High-sensitivity dual-fluorescence signal detection system and detection method for CD4 + cells in HIV whole blood

Abstract

The invention belongs to the technical field of biomedical detection, and particularly relates to a high-sensitivity dual-fluorescence signal detection system and detection method for CD4 + cells in HIV whole blood, and the detection system comprises four hairpin structures of HP1, HP2, Helper1 and Helper2 and a CD4 protein aptamer. According to the detection method disclosed by the invention, the aptamer and the double-signal molecule are bridged and integrated into the aptamer sensor by utilizing parallel catalytic hairpin assembly (CHA), a method for detecting the CD4 protein and the CD4 + cells in the whole blood of the HIV positive patient by using high-sensitivity homogeneous double-fluorescence signals is constructed, the detection limits respectively reach fg / mL and a single cell level, the detection sensitivity is high, and the specificity is strong.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedical detection, and in particular relates to a high-sensitivity dual-fluorescence signal detection system and a detection method for CD4+ cells in HIV whole blood. Background Art

[0002] Aptamer is a single-stranded oligonucleotide that can specifically bind to a target and can be used for cell quantification. It has the advantages of high affinity, simple synthesis steps, small batch effect, low production cost, low immunogenicity and toxicity, and easy chemical modification. Luminescent materials such as quantum dots (QDs), calcein, and metal nanoparticles can react with certain chemicals to cause changes in fluorescent signals, which not only helps signal amplification, but also can serve as signal reporter molecules. + It can undergo cation exchange reaction (CER) with CdTeQDs, enabling it to selectively recognize “C-Ag + -C" structure and Ag + Methylene blue (MB) reacts with single-stranded DNA rich in cytosine (C), but not with "C-Ag + -C" structure reaction, thereby distinguishing the two. In addition, using aptamers as recognition probes can introduce various nucleic acid amplification strategies to improve sensitivity. DNA amplification technology generally consists of thermal cycling and isothermal amplification technology. In order to simplify the steps, reduce costs, and achieve homogeneous detection, isothermal enzyme-free amplification is preferred, including hybridization chain reaction, catalytic hairpin self-assembly (CHA) reaction, etc., especially CHA uses two DNA hairpins with complementary sequences to initiate cyclic signal amplification, with high catalytic efficiency, simple design and stable system.

[0003] Human immunodeficiency virus (HIV) is the pathogen of acquired immunodeficiency syndrome (AIDS), which attacks the CD4+ cells of the immune system and causes the disease, with high morbidity and mortality. Detection of CD4+ cells can reflect the degree of damage to the immune system by HIV, and help indicate the effectiveness of treatment, prevent opportunistic infections, and predict prognosis. Patients with tumors and chronic infections also need to regularly test CD4+ cells to monitor immune status. The standard method for clinical detection of CD4+ cells is flow cytometry, but due to the complex operation, high cost of the instrument itself and maintenance, and large footprint, it is limited in resource-limited areas. Currently, some alternative methods including immunochromatography, double antibody sandwich method, and microfluidic chip counting have been reported. Summary of the invention

[0004] In order to solve the above problems existing in the prior art, the present invention provides a high-sensitivity dual-fluorescence signal detection system and method for CD4+ cells in HIV whole blood. The detection system of the present invention is constructed based on nucleic acid amplification technology and selective recognition of luminescent materials, and is used to detect dual-fluorescence signals of CD4+ cells in whole blood of HIV-positive patients, with high detection sensitivity and strong specificity.

[0005] The technical solution adopted by the present invention is:

[0006] A highly sensitive dual fluorescence signal detection system for CD4+ cells in HIV whole blood, comprising four hairpin structures and CD4 protein aptamers;

[0007] The four card issuing structures are HP1, HP2, Helper1 and Helper2 respectively.

[0008] The nucleotide sequence of HP1 is shown in SEQ ID No.1.

[0009] The nucleotide sequence of HP2 is shown in SEQ ID No.2.

[0010] The nucleotide sequence of Helper1 is shown in SEQ ID No.3.

[0011] The nucleotide sequence of Helper2 is shown in SEQ ID No.4.

[0012] The nucleotide sequence of the CD4 protein aptamer is shown in SEQ ID No.5.

[0013] A high-sensitivity dual-fluorescence signal detection method for CD4+ cells in HIV whole blood comprises the following steps:

[0014] (1) Take HP1 or HP2 respectively and mix with Ag + After mixing, add them to the buffer solution and incubate at room temperature in the dark to form a + -C" structure and the first mixed solution containing "C-Ag + -C" structure of the second mixed solution;

[0015] (2) taking CD4+ cells, CD4 protein aptamer, and buffer, mixing them thoroughly, and incubating them at room temperature to obtain a third mixed solution;

[0016] (3) After the first mixed solution, the second mixed solution and the third mixed solution are mixed, Helper 1 and Helper 2 are added, and the mixture is incubated at room temperature. CdTe QDs and MB are added, and the fluorescence intensity is detected at the excitation wavelength.

[0017] In step (1) and step (2), the buffer is a MOPS buffer composed of 10 mM MOPS, 2.5 mM Mg(NO3)2 and 100 mM NaNO3, pH = 7.2.

[0018] In step (1), the HP1 or HP2 and Ag + The molar ratio of the substance was 1:6, and the mixture was incubated at room temperature in the dark for 45 minutes;

[0019] In step (2), the molar ratio of the CD4+ cells to the CD4 protein aptamer is 1:1, and the cells are incubated at room temperature in the dark for 60 minutes;

[0020] In step (3), the molar ratio of Helper1 to Helper2 is 1:1, and the incubation is performed at room temperature in the dark for 90 minutes.

[0021] In step (3), the CdTe QDs are prepared by the following method:

[0022] (a) Take 0.5 mmol of CdCl2 and 0.2 g of trisodium citrate, add them into water, and after fully dissolving, prepare 50 mL of solution;

[0023] (b) adding 52 μL MPA to the solution obtained in step (a), and adjusting the pH of the solution to 10.5 with NaOH;

[0024] (c) 0.1 mmol of Na2TeO3 and 50 mg of KBH4 were added to the solution obtained in step (b) respectively, and refluxed for 1 hour to obtain CdTe QDs.

[0025] The beneficial effects of the present invention are:

[0026] The present invention provides a highly sensitive dual fluorescence signal detection system for CD4+ cells in HIV whole blood, comprising four hairpin structures HP1, HP2, Helper1, and Helper2 and a CD4 protein aptamer. The detection system of the present invention integrates an aptamer sensor by bridging the aptamer and the dual signal molecule using parallel catalytic hairpin assembly (CHA), and constructs a highly sensitive and homogeneous dual fluorescence signal method for detecting CD4 protein and CD4+ cells in whole blood of HIV-positive patients, with detection limits reaching fg / mL and single cell levels, respectively, with high detection sensitivity and strong specificity. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0028] Figure 1 A schematic diagram showing the structure of the high-sensitivity dual-fluorescence signal detection system of the present invention;

[0029] Figure 2 The feasibility of the method of the present invention for CD4+ cell analysis is demonstrated, wherein: Figure 2 A shows a transmission electron microscopy image of the CdTe QDs. Figure 2 B shows QDs and Ag + The SEM image of Ag2Te produced by CER is triggered by the reaction. Figure 2 C shows the UV-visible spectrum of the CdTe QDs. Figure 2 D shows the UV-visible spectrum of methylene blue (MB), Figure 2 E shows the X-ray photoelectron spectrum of the CdTeQDs. Figure 2 F shows Ag + Fluorescence signal change diagram of quenched QDs, Figure 2 G shows the addition of HP1 to form “C-Ag + -C” after the QDs fluorescence signal changes, Figure 2 H shows the fluorescence signal change of MB after adding HP1 only. Figure 2 I shows in HP1, Ag + The fluorescence signal change diagram of MB in the system, Figure 2 J shows the fluorescence signal changes of MB and QDs at different excitation wavelengths. Figure 2 K shows the fluorescence signal changes under different MB and QDs systems.

[0030] Figure 3 Display Ag + , the interaction between CdTe QDs and CD4+ cells; Figure 3 A and Figure 3 B shows Ag before and after adding CD4+ cells + Comparison of CER between CdTe QDs and Figure 3 C and Figure 3 D shows the CCK-8 detection of CdTe QDs and Ag, respectively. + Cytotoxicity to CD4+ cells;

[0031] Figure 4The optimization of the analysis conditions of the method for detecting CD4 protein with dual fluorescence signals described in the present invention is shown; wherein, Figure 4 A shows that HP1 and HP2 formed a stable “C-Ag + -C" hairpin structure, Figure 4 B shows Ag + The optimal ratio with HP DNA is 1:6. Figure 4 C shows that CD4 protein completely binds to CD4 protein aptamer within 60 minutes. Figure 4 D shows that the CHA amplification reaction is sufficient after adding Helper1 and Helper2 and incubating for 1.5 hours. Figure 4 E and Figure 4 F shows that the signal-to-noise ratio of CdTe QDs diluted 10 times by 9.4 μL ultrapure water and 20 μL MB solution (21 μM) is the best; Figure 4 GJ shows that the above CdTe QDs and MB solutions are respectively reacted with Ag in 1 minute. + It reacts fully with DNA chains containing C-rich sticky ends;

[0032] Figure 5 The sensitivity and specificity of the quantitative CD4+ cell detection method of the present invention are shown; wherein, Figure 5 A shows that with the increase of CD4 protein, the fluorescence signals of CdTe QDs and MB gradually increase; Figure 5 B and Figure 5 C shows that there is a good linear relationship between the concentration range of CD4 protein from 0.1 to 100 fg / mL and the fluorescence signals of QDs and MBs; Figure 5 D shows the dual-signal strategy for detecting CD4 protein using 11 coexisting proteins in serum; Figure 5 E to 5G show that there is a significant linear relationship between the concentration range of CD4+ cells from 1 / mL to 1000 / mL and the fluorescence signals of QDs and MBs;

[0033] Figure 6 Demonstrating analytical performance of detecting CD4 protein using a single fluorescence signal;

[0034] Figure 7 Shown is the analytical performance of detecting CD4+ cells as a single fluorescent signal. DETAILED DESCRIPTION

[0035] To make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described in detail below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other implementation methods obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention.

[0036] Unless otherwise specified in the following examples, the solvents of the solutions are all ultrapure water.

[0037] Example 1

[0038] This embodiment constructs a high-sensitivity dual fluorescence signal detection system for CD4+ cells in HIV whole blood, and its structure is as follows: Figure 1 As shown, it includes four hairpin structures and a CD4 protein adaptor; the four hairpin structures are HP1, HP2, Helper1, and Helper2.

[0039]

[0040] HP1 and HP2 each contain 6 consecutive C bases at both ends, which can bind to Ag. + Combined to form "C-Ag + -C" structure, and the rest of the structure is complementary to the CD4 protein aptamer. Helper1 and Helper2 both contain 6 consecutive C bases and G bases at both ends, and the rest of the structure is complementary to HP1 and HP2. When the solution containing CD4+ cells is not added, the CD4 protein aptamer cannot recognize CD4+ cells, and thus recognizes and binds to HP1 and HP2, releasing Ag. + After Helper1 and Helper2 are added, HP1 and HP2 are recognized and bound by Helper1 and Helper2 respectively, and the CD4 protein aptamer is released at the same time, achieving parallel CHA amplification and forming double-stranded DNA containing C-rich sticky ends. + The double-stranded DNA containing exposed C-rich sticky ends reacted with CdTe QDs and MB, respectively, resulting in a decrease in the fluorescence signal. On the contrary, when a solution containing CD4+ cells was added, the CD4 protein aptamer specifically recognized and bound to CD4+ cells, thus failing to bind to HP1 and HP2. + Release, Helper1 and Helper2 cannot recognize HP1 and HP2, the subsequent reaction cannot proceed, and the change of fluorescence signal can be ignored.

[0041] HP1 and HP2 are recognized by Helper1 and Helper2 respectively to form double-stranded DNA with exposed C-rich base sticky ends, and trigger parallel CHA at the same time. This is the first stage of amplification, which improves the sensitivity. CdTe QDs and MB finally act as signal reporter molecules, selectively reacting with Ag +The reaction with single-stranded DNA containing continuous C bases leads to a decrease in the fluorescence signal. The introduction of these two luminescent materials further improves the sensitivity, which is the second-level amplification. The fluorescence signal of CdTe QDs corresponds to the concentration of CD4+ cells. By judging and calculating the changes in the fluorescence signal, the quantitative analysis of CD4+ cells in the whole blood of HIV-positive patients can be achieved. Figure 1 shown.

[0042] Example 2

[0043] This embodiment provides a high-sensitivity dual-fluorescence signal detection method for CD4+ cells in HIV whole blood, comprising the following steps:

[0044] (1) 20 μL HP1 or HP2 (1 μM) was mixed with 24 μL AgNO3 (5 μM), and then added to 50 μL 3-(N-morpholino)propanesulfonic acid (MOPS) buffer (10 mM MOPS, 2.5 mM Mg(NO3)2, 100 mM NaNO3, pH 7.2). The mixture was incubated at room temperature in the dark for 45 min to form a “C-Ag”-containing + -C" structure and the first mixed solution containing "C-Ag + -C" structure; wherein the second mixed solution containing "C-Ag + -C” structure first mixed solution refers to: HP1, Ag + , buffer solution; the "C-Ag + -C" structure refers to the second mixed solution of HP2, Ag + , buffer solution;

[0045] (2) incubating a mixed solution consisting of 20 μL of CD4+ cells of different concentrations (10, 100, 500, and 1000 cells / mL), 20 μL of CD4 protein aptamer, and 50 μL of MOPS buffer at room temperature for 60 minutes to obtain a third mixed solution;

[0046] (3) Then the first mixed solution, the second mixed solution and the third mixed solution were mixed, 20 μL Helper1 (1 μM) and 20 μL Helper2 (1 μM) were added to the mixture, and incubated in the dark at room temperature for 90 minutes; finally, 9.4 μL ultrapure water diluted 10 times CdTe QDs and 20 μL MB (21 μM) were added; after 1 minute, the fluorescence intensity was measured at an excitation wavelength of 540 nm.

[0047] Wherein, the CdTe QDs were prepared by the following method: first, a 50 mL solution containing CdCl2 (0.5 mmol) and trisodium citrate (0.2 g) was prepared. Then, MPA (52 μL) was immediately added to the above solution, and the pH of the solution was adjusted to 10.5 with NaOH. Then, Na2TeO3 (0.1 mmol) and KBH4 (50 mg) were added to the above solution and refluxed for 1 hour to obtain CdTeQDs. Subsequently, high-purity CdTe QDs were obtained by precipitation with n-propanol and centrifugation (11000 rpm). The purified yellow CdTe QDs were redispersed in ultrapure water before use.

[0048] Example 3

[0049] The only difference between this embodiment and embodiment 2 is that the CD4+ cells in step (2) are replaced by CD4 protein, and the different concentrations of CD4 protein are 1, 10, and 100 fg / mL respectively.

[0050] Experimental example

[0051] 1. Feasibility verification of CD4+ cell analysis

[0052] First, the synthesized CdTe QDs were characterized to verify their feasibility. CdTe QDs are spherical particles with a diameter of about 4 nm ( Figure 2 A), its UV-visible spectrum shows an absorption peak at 530 nm ( Figure 2 C) QDs and Ag + The reaction of CER induces the production of Ag2Te and produces a significant agglomeration effect ( Figure 2 B). The characteristic ultraviolet absorption peaks of MB are 605nm and 660nm ( Figure 2 D). The elemental composition and surface states of CdTe QDs were analyzed by X-ray photoelectron spectroscopy (XPS). Figure 2 E). The binding energies of Cd 3d are around 404.0eV and 410.7eV, and those of Te 3d are 581.6eV and 572.5eV, respectively. Figure 2 F verified the existence of CER, indicating that Ag + It can quench QDs. When HP1 is added, “C-Ag + -C" structure, free Ag + Less, the QDs fluorescence signal gradually increased ( Figure 2 G). The decrease in MB fluorescence signal is due to the fact that the C-rich single-stranded DNA hinders the electron transition ( Figure 2 H), and not with "C-Ag + -C" structure reacts ( Figure 2I). The difference between the fluorescence signals of QDs and MB at 365nm excitation wavelength is too large, much larger than the fluorescence signal generated at 540nm, and the maximum emission wavelength of MB and QDs is around 660nm, so 540nm is selected as the common excitation wavelength ( Figure 2 J).

[0053] Based on the above results, the feasibility of this strategy is verified ( Figure 2 K). HP1 and HP2 bind to CD4 protein aptamers, resulting in a decrease in the fluorescence signals of QDs and MBs ( Figure 2 Ka to b and 2K-g to h) suggest Ag + When different concentrations of CD4 protein (1, 10, 100 fg / mL) were added, the fluorescence intensity of QDs and MB gradually increased ( Figure 2 Kc to f and 2K-g to l), indicating that the CHA-assisted strategy is effective. Adding fg / mL of CD4 protein to the system can significantly enhance the fluorescence intensity, indicating that the method has high sensitivity.

[0054] In view of the high expression of CD4 protein on CD4+ cells, the detection of CD4 protein can indirectly quantify CD4+ cells. Before using the system of the present invention to quantify CD4+ cells, we first explored the Ag + , the interaction between CdTe QDs and CD4+ cells. CD4+ cells to Ag + The CER between CdTe QDs is not significantly affected ( Figure 3 A and B). CdTe QDs were detected using CCK-8 ( Figure 3 C) and Ag + ( Figure 3 D) Cytotoxicity to CD4+ cells. Calculation of CdTe QDs and Ag + IC for CD4+ cell viability 50 , 0.62μM and 27.4μM respectively. In the detection system of the present invention, CdTe QDs and Ag + The concentrations of the two compounds in the detection of CD4+ cells were 0.04 μM and 0.8 μM, respectively, which are far lower than their IC 50 Therefore, a strategy for simultaneously directly quantifying CD4 protein and indirectly quantifying CD4+ cells can be achieved.

[0055] 2. Optimization of CD4+ cell analysis conditions

[0056] The experimental conditions involved in the experiment were investigated, such as Figure 4 As shown. 45 minutes is sufficient for HP1 and HP2 to form a stable “C-Ag + -C" hairpin structure, Ag +The optimal ratio with HP DNA is 1:6 ( Figure 4 A and B), CD4 protein completely binds to CD4 protein aptamer within 60 min ( Figure 4 C) For CHA amplification, Helper1 and Helper2 were added and incubated for 1.5 hours, and the reaction was complete ( Figure 4 D). Select 9.4 μL ultrapure water to dilute CdTe QDs 10 times ( Figure 4 E) and 20 μL MB solution (21 μM, Figure 4 F) To optimize the signal-to-noise ratio, and both were separated by Ag for 1 minute. + The DNA chain with C-rich sticky ends reacts well ( Figure 4 G to J).

[0057] 3. Sensitivity and specificity of quantitative CD4+ cell detection methods

[0058] like Figure 5 As shown. Under the optimized experimental conditions, the analysis performance of this strategy is good. With the increase of CD4 protein, the fluorescence signals of CdTe QDs and MB gradually increase ( Figure 5 A), CD4 protein has a good linear relationship with the fluorescence signals of QDs and MB in the concentration range of 0.1 to 100 fg / mL, the linear equations are Y=172LogC+1130 and Y=63LogC+416, and the detection limits are 0.02 and 0.03 fg / mL, respectively (based on triple signal-to-noise ratio calculation, Figure 5 B and C). Using QDs or MB as single signal reporter molecules, the detection limits were approximately 2.5 and 4 fg / mL, respectively ( Figure 6 There is a significant linear relationship between the concentration of CD4+ cells in the range of 1 cell / mL to 1000 cells / mL and the fluorescence signals of QDs and MBs ( Figure 5 E to G), the linear equations were Y = 136LogC + 849 and Y = 71LogC + 313, respectively, with detection limits of 0.2 and 0.3 / mL, respectively. When QDs or MB were used as single signal reporter molecules, the detection limits were approximately 3 and 5 / mL, respectively. Figure 7 ).

[0059] Eleven coexisting proteins in serum, including glucose oxidase (GOD), CD3, interferon-γ (IFN-γ), mucin 1, papain, pepsin, prostate-specific antigen (PSA), streptavidin (SA), transferrin, tetanus toxoid, and immunoglobulin G (IgG), were used as potential interfering molecules to evaluate the dual-signal strategy for detecting CD4 protein. Figure 5D shows that the fluorescence intensity change caused by the above protein (10fg / mL) is similar to that of the blank solution. Low concentrations of CD4 protein (1 and 10fg / mL) cause a significant increase in fluorescence signal. These results show that the detection system of the present invention has strong targeting selectivity with the help of signal amplification technology due to the high affinity between the CD4 protein aptamer and the CD4 protein. The sensitivity of the dual-signal strategy is significantly higher than that of the single-signal strategy due to the synergistic effect.

[0060] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art can easily think of changes or substitutions within the technical scope disclosed by the present invention, which should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. A high-sensitivity dual-fluorescence signal detection system for CD4+ cells in HIV whole blood, characterized in that: It includes four hairpin structures and CD4 protein aptamer; The four card issuing structures are HP1, HP2, Helper1 and Helper2 respectively.

2. The detection system according to claim 1, characterized in that: The nucleotide sequence of HP1 is shown in SEQ ID No.

1.

3. The detection system according to claim 1, characterized in that: The nucleotide sequence of HP2 is shown in SEQ ID No.

2.

4. The detection system according to claim 1, characterized in that: The nucleotide sequence of Helper1 is shown in SEQ ID No.

3.

5. The detection system according to claim 1, characterized in that: The nucleotide sequence of Helper2 is shown in SEQ ID No.

4.

6. The detection system according to claim 1, characterized in that: The nucleotide sequence of the CD4 protein aptamer is shown in SEQ ID No.

5.

7. A high-sensitivity dual-fluorescence signal detection method for CD4+ cells in HIV whole blood, characterized in that: The steps include: (1) Take HP1 or HP2 respectively and mix with Ag + After mixing, add them to the buffer solution and incubate at room temperature in the dark to form a + -C" structure and the first mixed solution containing "C-Ag + -C" structure of the second mixed solution; (2) taking CD4+ cells, CD4 protein aptamer, and buffer, mixing them thoroughly, and incubating them at room temperature to obtain a third mixed solution; (3) After the first mixed solution, the second mixed solution and the third mixed solution are mixed, Helper 1 and Helper 2 are added, and the mixture is incubated at room temperature. CdTe QDs and MB are added, and the fluorescence intensity is detected at the excitation wavelength.

8. The high-sensitivity dual-fluorescence signal detection method for CD4+ cells according to claim 7, characterized in that: In step (1) and step (2), the buffer is a MOPS buffer composed of 10 mM MOPS, 2.5 mM Mg(NO3)2 and 100 mM NaNO3, pH = 7.

2.

9. The high-sensitivity dual-fluorescence signal detection method for CD4+ cells according to claim 7, characterized in that: In step (1), the HP1 or HP2 and Ag + The molar ratio of the substance was 1:6, and the mixture was incubated at room temperature in the dark for 45 minutes; In step (2), the molar ratio of the CD4+ cells to the CD4 protein aptamer is 1:1, and the cells are incubated at room temperature in the dark for 60 minutes; In step (3), the molar ratio of Helper1 to Helper2 is 1:1, and the incubation is performed at room temperature in the dark for 90 minutes.

10. The high-sensitivity dual-fluorescence signal detection method for CD4+ cells according to claim 7, characterized in that: In step (3), the CdTe QDs are prepared by the following method: (a) Take 0.5 mmol of CdCl2 and 0.2 g of trisodium citrate, add them into water, and after fully dissolving, prepare 50 mL of solution; (b) adding 52 μL MPA to the solution obtained in step (a), and adjusting the pH of the solution to 10.5 with NaOH; (c) 0.1 mmol of Na2TeO3 and 50 mg of KBH4 were added to the solution obtained in step (b) respectively, and refluxed for 1 hour to obtain CdTe QDs.

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