaks when the sample contains only double-stranded DNA without enzyme or contains enzyme but no double-stranded DNA. Sample 5 shows a high signal peak when both double-stranded DNA and enzyme are present in the system, indicating that both double-stranded DNA and enzyme are necessary conditions for signal peak generation. Sample 4 shows that when CRP is present in the system, the signal peak decreases due to the specific binding of CRP and aptamers, indicating that this method is feasible for detecting CRP. Furthermore, this electrophoresis condition has good separation effect on single-stranded DNA with a length of less than 11 bases.

[0051] Example 3: Capillary Electrophoresis Condition Optimization Experiment

[0052] 3.1 Sample preparation: aptamer (20 nM), complementary DNA (1 μM), signal DNA2 (1 μM) and NEB buffer 2 (1×).

[0053] 3.2 Capillary electrophoresis analysis:

[0054] (A) 110mM Tris; injection time: 4min; application voltage: 12kV; signal DNA length: 9bases; detection wavelength: 260nm;

[0055] (B) 45mM borax, other conditions are the same as (A);

[0056] (C) 45mM borax, 70mM Tris, other conditions the same as (A);

[0057] (D) 45mM borax, 70mM Tris, electrophoresis buffer pH 9.5, other conditions same as (A);

[0058] (E) 45mM borax, 70mM Tris, electrophoresis buffer pH: 9.5, injection time: 4min;

[0059] (F) 45mM borax, 70mM Tris, electrophoresis buffer pH: 9.5, injection time: 4min, application voltage: 14kV.

[0060] See results Figure 3 , Figure 3 Figures A and 3B show the optimization of borax and Tris concentrations in the electrophoresis buffer. The peak heights reached their maximum at borax concentrations of 45 mM and Tris concentrations of 70 mM, respectively, and decreased with further increases in concentration. In capillary zone electrophoresis, low-conductivity solutions at high concentrations can improve column efficiency and resolution. However, Joule heating gradually increases with increasing electrophoresis buffer concentration. Excessive Joule heating broadens the peaks and reduces sensitivity. Therefore, subsequent experiments selected borax and Tris concentrations of 45 mM and 70 mM, respectively. Figure 3 The peak height and resolution were better at pH 9.5, while the peak height was highest at pH 8.5 due to poor separation of signal DNA and other DNA. In capillary zone electrophoresis, the degree of analyte dissociation is affected by the pH of the electrophoresis buffer, thus influencing resolution and enrichment efficiency. As pH increases, the current and the degree of DNA dissociation increase, leading to increased column efficiency and thus higher peak height and resolution. However, excessively high pH results in excessive current and Joule heating, reducing column efficiency. Therefore, pH 9.5 was selected as the optimal pH for the electrophoresis buffer. Figure 3 The D-display shows that peak height increases with increasing sample injection time, but excessive injection volume leads to peak broadening and reduced resolution. To obtain better peak height and resolution, an injection time of 4 minutes was selected. Figure 3 E shows that above 14 kV, the peak height gradually decreases as the voltage increases, and the elution time also decreases with increasing voltage. Joule heating increases with increasing voltage, and excessive Joule heating reduces column efficiency. However, too low a voltage results in too low an electroosmotic current, leading to a rapid increase in elution time. Therefore, 14 kV was chosen for subsequent experiments to obtain a higher peak height and a shorter analysis time. Figure 3 F shows the effect of the signal DNA length after enzyme digestion on peak height and separation. As the signal DNA base length increases, the peak height gradually increases, and at 11 bases, the signal DNA peaks overlap with other DNA peaks. Therefore, a signal DNA base length of 9 bases was chosen. The cleavage site of the restriction enzyme is fixed, i.e., the complementary DNA breaks at the arrow in the sequence AAAAAATGC↑TGAGGCAGTTAAAAAAAAAA. After enzyme digestion, the left part (9 bases long AAAAAATGC) can be separated, and this is used as the quantitative signal.

[0061] Example 4 Enzyme digestion cycle signal amplification experiment

[0062] 4.1 Sample Preparation:

[0063] 4.1.1: Samples with CRP: 1×NEB buffer, aptamer (20 nM), cDNA (1 μM), CRP (2 μg / mL) -1) and cleavage endonuclease, digestion time: 45 min;

[0064] CRP-free sample: 1×NEB buffer, aptamer (20 nM), cDNA (1 μM) and nicking restriction enzyme, digestion time: 45 min;

[0065] The enzyme concentration gradient was set to: 0, 5, 10, 15, 20 U.

[0066] 4.1.2: Same as 4.1.1.

[0067] 4.1.3: Samples containing CRP: 1×NEB buffer, aptamer (20 nM), cDNA (1 μM), CRP (2 μg / mL) -1 ) and nicking endonuclease (10U);

[0068] CRP-free samples: 1×NEB buffer, aptamer (20 nM), cDNA (1 μM) and nicking endonuclease (10 U), other conditions are the same as in 1.1;

[0069] The enzyme digestion time gradient was set to: 0, 15, 30, 45, 60, 75 min.

[0070] 4.1.4: Same as 4.1.3.

[0071] 4.2 Electrophoresis conditions: Same as 1.2.

[0072] The optimization results of enzyme digestion cycle signal amplification conditions are shown in the figure. Figure 4 , Figure 4 A shows that as the amount of enzyme increases, the signal DNA changes in the presence or absence of 2 μg / mL. -1 CRP is enhanced. Figure 4 B shows that the peak height difference reaches its maximum when the enzyme amount is 10U, indicating that 10U of nicking endonuclease can maximize the change in signal difference; Figure 4 C and 4D show that the signal DNA is enhanced with increasing enzyme digestion time, both in the presence and absence of CRP. The peak height difference reaches its maximum at a digestion time of 60 min, indicating that a digestion time of 60 min can maximize the change in peak height difference.

[0073] Example 5 Aptamer and CRP Affinity Determination

[0074] 5.1 Sample preparation: The sample contains 1×NEB buffer, aptamer (20nM), cDNA (1μM), different concentrations of CRP (0, 8, 32, 64, 96, 128, 160, 192, 256, 320, 384nm) and nicking endonuclease (10U). The digestion time is 60min. Other conditions are the same as in 1.1.

[0075] 5.2 Electrophoresis conditions: Same as 1.2.

[0076] See results Figure 5 According to the formula Y=C CRP *B max / (K d +C CRP The measured Kd value was 83 nM, indicating that the aptamer has a high affinity for CRP. Adding three bases to the aptamer resulted in the inclusion of a nicking endonuclease (Nb.BbvCI) recognition site; thus, the aptamer possesses both CRP-specific recognition capability and a nicking endonuclease recognition site. Its aptamer sequence is as follows: The bold text indicates the three added bases that form the recognition sequence for Nb.BbvCI.

[0077] Example 6: Specificity determination of CRP detection

[0078] 6.1 Sample Preparation: The samples contained CRP (8 μg / mL) -1 ), IL-6 (40 μg mL) -1 GPC-3 (40 μg mL) -1 ), AFP (40 μg mL -1 ), TB (40 μg mL) -1 ), IgG (100 μg mL) -1 ) and HSA (400 μg mL -1 Other conditions are the same as in 1.1.

[0079] 6.2 Electrophoresis conditions: Same as 1.2.

[0080] See results Figure 6 The samples contained CRP (8 μg / mL). -1 ), IL-6 (40 μg mL) -1 GPC-3 (40 μg mL) -1 ), AFP (40 μg mL -1 ), TB (40 μg mL) -1 ), IgG (100 μg mL) -1 ) and HSA (400 μg mL -1 The peak area difference is shown in the figure. As can be seen, a significant change in the peak area difference only occurs when CRP is present, indicating that the aptamer has a high specificity for CRP.

[0081] Example 7: Determination of linearity

[0082] 7.1 Sample Preparation: 0, 0.0125, 0.125, 2.5, 5, 7.5, 10, 12.5, and 15 μg / mL of human serum sample buffer were prepared.-1 The CRP, other conditions are the same as in 1.1.

[0083] 7.2 Electrophoresis conditions: Same as 1.2.

[0084] The results showed a linear range of 0.0125-15 μg / mL, with the linear equation y = -3.5038x + 61.551 (where y is the peak area (mV×s); x is the CRP standard solution concentration (μg / mL)). -1 r = 0.994, detection limit 4 ng / mL (35 pM) (S / N = 3).

[0085] Example 8: Determination of intraday and interday precision

[0086] 8.1 Sample Preparation: 2 μg / mL of 1% human serum sample buffer was prepared. -1 The CRP levels were measured under the same conditions as in 1.1. The measurements were repeated 6 times each during the day and between days.

[0087] 8.2 Electrophoresis conditions: Same as 1.2.

[0088] The results were: intra-day precision 2.5% and inter-day precision 3.7%.

[0089] Example 9: Determination of a mouse model of sepsis

[0090] 9.1 Sepsis Mouse Model: The mice used were C57 mice, weighing 22g, that had been fed for six weeks and were specific pathogen-free (SPF). 1mg / mL LPS was dissolved in sterile saline, and the corresponding amount of LPS solution was injected intraperitoneally at a dose of 20mg LPS per kilogram of mouse body weight. 0.3mL of blood was collected from the orbital fossa of healthy mice, and another 0.3mL of blood was collected from the orbital fossa 6 hours after LPS injection. After natural clotting, the blood samples were centrifuged at 3000 rpm for 20 min, and the supernatant serum sample was stored at -20℃.

[0091] 9.2 Sample Preparation:

[0092] Sample 1: Serum from healthy mice, 1×NEB buffer;

[0093] Sample 2: 300 μg mL was added to the serum of septic mice. -1 CRP standard solution, 1×NEB buffer, aptamers, cDNA, and nicking endonuclease;

[0094] Sample 3: Serum from septic mice, 1×NEB buffer, aptamers, cDNA, and nicking endonuclease;

[0095] Sample 4: Healthy mice, 1×NEB buffer, aptamers, cDNA, and nicking endonuclease. Other conditions were the same as in 1.1.

[0096] 9.3 Electrophoresis conditions: Same as 1.2.

[0097] See results Figure 7 Sample 1 shows that the signal peak in the sample containing 1% mouse serum is less affected by serum interference. Sample 4 shows that a high signal peak is obtained after the reaction in normal mouse serum. Sample 3 shows that the signal peak becomes smaller after the reaction in septic mouse serum, indicating that the CRP content in septic mouse serum is increased. Sample 2 shows that the signal peak value measured after adding standard CRP diluent to septic mouse serum is further reduced. It can be seen that this method has application prospects in actual samples.

[0098] Example 10: Determination of recovery rate in serum of septic mice

[0099] 10.1 Sample Preparation: The septic mouse model was prepared in the same manner as in 9.1. 150 μg / mL of CRP standard solution was added to the serum of the septic mice. -1 300μg mL -1 450μg mL -1 Other conditions are the same as in 1.1.

[0100] 10.2 Electrophoresis conditions: Same as 1.2.

[0101] Table 2 Recovery Rate Statistics

[0102]

[0103] The results are shown in Table 2. The recovery rate ranged from 94% to 101%, indicating that the method has a good recovery rate.

[0104] The above detailed embodiments have provided a comprehensive description of the present invention. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention. Furthermore, unless otherwise specified, the embodiments of the present invention and the features thereof can be combined with each other. SEQUENCE LISTING <110> Southern Medical University <120> A method for detecting C-reactive protein based on aptamers <130> <160> 3 <170> PatentIn version 3.5 <210> 1 <211> 43 <212> DNA <213> Artificial sequence <400> 1 cgaaggggat tcgaggggtg attgcgtgcc tcagcatttg gtg 43 <210> 2 <211> 29 <212> DNA <213> Artificial sequence <400> 2 aaaaaatgct gaggcagtta aaaaaaaaa 29 <210> 3 <211> 11 <212> DNA <213> Artificial sequence <400> 3 aaaaaaaatg c 11

Claims

1. A method for detecting C-reactive protein for non-diagnostic purposes, comprising the following steps: S1: Mix the aptamer with the sample to be tested to obtain mixture A; S2: Add the complementary DNA of the nicking endonuclease and aptamer to mixture A for enzymatic digestion, and then centrifuge to obtain the supernatant; S3: Take the supernatant for capillary electrophoresis detection, which uses AAAAAATGC as the quantitative signal; The nucleotide sequence of the aptamer is shown in SEQ ID NO.1; The nucleotide sequence of the complementary DNA of the aptamer is shown in SEQ ID NO.2; The nicking endonuclease is Nb.BbvCI.

2. The method according to claim 1, characterized in that, The mixing conditions described in step S1 are 13–17°C and 27–33 min.

3. The method according to claim 1, characterized in that, The enzyme digestion reaction conditions described in step S2 are 35–39°C and 55–65 min.

4. The method according to claim 1, characterized in that, The enzyme activity of the endonuclease is 5–15 U.

5. The method according to claim 1, characterized in that, The electrophoresis conditions are: voltage 12-18 kV; injection time 3-5 min; UV detection wavelength: 250-300 nm.

6. The method according to claim 1, characterized in that, The buffer solution for the electrophoresis consisted of 40–50 mM borax and 60–80 mM Tris.

7. The method according to claim 6, characterized in that, The pH of the buffer solution is 8 to 10.

8. The method according to claim 1, characterized in that, The sample is injected by gravity during the detection process, with an injection height of 15–16 cm.

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