CN116027044A - PCSK9 detection method based on ortho induction effect and gate-controlled mesoporous silicon - Google Patents

Abstract

The invention discloses a PCSK9 detection method based on an ortho induction effect and gate-controlled mesoporous silicon, which is characterized in that when no target protein PCSK9 exists, two single-stranded DNAs cannot be close to each other, and no subsequent reaction exists by designing and synthesizing a pair of PCSK9 antibody-DNAs; if and only if two antibodies simultaneously recognize and bind to the same target protein PCSK9, the DNA on the antibodies are sufficiently close to each other, then a subsequent hybridization reaction is carried out, and the change of system signals is initiated; and (3) quantifying the PCSK9 protein into quantifying DNA small molecules, and finally finishing PCSK9 detection in one step in a homogeneous solution. The detection method has high sensitivity and wide dynamic range, and can meet the specific detection of target proteins in high-dilution samples.

Description

PCSK9 detection method based on ortho induction effect and gate-controlled mesoporous silicon

Technical Field

The invention belongs to the technical field of immunoassay, and particularly relates to a PCSK9 detection method, in particular to a PCSK9 detection method based on an ortho induction effect and gate-controlled mesoporous silicon.

Background

Proprotein convertase subtilisin-9 (PCSK 9), a secreted serine protease synthesized by the liver, is one of the targets for lowering low density lipoprotein cholesterol (LDL-C) and for treating atherosclerosis cardiovascular disease (ASCVD). Accurate quantification of PCSK9 facilitates predictive diagnosis of ASCVD and guides clinical medication.

Traditional methods employ enzyme-linked immunosorbent assay (ELISA) or Western Blot (WB) to quantify PCSK9 protein in serum or cell lysates. WB usually quantifies proteins by the biquinolinecarboxylic acid method with detection limits at nM level, ELISA detection limits at pM-nM level, while PCSK9 is slightly expressed in serum, making lower sensitivity and limited dynamic range the main technical bottleneck of the existing detection methods; WB involves steps such as electrophoresis, membrane transfer, ELISA involves steps such as coating, plate washing, etc., is tedious and time-consuming, and is prone to human error.

The recent approach to the approach of using a wash-free approach overcomes the above drawbacks, but the approach is mostly based on fluorescence and electrochemistry as signal output, and the progress in Chemiluminescence (CL) is slow, mainly because the CL indicator is difficult to label, purify and enrich on DNA.

Disclosure of Invention

The invention aims to: aiming at the defects of the prior art, the invention aims to provide the PCSK9 detection method with short time, high sensitivity and wide dynamic range.

The technical scheme is as follows: in order to solve the technical problems, the invention provides a PCSK9 detection method based on an ortho induction effect and gate-controlled mesoporous silicon, which comprises the following steps:

(1) Preparing a gate-control mesoporous silicon CL probe MSN-hemin@DNA3 by using aminated mesoporous silicon, hemin and DNA3; the nucleotide sequence of the DNA3 is shown as SEQ ID NO. 7;

(2) Preparing PCSK9 antibody-DNA conjugates Ab1-DNA1 and Ab2-DNA2 using a PCSK9 antibody with an amino group, a DNA1 with a thiol-modified 3 'end, and a DNA2 with a thiol-modified 5' end; the sulfhydryl modified DNA1 nucleotide sequence is shown in SEQ ID NO. 1; the sulfhydryl modified DNA2 nucleotide sequence is shown in SEQ ID NO. 3;

(3) Uniformly mixing the prepared MSN-hemin@DNA3, ab1-DNA1 and Ab1-DNA2 with NEB buffer 4buffer solution, mixing with PCSK9 standard solutions with different concentrations or serum samples to be tested, incubating at room temperature, adding luminol-hydrogen peroxide, shooting a luminous image, reading the image gray value of the PCSK9 standard solution, drawing a standard curve, and converting the standard curve to obtain the concentration value of the PCSK9 in the serum samples to be tested.

The detection method of the invention is a hypersensitive wash-free chemiluminescent immunoassay (PCLIA) method for PCSK9 protein. According to the invention, a bright CL system of hemin (hemin) -luminol-hydrogen peroxide is used as signal output, mesoporous Silicon (MSN) with porous structure property is used as a nano carrier, hemin is conveniently enriched in MSN pore channels as a CL indicator, and DNA3 chain electrostatic adsorption is used as a biological gate on the MSN surface, so that a CL probe based on gate-controlled MSN is constructed. Designing and synthesizing a pair of PCSK9 antibody-DNA (Ab 1-DNA1 and Ab2-DNA 2), when no target protein PCSK9 exists, two single-stranded DNA on the Ab1-DNA1 and Ab2-DNA2 cannot be close, a CL indicator hemin is blocked in an MSN pore canal, luminol hydrogen peroxide outside the MSN cannot be catalyzed to generate CL, and a system signal is closed without subsequent reaction; with and only when the target protein PCSK9 is present, the two antibodies on Ab1-DNA1 and Ab2-DNA2 simultaneously recognize and bind to the same target protein PCSK9, forming an orthocomplex (proximity complex), DNA1 and DNA2 on the antibodies are brought close together, co-generating complementary strands sufficient to hybridize with the biological DNA3, the double-stranded structure carries the DNA3 off the MSN surface, MSN internal hemin is brought into contact with external luminol hydrogen peroxide, CL is turned on.

Specifically, the step (1) further includes a step of aminating mesoporous silicon: dissolving hexadecyl trimethyl ammonium bromide in sodium hydroxide aqueous solution at 70-90 ℃ under stirring, sequentially adding tetraethoxysilane and 3-aminopropyl triethoxysilane-ethanol solution, stirring, centrifuging, and precipitating to obtain aminated mesoporous silicon.

Specifically, the specific steps of step (2) include: adding an SMCC coupling agent with the quantity of the PCSK9 antibody and substances exceeding 20 times of that of the PCSK9 antibody into a PBE1 buffer solution for reaction for 1-3 hours to obtain an activated PCSK9 antibody; adding sulfhydryl modified DNA1/DNA2 and dithiothreitol into PBS buffer solution to react for 50-70 min at 37 ℃ to obtain reduced DNA1/DNA2; adding the activated PCSK9 antibody and excessive reduced DNA1/DNA2 into a PBE2 buffer solution, and incubating overnight at 4 ℃ to obtain Ab1-DNA1/Ab2-DNA2.

Specifically, the molar concentration ratio of PBS, naCl, EDTA in the PBE1 buffer is 11:30:4; the molar concentration ratio of PBS, naCl, EDTA in the PBE2 buffer was 11:30:1.

Specifically, the addition amount of MSN-hemin@DNA3, ab1-DNA1 and Ab1-DNA2 described in the step (3) was 8.2. Mu.g: 0.12pmol:0.12pmol.

Specifically, the step (3) further comprises adding T7 exonuclease into NEB buffer 4, wherein the use concentration of the T7 exonuclease is 2U.

T7 exonuclease is a product of T7 phage gene 6, acts on double-stranded DNA, and catalyzes removal of 5' single nucleotide in the 5 '. Fwdarw.3 ' direction. All the DNA designed by the invention is single-stranded, and the T7 exonuclease is inactive at the beginning. Once the DNA hybridizes, double strand is present, T7 can begin digestion from the 5' end of DNA3 until DNA3 is completely sheared. The DNA1 designed by the invention reserves a plurality of protecting bases at the 5' end, proximity complex is free from T7 shearing, and more DNA3 in the system can be obtained for hybridization, shearing and reciprocating circulation. The single PCSK9 triggers unlocking of a plurality of DNA3 and release of hemin a plurality of MSN pore canals, so that the CL signal is amplified under the same analysis condition, the detection sensitivity is improved, and meanwhile, the analysis time can be effectively shortened.

Specifically, the room temperature incubation time in step (3) is 50min.

Specifically, the PCSK9 standard solution or the serum sample to be tested in the step (3) is added in an amount of 0.8. Mu.L.

Specifically, the exposure time after the addition of luminol-hydrogen peroxide in step (3) was 5min.

The beneficial effects are that: compared with the prior art, the invention has the following remarkable advantages: (1) According to the detection method, the gated mesoporous silicon CL probe is synthesized and combined with the ortho-position induction effect, so that PCSK9 protein recognition and CL signal opening are completed in one step in a homogeneous solution. The invention has the advantages of no need of washing, no need of coating, no need of separation, time and cost saving, no need of external light source or electrode, constant temperature in the reaction process, no need of special instrument and equipment, no need of specific recognition site, and simple operation. (2) The invention selects the T7 nucleic acid signal amplification strategy, has high sensitivity and wide dynamic range, and meets the specific detection of target protein in high dilution samples. The detection range is as wide as 5 orders of magnitude, and the detection limit is as low as 0.7 pg/mL. The whole detection reaction can be controlled to be 55 minutes at the fastest speed. (3) multiple samples may be detected simultaneously.

Drawings

FIG. 1 is a reaction scheme of the detection method of the present invention; wherein A is a reaction route diagram when PCSK9 is detected in a sample or not, and B is a step diagram when a plurality of samples are detected simultaneously;

FIG. 2 is a graph of analytical characterization results for MSNs; wherein A is a transmission electron microscope representation graph, B is a nitrogen adsorption/desorption result graph, C is a dynamic light scattering result graph, D is a Zeta potential representation (a is bare MSN, B is aminated MSN, C is MSN-hemin@DNA3);

FIG. 3 is a graph of UV-induced results of MSN-hemin@DNA3 and Ab1-DNA 1; wherein A is the ultraviolet characterization of DNA3, hemin, MSN-hemin@DNA3, and B is the ultraviolet characterization of DNA1, ab1-DNA 1;

FIG. 4 is a graph of characterization results of MSN loading hemin; wherein A is a graph of the result of the change of the CL intensity of MSN-hemin catalyzed luminol-hydrogen peroxide with time, and B is a standard curve of the relation between the CL intensity and hemin concentration;

FIG. 5 is a graph of characterization results of MSN-hemin@DNA3; wherein A is a fluorescence spectrum of 3' of DNA with different concentrations, B is a standard curve of the relationship between the fluorescence intensity and the 3' concentration of DNA (dots represent the fluorescence intensity of MSN-hemin@DNA3 ') centrifugal supernatant);

FIG. 6 is a graph of DNA3 gating verification results; wherein A is MSN-hemin, B is MSN-hemin@DNA3, and C is MSN-hemin@DNA3 and a complementary strand thereof;

FIG. 7 is a fluorescence spectrum of AMCA and hemin; wherein A is AMCA, B is hemin; (the samples to be tested were supernatants after incubation of MSN-hemin@DNA3 with a, NEB 4buffer, b, NEB 4buffer (5 nM Ab1-DNA1, 5nM Ab2-DNA2 and 0.05U T7 in the buffer), c, NEB 4buffer (5 nM Ab1-DNA1, 5nM Ab2-DNA2 and 0.ng/mL PCSK9 in the buffer), d, NEB 4buffer (5 nM Ab1-DNA1, 5nM Ab2-DNA2, 0.ng/mL PCSK9 and 0.05U T7 in the buffer)); c is an agarose electrophoresis characterization result diagram, wherein lane 1 is a DNA ladder, lane 2 is 100 mu M DNA3, lane 3 is 10 mu M DNA1, lane 4 is 10 mu M DNA1+10 mu M DNA2 (6-bp) +10 mu M DNA3, lane 5 is 10 mu M DNA1+10 mu M DNA2 (6-bp) +10 mu M Ref DNA, lane 6 is a mixed solution of lane 5+10 mu M DNA3, and lane 7 is a mixed solution of lane 6+2U T.7;

FIG. 8 is a graph of the reaction kinetics of MSN-hemin and MSN-catalyzed luminol-hydrogen peroxide under identical conditions; wherein a is MSN-hemin, and b is MSN;

FIG. 9 is a graph showing the results of optimizing experimental conditions, wherein A is a graph showing the result of the CL signal-to-noise ratio when DNA1 reacts with DNA2 containing different numbers of complementary bases, B is a graph showing the result of the CL intensity when the amount of T7 exonuclease is different, and C is a graph showing the result of the CL intensity when the reaction time is different;

FIG. 10 is a graph of sensitivity and dynamic range detection results of the detection method and the detection method of the invention, wherein A is a CL imaging photo and a standard curve triggered by PCSK9 with different concentrations in a homogeneous solution (a first row of photos are the results of adding T7 exonuclease, 0.001, 0.01, 0.1, 1.0, 10 and 100ng/mL of PCSK9 standard substances are sequentially corresponding from left to right in a centrifuge tube, a second row of photos are the results of not adding T7 exonuclease, and 0.01, 0.1.0, 10 and 100ng/mL of PCSK9 standard substances are sequentially corresponding from left to right in the centrifuge tube); b is a specific test result diagram of the immunosensor method;

FIG. 11 is a graph of stability test results of immunosensory methods in the presence of 100pg/mL PCSK 9;

FIG. 12 is a graph of the results of testing consistency of identical samples using ELISA and the detection method of the present application.

Detailed Description

The technical scheme of the invention is further described below with reference to the accompanying drawings.

Reagents used in the examples: cetyl trimethylammonium bromide (CTAB) is available from Acros Organics; tetraethyl orthosilicate (TEOS), 3-aminopropyl triethoxysilane (APTES), hemin (hemin), ethylenediamine tetraacetic acid (EDTA) were purchased from Sigma-Aldrich corporation; PCSK9 protein and PCSK9 monoclonal antibodies (with amino groups) were purchased from Abcam corporation; coupling reagent 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid succinimidyl ester (SMCC), dithiothreitol (DTT) were purchased from Biotechnology (Shanghai) Co., ltd; t7 exonuclease and NEB 4buffer were purchased from New England Biolabs; luminol and hydrogen peroxide were purchased from the midwifery biotechnology company of beijing.

Kit used in the examples: the PCSK9 ELISA kit was purchased from Shanghai enzyme-linked biotechnology limited; total Cholesterol (TC), triglycerides (TG), high density lipoprotein cholesterol (HDL-C) and low density lipoprotein-cholesterol (LDL-C) test kits were purchased from the institute of bioengineering, built in south-kyo.

The DNA sequence listing used in the examples is as follows:

EXAMPLE 1 preparation of MSN-hemin@DNA3 and PCSK9 antibody-DNA

1. Preparation of aminated mesoporous silicon

Aminated MSN was synthesized by surfactant-assisted self-assembly: 100mg of CTAB was first dissolved in 48mL of aqueous NaOH (14.5 mM) with vigorous stirring at 80 ℃. Then 500. Mu.L TEOS and 100. Mu.LAPTES-ethanol solution (1/4, v/v) were added in this order, and the mixed solution was stirred for about 2 hours. And centrifuging the stirred mixed solution for 10 minutes at 10000rpm, and washing the obtained precipitate by using absolute ethyl alcohol to obtain the nano particles. The resulting nanoparticles were refluxed 2 times in concentrated hydrochloric acid-ethanol solution (1.5/98.5, v/v) to remove the surfactant from the wells. Evaporating and drying to obtain the aminated MSN.

2. Preparation of Biophylum DNA-terminated heme mesoporous silica (MSN-hemin@DNA3)

1mg of the aminated MSN prepared above was sonicated in 1mL of PBS buffer to obtain a homogeneous suspension. 200. Mu.L of hemin stock solution (1.2 mM) was added dropwise to the suspension under magnetic stirring. The resulting mixture was stirred at room temperature overnight until it turned clear dark brown. The strong interaction between Hemin and the silicon wall results in a significant concentration gradient within the pores, and large amounts of Hemin can be loaded into MSN to form MSN-Hemin.

10. Mu.L of 100. Mu.M DNA3 and DNA3' (DNA 3 labeled with the fluorescent dye aminomethylcoumarin AMCA) were each directly dispersed in the above mixture containing MSN-hemin, incubated at 37℃for 1h, during which the pores of aminated MSN could be blocked by DNA3 based on electrostatic interactions between electropositive MSN and electronegative DNA3/DNA3', centrifuged at 1000rpm for 3min to give MSN-hemin@DNA3 and MSN-hemin@DNA3'. The mixture was washed 3 times with ultrapure water to remove excess hemin and DNA strands, and the resulting brown precipitate was stored in 1mL of PBS.

3. Preparation of DNA-coupled PCSK9 antibody

The PCSK9 antibody (with amino group) is covalently coupled with DNA1 or DNA2 (with sulfhydryl group) through SMCC, and the excessive DNA is ensured in the process.

1mg/mL of PCSK9 antibody was reacted with SMCC coupling agent in an amount of 133.4. Mu. Mol or more in PBE1 buffer (55mM PBS,pH 7.4,150mM NaCl,20mM EDTA) at room temperature for 2 hours, followed by purification with a 100KD ultrafiltration tube to remove unbound DNA.

Reduction method of DNA1 and DNA 2: mu.L of 100. Mu.M thiol-modified DNA1/DNA2 was reacted with 16. Mu.L of 100mM DTT in PBS at 37℃for 1 hour, followed by purification with a 10KD ultrafiltration tube, respectively.

The activated antibodies and reduced DNA1/DNA2 were added to PBE2 buffer (55mM PBS,pH 7.4,150mM NaCl,5mM EDTA) and incubated overnight at 4℃and unreacted DNA was purified by 100KD ultrafiltration tube. Thus obtaining Ab1-DNA1 and Ab2-DNA2.

Example 2 characterization analysis

1. Characterization of MSN

By transmission electron microscopy(TEM) images the aminated MSN nanoparticles were characterized (fig. 2A), with MSN nanoparticles having a particle size of about 120nm having a porous structure. The nitrogen adsorption/desorption isotherms (fig. 2B) indicate: the average pore diameter of MSN nano particles is 3nm; the total volume and specific surface area of MSN nano particles obtained by theoretical calculation of BET, BJH and the like are respectively 0.96cm ³ /g and 1085.9590m ² And/g. The size of MSN nanoparticles can be further characterized by measuring Dynamic Light Scattering (DLS) (fig. 2C), indicating that they possess a relatively concentrated hydrated particle size distribution in a liquid phase medium. Zeta potential (fig. 2D) shows that bare MSN (a) is negatively charged, aminated MSN (b) has a positive charge on the surface, and according to literature reports that hemin is loaded into MSN by hydrophobic effect and is not on the surface, so when MSN-hemin is combined with DNA3 to form MSN-hemin@dna3 (c), the surface shows negative charge, which indicates that positive amino groups on the surface of MSN electrostatically adsorb negatively charged DNA3, and the surface is finally covered with a large amount of DNA3.

2. Characterization of MSN-hemin

When no DNA3 is coated on MSN, hemin in the MSN pore channel will leak spontaneously over time. Assuming that the maximum leakage amount of hemin is equal to the amount of all hemins in MSN, the CL intensity of the MSN-hemin prepared in example 1 (FIG. 4A) for catalyzing luminol hydrogen peroxide at 120min is substituted into the free hemin catalyzing CL calibration curve (FIG. 4B) under the same conditions, and the amount of hemin loaded in MSN-hemin is calculated to be 5.797mg/g. This result demonstrates that MSN has an effective enrichment capacity. Wherein, the reaction conditions of FIGS. 4A and 4B were each diluted with 110. Mu.L PBS for 10. Mu.L hemin or MSN-hemin, and then mixed with 10. Mu.L luminol hydrogen peroxide. The free hemin catalytic CL calibration curve of fig. 4B is y=6320.551x+0.778, r ² ＝0.999。

3. Characterization of MSN-hemin@DNA3

MSN-hemin@DNA3 was centrifuged at 1000rpm for 3min and the supernatant was taken for UV-vis measurement. As shown in FIG. 3A, comparing the UV-vis curves for MSN-hemin@DNA3, DNA3, hemin, the characteristic peak for hemin was found to be significantly reduced, while the characteristic peak for DNA was almost vanished, indicating that hemin had been encapsulated by DNA3 in the wells of MSN.

MSN-hemin@DNA3 'was centrifuged at 1000rpm for 3min, and the fluorescence value of DNA3' in the supernatant (FIG. 5B) was substituted into the FL calibration curve of free DNA3 '(FIG. 5A) to give the amount of DNA3' in the supernatant. Assuming that DNA3 'and MSN-hemin achieve the most efficient adsorption, the amount of DNA3' adsorbed was calculated to be 0.73. Mu. Mol/g by subtracting the amount of DNA 'in the supernatant from the total amount of DNA3' added.

4. DNA3 gating validation

After 10. Mu.L (1 mg/mL) of MSN-hemin, MSN-hemin@DNA3 and its complementary strand (3.3 nM, SEQ ID NO: 5'-GCT GAG GTT GCT GAG GAT-3', available from Biotechnology (Shanghai) Co., ltd.) were left to stand for different times, diluted with 110. Mu.L of PBS and mixed with 10. Mu.L of luminol-hydrogen peroxide, the changes in their CL intensities with time were monitored with a photomultiplier, and the results are shown in FIG. 5. MSN-hemin showed a rapid increase in CL value relative to initial time, with about 100min reaching the plateau (fig. 6A), indicating that hemin was more contacted with luminol hydrogen peroxide over time; after blocking DNA3 on its surface, CL relative intensity was always low (fig. 6B), indicating that hemin and luminol contact was hindered by DNA3; in the presence of the complementary strand of DNA3 within the system, CL restored the tendency to rise gradually over time, reaching the plateau for about 100min (fig. 6C), indicating increased exposure of hemin and luminol hydrogen peroxide in the MSN. The above phenomenon demonstrates that DNA3 can act as a switch for the "biological gate".

5. Characterization of PCSK9 antibody-DNA conjugates

DNA1, ab1-DNA1 were characterized by UV-vis. As shown in FIG. 3B, the UV absorption peaks of DNA1 and Ab1 are consistent with those of 260 (DNA characteristic peak) and 280nm (protein characteristic peak) reported in the literature, and Ab1-DNA1 shows a combined peak at 270nm, which proves the successful synthesis of PCSK9 antibody-DNA.

6. Feasibility verification of signal amplification by opening MSN biological gate through ortho-induction effect

6.1 testing the fluorescence intensity of MSN-hemin@DNA3', verifying the feasibility of opening MSN biological gate by ortho-induction effect by fluorescence experiment, and the results are shown in FIG. 7A (AMCA fluorescence spectrum) and B (hemin fluorescence spectrum). After MSN-hemin@DNA3 'is dispersed in the solution and incubated for 50min, fluorescence intensities of both the AMCA and hemin in the supernatant are weak (curve a), which proves that DNA3' can be blocked on the surface of MSN-hemin, and the MSN-hemin has good stability. After incubation of MSN-hemin@DNA3 'with the mixture of Ab1-DNA1, ab2-DNA2 and T7 for 50min, the fluorescence intensity of the supernatant increased slightly, probably because a small amount of DNA3' left the MSN surface, releasing a small amount of hemin (curve b) with negligible side reactions. After incubation of MSN with Ab1-DNA1, ab2-DNA2 and the mixture containing 0.33ng/mL PCSK9 for 50min, fluorescence intensity of AMCA and hemin in the supernatant was significantly increased (curve c), indicating that PCSK9 was recognized by Ab1-DNA1 and Ab2-DNA2 simultaneously, forming an ortho complex, enabling DNA3' to be detached from the surface of MSN. After addition of T7, the fluorescence intensity of AMCA and hemin (curve d) was much greater than that without T7 added to the mixture, indicating that T7 could initiate cyclic amplification.

6.2 further validation of the ortho-inducible regulatory signal switch by agarose gel electrophoresis techniques.

A4% agarose gel was prepared from 1 XTBE buffer. The loading was 7. Mu.L of sample mixed with 1.5. Mu.L of 6 Xloading buffer and 1.5. Mu.L of UltraPower dye. The sample was placed for 3min and then injected into agarose gel, and gel electrophoresis was performed in 1 XTBE buffer and photographed after running at 90V for 20 min.

The orthocomplex was simulated using long-chain ref DNA of 80 nucleotides (nucleotide sequence of ref DNA is shown in SEQ ID NO. 6) and DNA2 without thiol group (SEQ ID NO. 3). The results are shown in fig. 7C: the mixture of DNA1, DNA2 and DNA3 (lane 4) showed two bands at the same positions as DNA3 (lane 2) and DNA1 (lane 3), indicating that no hybridization between DNA1, DNA2 and DNA3 occurred. When ref DNA was added to the mixture, a new band appeared at about 200bp (lane 6), indicating that hybridization between DNA1, DNA2, DNA3 and ref DNA occurred, resulting in the formation of a DNA1/ref DNA/DNA2/DNA3 complex. After addition of T7 exonuclease to the mixture, a new band was observed at about 180bp (lane 7), consistent with the position of DNA1/ref DNA/DNA2 (lane 5), demonstrating that DNA3 was cleaved from DNA1/ref DNA/DNA 2.

Example 3 optimization of detection conditions

1. Optimization of exposure time

mu.L of 1mg/mL MSN-hemin and aminated MSN were mixed with 10. Mu.L of luminol-hydrogen peroxide, respectively, and their CL intensities were monitored with a photomultiplier tube over time. As a result, as shown in FIG. 8, MSN-hemin (line a) catalyzed luminol-hydrogen peroxide CL first tended to rise and then fall, peaking at about 50 seconds. After 5 minutes of reaction, the CL strength can still be maintained at 80% of the maximum. This CL dynamics demonstrates the feasibility of imaging readout. 5 minutes was chosen as the exposure time for the CCD camera. As a control, MSN itself cannot induce CL under the same conditions (line b).

2. Optimization of DNA2 nucleotide sequence

The ortho-position induction reaction utilizes the principle that Ab1-DNA1 and Ab2-DNA2 are driven by target protein PCSK9 to be sufficiently close to each other and then hybridize, which means that DNA1 and DNA2 have a certain number of complementary bases. However, if there are too many complementary bases, the noise is too loud, i.e., the target proteins can hybridize to each other without the need for them; if the number of complementary bases is too small, hybridization is not possible even if it is close, and no signal is generated. Thus, we used a long strand of 80 nucleotide DNA ref DNA to mimic Ab1/PCSK9/Ab2 sandwich complex structure to optimize complementary bases of DNA1 and DNA2. Experimental conditions: 5nM DNA1, 5nM DNA2, 10nM ref DNA or blank solution (NEB 4 buffer) were mixed. As a result, as shown in FIG. 9A, in the presence of 10nM ref DNA, the signal-to-noise ratio increased with an increase in the number of complementary bases, from 4 to 6, and then decreased from 6 to 10, indicating that DNA2 having a smaller number of bases complementary to DNA1 cannot form a proximity complex with DNA1, and that DNA2 having a larger number of bases complementary to DNA1 may have nonspecific hybridization with DNA 1. Based on the optimal signal to noise ratio, DNA2 complementary to DNA1 of 6 bases was selected for the experiment.

3. Optimization of T7 usage and incubation time

Because PCLIA adopts an exonuclease circulation strategy to improve the detection sensitivity, the dosage of T7 also needs to be optimized. Experimental conditions: after all materials including 8.2. Mu.g MSN-hemin@DNA3, 0.12pmol Ab1-DNA1, 0.12pmol Ab2-DNA2, different amounts of T7 were dispersed in 9.2. Mu.L of LNEB buffer 4buffer, and 0.8. Mu.L of 20ng/mL PCSK9 were mixed, the CL intensities of the different amounts of T7 (0.0, 0.5, 1.0, 1.5, 2.0, 2.5U) were measured. As a result, as shown in fig. 9B, CL strength increased with increasing T7 usage and tended to stabilize at 2U. Therefore, 2U was chosen as the optimal amount of T7.

Reaction time is another important parameter affecting analytical performance. Experimental conditions: all materials including 8.2. Mu.g of MSN-hemin@DNA3, 0.12pmol of Ab1-DNA1, 0.12pmol of Ab2-DNA2, 2U T7 were dispersed in 9.2. Mu.L NEB buffer 4buffer, mixed with 0.8. Mu.L 20ng/mLPCSK9, and the CL intensities were measured for the different times of the reaction (20, 40, 60, 80, 100 min). As a result, as shown in fig. 9C, CL intensity increased with an increase in reaction time and tended to be maximum at 50min. Therefore, the optimal reaction time is 50min.

EXAMPLE 4 Performance analysis of PCLIA

1) The operation steps are as follows: all materials including 8.2. Mu.g MSN-hemin@DNA3, 0.12pmol Ab1-DNA1 and 0.12pmol Ab2-DNA2 and 2U T7 exonuclease were dispersed in 9.2. Mu.L NEB buffer 4buffer (control without T7 exonuclease) at a different concentration from 0.8. Mu.L PCSK9 (10 ^-3 、10 ^-2 、10 ^-1 、10 ⁰ 、10 ¹ 、10 ² ng/mL, control group did not have 10 ^-3 ng/mL) was mixed in one EP tube and after incubation for 50min at room temperature 10 μl luminol-hydrogen peroxide was added. The luminous image was immediately photographed without washing, and the exposure time was 5min.

As a result, as shown in fig. 10A, as the PCSK9 concentration in the sample increases, the CL intensity of the homogeneous system increases accordingly. When T7 is not used, the CL intensity and the PCSK9 concentration logarithm are in a linear relation in the range of 0.01-100 ng/mL, the correlation coefficient is 0.9995, when T7 is used, the CL intensity and the PCSK9 concentration logarithm are in a linear relation in the range of 0.001-100 ng/mL (this is a standard curve used in detection), and the correlation coefficient is 0.9999. The detection limits corresponding to the 3SD signal were 9.3pg/mL (T7 was not used) and 0.7pg/mL (T7 was used), respectively.

Compared with the method without using T7, the sensitivity (slope) of the ortho induction method using the T7 exonuclease cycle amplification strategy is improved by 2.4 times, the dynamic range is increased from 4 orders of magnitude to 5, and the analysis time is shortened from 100min to 50min. The method is based on the fact that under the condition that PCSK9 with the same concentration exists due to the cyclic amplification of T7 exonuclease, the hemin released by MSN is increased within 50min, the detection sensitivity is improved, and the dynamic range is widened. Compared with the traditional ELISA method, the method has longer incubation time (5-8 h), lower sensitivity (pM-nM level) and limited dynamic range (2-3 orders of magnitude), and the hypersensitive wash-free CL imaging analysis method provides higher protein detection application prospect.

2) The operation method is the same as 1), PCSK9 with different concentrations is changed into the following substances which are respectively blank (NEB 4 buffer); 1 μg/mL Total Cholesterol (TC); 1 μg/mL Triglyceride (TG); 1 μg/mL high density lipoprotein cholesterol (HDL-C); 1 μg/mL low density lipoprotein cholesterol (LDL-C); 1. Mu.g/mL of a mixture of TC, TG, HDL-C, LDL-C and 10ng/mL of PCSK 9; 10ng/mL PCSK9.

As shown in fig. 10B, the CL intensity caused by PCSK9 at 10ng/mL was significantly higher than that caused by other interferents, confirming that only PCSK9 and its antibody-specific recognition can produce CL response.

3) The entire test procedure in the EP tube can be completed within 55 minutes. Wherein the incubation time including the one-step sandwich immune response was 50 minutes and the CL imaging exposure time was 5 minutes. Since up to 24 tubes can be incubated simultaneously, the method can achieve a throughput of about 26 tests/h.

4) Stability is an important factor in evaluating the performance of immunosensory methods. In order to examine the stability of the hypersensitive wash-free CL immunosensory method, prepared MSN-hemin@DNA3, ab1-DNA1, ab2-DNA2 were stored at 4 ℃. After 31 days, immunosensor retained 92.3% of initial CL response (fig. 11). The results show that the immunosensor prepared has good long-term stability.

Example 5 detection of PCSK9 in actual samples

Different PCSK 9-containing serum samples were tested using the established optimal conditions for the PCLIA procedure and tested using the ELISA procedure. As shown in fig. 12, the PCSK9 level in serum had satisfactory agreement between the reference method ELISA and PCLIA, verifying that the immunosensor had good reliability for detection of PCSK9 in actual samples.

Claims (9)

1. The PCSK9 detection method based on the ortho induction effect and the gate-controlled mesoporous silicon is characterized by comprising the following steps of:

preparing a gate-control mesoporous silicon CL probe MSN-hemin@DNA3 by using aminated mesoporous silicon, hemin and DNA3; the nucleotide sequence of the DNA3 is shown as SEQ ID NO. 7;

preparing PCSK9 antibody-DNA conjugates Ab1-DNA1 and Ab2-DNA2 using a PCSK9 antibody with an amino group, a DNA1 with a thiol-modified 3 'end, and a DNA2 with a thiol-modified 5' end; the sulfhydryl modified DNA1 nucleotide sequence is shown in SEQ ID NO. 1; the sulfhydryl modified DNA2 nucleotide sequence is shown in SEQ ID NO. 3;

uniformly mixing the prepared MSN-hemin@DNA3, ab1-DNA1 and Ab1-DNA2 with NEB buffer 4buffer solution, mixing with PCSK9 standard solutions with different concentrations or serum samples to be tested, incubating at room temperature, adding luminol-hydrogen peroxide, shooting a luminous image, reading the image gray value of the PCSK9 standard solution, drawing a standard curve, and converting the standard curve to obtain the concentration value of the PCSK9 in the serum samples to be tested.

2. The method of claim 1, wherein step (1) further comprises the step of preparing aminated mesoporous silicon: and (3) dissolving hexadecyl trimethyl ammonium bromide in a sodium hydroxide aqueous solution at the temperature of 70-90 ℃ under the stirring condition, sequentially adding tetraethoxysilane and 3-aminopropyl triethoxysilane-ethanol solution, stirring, centrifuging, and precipitating to obtain the aminated mesoporous silicon.

3. The method according to claim 1, wherein the specific step of step (2) comprises: adding SMCC coupling agent with the quantity of PCSK9 antibody and substance exceeding 20 times of that of the PCSK9 antibody into PBE1 buffer solution to react for 1-3 hours to obtain activated PCSK9 antibody, adding sulfhydryl modified DNA1/DNA2 and dithiothreitol into PBS buffer solution to react for 50-70 minutes at 37 ℃ to obtain reduced DNA1/DNA2; adding the activated PCSK9 antibody and excessive reduced DNA1/DNA2 into a PBE2 buffer solution, incubating overnight at 4 ℃, and ultrafiltering and purifying to obtain Ab1-DNA1/Ab2-DNA2.

4. The method according to claim 3, wherein the molar concentration ratio of PBS, naCl, EDTA in the PBE1 buffer is 11:30:4; the molar concentration ratio of PBS, naCl, EDTA in the PBE2 buffer was 11:30:1.

5. The method according to claim 1, wherein the MSN-hemin@DNA3, ab1-DNA1 and Ab1-DNA2 in step (3) are added in an amount of 8.2 μg:0.12pmol:0.12pmol.

6. The method according to claim 1, wherein step (3) further comprises adding T7 exonuclease to NEB buffer 4, wherein the amount of T7 exonuclease is 2U.

7. The method according to claim 1, wherein the incubation time at room temperature in step (3) is 50min.

8. The method according to claim 1, wherein the PCSK9 standard solution or the serum sample to be tested in the step (3) is added in an amount of 0.8. Mu.L.

9. The method according to claim 1, wherein the exposure time after the addition of luminol-hydrogen peroxide in step (3) is 5min.

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