Compositions, working solutions and products for biosensors for detecting ape1

Abstract

The application belongs to the field of biology and particularly relates to a composition of a biosensor for detecting APE1, a working solution and a product, the composition comprising a double-stranded promoter, an H1 hairpin, an H2 hairpin and an FQ reporter molecule, the double-stranded promoter comprising a promoter I and a blocking chain B, and the FQ reporter molecule comprising a RepF chain and a RepQ chain, the biosensor formed by the composition of the application has high sensitivity and strong specificity for detecting APE1.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to compositions, working solutions, and products of biosensors for detecting APE1. Background Technology

[0002] Apurinic / apyrimidinic endonuclease 1 (APE1) is a protease widely distributed in organisms that recognizes and cleaves DNA at base deletion sites (AP sites) during base excision repair (BER). It exhibits high efficiency and specificity. Furthermore, APE1 activity is significantly higher in some cancer cells than in normal cells, thus making it a potential cancer biomarker.

[0003] Traditional detection methods include gel electrophoresis, enzyme-linked immunosorbent assay (ELISA), high-performance liquid chromatography (HPLC), and colorimetric methods. However, these methods have the following drawbacks: they require complex systems and cumbersome sample / reagent processing, which depends on well-established laboratories with specialized instruments or well-trained operators; they have low sensitivity, are time-consuming, prone to false positives, and require expensive equipment.

[0004] Therefore, there is an urgent need to develop new strategies with high sensitivity and specificity to detect APE1 levels, especially those that can be used for rapid and universal point-of-care testing (POCT) diagnostic techniques. Summary of the Invention

[0005] In view of this, the present invention provides compositions, working solutions, and products for detecting APE1 in a biosensor.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows:

[0007] In some embodiments, this application provides a composition for detecting an APE1 biosensor, the composition comprising a double-stranded promoter, an H1 hairpin, an H2 hairpin, and an FQ reporter molecule, the double-stranded promoter comprising promoter I and a blocking strand B, and the FQ reporter molecule comprising a RepF strand and a RepQ strand.

[0008] In some embodiments, the nucleotide sequence of promoter I is shown in SEQ ID NO.1.

[0009] In some embodiments, the nucleotide sequence of the blocking chain B is shown in SEQ ID NO.2.

[0010] In some embodiments, the nucleotide sequence of the RepF chain is shown in SEQ ID NO.3.

[0011] In some embodiments, the nucleotide sequence of the RepQ chain is shown in SEQ ID NO.4.

[0012] In some embodiments, the nucleotide sequence of the H1 hairpin is as shown in SEQ ID NO.5.

[0013] In some embodiments, the nucleotide sequence of the H2 hairpin is as shown in SEQ ID NO.6.

[0014] This application also provides the use of the composition described above in the working solution for preparing a biosensor or in a biosensor.

[0015] This application also provides a working fluid for a biosensor, comprising the composition described above and acceptable excipients or additives.

[0016] This application also provides the use of the working solution of the biosensor described above in the preparation of products for detecting APE1.

[0017] This application also provides a product for detecting APE1, which includes the working solution of the biosensor as described above.

[0018] In some embodiments, the product includes a biosensor or a kit.

[0019] The present invention has the following beneficial effects:

[0020] The sensor formed by the composition of this application has high sensitivity and strong detection specificity for APE1.

[0021] Due to the integration of the catalytic hairpin assembly, the sensor formed by the composition of this application can significantly amplify the detection signal of APE1.

[0022] The sensor formed by the composition of this application has good clinical applicability for the detection of APE1 in serum samples.

[0023] The sensor formed by the composition of this application has successfully and simply detected low abundance of APE1 in a one-step method without the need for additional enzyme separation steps. Attached Figure Description

[0024] Figure 1 This is a schematic diagram of the principle of the method of this application, wherein Target Recognition is target recognition, Signal Amplification is signal amplification, Signal transduction is signal transduction, and FQ-Reporters are FQ reporter molecules;

[0025] Figure 2The electrophoresis results are shown to verify the feasibility of this application. Initaitor strand (I) is promoter I, Blocker strand (B) is blocking strand B, Hairpin is hairpin, Ampliafication products are signal amplification products, and Cleavage products are cleavage products.

[0026] Figure 3 Figure A shows the detection results for the optimal base length of the blocking chain; Figure 3 Figure B shows the detection results for the optimal location of the AP site in the optimized double-stranded promoter; Figure 3 C is the detection result of the optimized reaction substrate ratio, and Ratio of I / B is the I / B ratio; Figure 3 D represents the optimal substrate concentration for the reaction. Figure 3 E represents the detection results of the optimized reaction time, and Concentration represents the concentration.

[0027] Figure 4 Figure A shows the results of the linear analysis in this application. Figure 4 Figure B shows the results of the specificity test for this application. Detailed Implementation

[0028] The present invention will be further illustrated by specific examples below. However, it should be noted that the specific material ratios, process conditions and results described in the embodiments of the present invention are only for illustrating the present invention and cannot be used to limit the scope of protection of the present invention. All equivalent changes or modifications made in accordance with the spirit and essence of the present invention should be covered within the scope of protection of the present invention.

[0029] This application provides a composition for a biosensor used to detect APE1, the composition comprising a double-stranded promoter, an H1 hairpin, an H2 hairpin, and an FQ reporter molecule. The double-stranded promoter comprises promoter I and a blocking strand B, the nucleotide sequence of promoter I being shown in SEQ ID NO.1, the nucleotide sequence of blocking strand B being shown in SEQ ID NO.2, the FQ reporter molecule comprising a RepF strand and a RepQ strand, the nucleotide sequence of the RepF strand being shown in SEQ ID NO.3, the nucleotide sequence of the RepQ strand being shown in SEQ ID NO.4, the nucleotide sequence of the H1 hairpin being shown in SEQ ID NO.5, and the nucleotide sequence of the H2 hairpin being shown in SEQ ID NO.6.

[0030] This application innovatively proposes a biosensor composition that combines the specific site recognition function of APE1 with local DNA self-assembly technology (DNA nanotechnology utilizes base complementarity to precisely self-assemble, forming various geometric structures and combinations to form DNA nanostructures). APE1 recognizes AP sites and performs double-stranded DNA cleavage to form single-stranded DNA as the introductory strand for subsequent reactions. Simultaneously, it initiates a footpoint-mediated dynamic catalytic hairpin assembly (CHA) reaction to generate a fluorescent signal output. By combining the programmability of catalytic hairpin assembly with the function of APE1, an isothermal nucleic acid detection platform is successfully constructed.

[0031] This application combines APE1 and CHA technologies, offering the following advantages: (1) High-fidelity recognition: APE1-specific recognition ensures highly specific target detection; (2) High sensitivity: the CHA reaction can recycle the introduced chain to amplify the output signal; (3) Isothermal reaction: suitable for on-site, real-time detection; (4) One-step reaction: simple and easy to operate, suitable for standardized procedural management and application in primary healthcare institutions. In summary, this application constructs an enzyme-free, isothermal, simple, and efficient detection method that reduces background signal and improves detection sensitivity, providing new technical support for constructing intelligent biomolecular sensors to detect APE1 and other bioanalyses.

[0032] This application also provides the use of the composition described above in the working solution for preparing a biosensor or in a biosensor.

[0033] This application also provides a working fluid for a biosensor, comprising the composition described above and acceptable excipients or additives.

[0034] This application also provides the application of the working solution of the biosensor described above in the preparation of products for detecting APE1.

[0035] This application also provides a product for detecting APE1, which includes the working solution of the biosensor as described above, and the product includes a biosensor or a kit.

[0036] The present invention will be illustrated below with specific embodiments. It should also be understood that the following embodiments are only for illustrative purposes and should not be construed as limiting the scope of protection of the present invention. Non-essential improvements and adjustments made by those skilled in the art based on the above description of the present invention are all within the scope of protection of the present invention. The specific process parameters, etc., in the following examples are merely examples within a suitable range; that is, those skilled in the art can make appropriate selections within the appropriate range based on the description herein, and are not intended to be limited to the specific values ​​in the examples below.

[0037] (I) Principle

[0038] This application utilizes an enzymatic DNA network (EDN) to directly detect APE1 activity. By leveraging the specific cleavage characteristics of the target APE1, an enzymatic DNA network reaction is formed through the integration of CHA (catalytic hairpin assembly). The entire process consists of three parts: target recognition (I), signal amplification (II), and signal transduction (III). Specifically, as follows... Figure 1 As shown, firstly, the target APE1 continuously recognizes the AP site of the double-stranded promoter, triggering a series of cleavage reactions. Acting as a signal input and amplifier, it induces the breaking of the blocking strand, releasing promoter-I from the double-stranded promoter. This promoter-I then acts as a signal input and amplifier, further triggering enzymatic DNA network reactions (I). The released I strand undergoes a CHA reaction with the H1 site via a foothold-mediated strand substitution reaction, producing a large amount of H1-H2 double-stranded product. Then, the released promoter strand can restart the CHA cycle, further amplifying the signal (II). Finally, to achieve signal transduction, the double-stranded product binds to the RepF foothold in the FQ reporter molecule, triggering a branching migration reaction that replaces RepQ, thereby emitting a strong fluorescent signal (III).

[0039] (II) Preparation of double-stranded promoters, FQ reporter molecules and hairpin substrates

[0040] Preparation of double-stranded promoter: DNA single strand I (i.e. promoter I) and DNA single strand B (i.e. blocking strand B) were annealed in 1×TNaK buffer (final concentration of NaCl was 140 mM, final concentration of Tris was 20 mM, final concentration of KCl was 5 mM, pH was 7.5) at a molar ratio of 1:1.5. The annealing process was denaturation at 95℃ for 5 min and annealing at 37℃ for 80 min to prepare a 1 μM double-stranded promoter for later use.

[0041] Preparation of FQ reporter molecules: To synthesize FQ reporter molecules, FAM fluorescent group-labeled chains (RepF) and quenched group-labeled chains (RepQ) were dissolved in 1×TNaK buffer at a molar ratio of 1:1.5 and annealed. The annealing process was denaturation at 95℃ for 5 min and annealing at 37℃ for 80 min to obtain a 2 μM FQ-reporter molecule solution.

[0042] Preparation of hairpin substrate: To form 5 μM hairpin substrate (H1 / H2), the DNA strand containing the stem loop (specifically H1 or H2) was mixed with 1×TNaK buffer to form a 5 μM substrate. The substrate was denatured in a 95°C water bath for 5 minutes and then slowly cooled to room temperature over 8 hours to obtain a 5 μM H1 solution and a 5 μM H2 solution.

[0043] Finally, all prepared DNA probes (i.e., double-stranded promoters, FQ reporter molecules, and hairpin substrates) were stored at -20°C for further use.

[0044] The nucleotide sequences (in this application, all sequences were designed by the applicant and synthesized by Sangon Biotech (Shanghai) Co., Ltd.) are shown in Table 1.

[0045] Table 1 Nucleotide sequence list

[0046]

[0047] (III) APE1 Activity Detection

[0048] The entire process was carried out in a 100 μL reaction mixture consisting of component A and component B. Component A contained 2 μL of a 1 μM promoter and 2 μL of 10×NEBuffer 4. Component B was an 80 μL reaction solution containing 4 μL of a 5 μM H1 solution, 4 μL of a 5 μM H2 solution, and 10 μL of a 2 μM FQ-reporter molecule solution, dissolved in 1×TNaK buffer.

[0049] Different concentrations of APE1 were mixed with components A and B and incubated simultaneously at 25°C for 3 hours. The fluorescence intensity was then immediately measured using a fluorophotometer.

[0050] The detailed process is as follows:

[0051] Effect verification

[0052] Constructing an integrated fluorescent EDN detection method

[0053] The specific steps of the one-step detection method are as follows: 2 μL of 1 μM double-stranded promoter, 2 μL of 10×NEBuffer 4, 1 μL of APE1 at different concentrations, 4 μL of 5 μM H1, 4 μL of 5 μM H2, 10 μL of 2 μM FQ-reporter molecule, and 1×TNaK buffer (total volume 100 μL, with enzyme-free water as the remainder) are mixed, vortexed, and incubated at 25°C for 3 h. The fluorescence intensity is then immediately measured using a fluorometer. A control group without APE1 is also included in the experiment. The parameters for fluorescence intensity measurement are as follows: scan speed 600 nm / min, excitation and emission slits 5 nm, excitation wavelength 490 nm, emission wavelength range 500-600 nm, and voltage 600 V.

[0054] Feasibility verification

[0055] To verify the feasibility of this method, polyacrylamide gel electrophoresis (PAGE) was first performed. The steps for polyacrylamide gel electrophoresis are as follows:

[0056] A 12% non-denaturing polyacrylamide gel (PAGE) was prepared. The reaction products were electrophoresed in 1×TBE buffer (2 mM EDTA, 90 mM Tris-boric acid, pH 8.4) at a constant voltage of 110 V for 45 min. The gel products were stained with Gel Red dye for 15 min and then imaged. The results are shown below. Figure 2 As shown, lanes 1 to 5 represent bands I, B, promoter, H1, and H2, respectively.

[0057] like Figure 2 As shown, no reaction products were observed in lane 6 for the double-stranded promoter and the two hairpin mixtures. However, in lane 7, when the target APE1 was added to the reactants, the double-stranded promoter band was degraded, and the H1-H2 complex was generated. This result confirms that the input target successfully led to the degradation of the double strand and the hybridization of the hairpins.

[0058] Optimization of experimental conditions

[0059] (1) The applicant optimized the promoter structure, including the optimal base length of the blocking strand and the optimal position of the AP site in the double-stranded promoter. The specific steps are as follows:

[0060] ① Optimize the optimal base length of the blocking chain

[0061] Double-stranded promoters with different base lengths were prepared (blocking strand B contained 13-17 bases and was complementary to strand I). The specific process was the same as above, and the specific sequences of each blocking strand B are shown below:

[0062] B sequence (13nt) (SEQ ID NO.7): GTCAGTAGCTAGG;

[0063] B sequence (14nt) (SEQ ID NO.8): GTCAGTAGCTAGGT;

[0064] B sequence (15nt) (SEQ ID NO.9): GTCAGTAGCTAGGTT;

[0065] B sequence (16nt) (SEQ ID NO.10): GTCAGTAGCTAGGTTA;

[0066] B sequence (17nt) (SEQ ID NO.11): GTCAGTAGCTAGGTTAG;

[0067] The test was performed using the one-step method described in this section (i.e., the effect verification section), and the results are as follows: Figure 3 As shown in Figure A.

[0068] Depend on Figure 3 As shown in A, the S / B ratio (signal-to-noise ratio) increases with the increase of complementary bases, up to 16 bp. After exceeding 16 bp, the S / B ratio (signal-to-noise ratio) decreases with the increase of complementary bases. This is because a short blocking strand B may bring high non-specific background signal through spontaneous hybridization between the promoter and hairpin; at the same time, an excessively long blocking strand B may reduce the dissociation of the blocking strand and hinder the effective release of the promoter.

[0069] ② Optimize the optimal location of the AP site in the double-stranded promoter:

[0070] Double-stranded promoters with AP sites at different base positions were prepared (AP sites in blocking strand B were located at positions 2, 4, 6, 8, and 10, respectively, complementary to strand I). The specific process was the same as above, and the details of each blocking strand B are shown below:

[0071] B sequence (site 2) (SEQ ID NO.12): GTAGTGAGCTAGGTTA;

[0072] B sequence (site 4) (SEQ ID NO.13): GTCATGAGCTAGGTTA;

[0073] B sequence (site 6) (SEQ ID NO.10): GTCAGTAGCTAGGTTA;

[0074] B sequence (site 8) (SEQ ID NO.2): GTCAGTGACTAGGTTA;

[0075] B sequence (site 10) (SEQ ID NO.14): GTCAGTGAGCAGGTTA;

[0076] The above one-step method (i.e., the effect verification part) was used for testing, and the results are as follows: Figure 3 As shown in B.

[0077] Depend on Figure 3 As shown in B, the background signal was completely suppressed, and only the AP site at position 6 or 8 of the 3' end caused a significant fluorescent signal. This is because the AP site is too close to the 3' end of the blocking strand, resulting in a short foothold for the enzyme reaction product, which cannot trigger the strand substitution reaction. At the same time, the AP site is too far from the 3' end of the blocking strand, which will hinder further hybridization by blocking the complementary region.

[0078] To improve the reactivity of APE1 and obtain better analytical sensitivity, the applicant selected the 8th AP spot at the 3' end (i.e., SEQ ID NO.2) for subsequent experiments.

[0079] (2) Optimize the ratio and concentration of the reaction substrate and the reaction time.

[0080] ① Optimize the reaction substrate ratio: Select the blocking strand B with the optimal length and optimal AP site.

[0081] Double-stranded promoters were prepared by mixing promoter I and blocking chain B in different ratios (I:B), with the specific process as described above, and the ratios being 1.25:1, 1:1, 1:1.25, 1:1.5 and 1:2 respectively.

[0082] The above one-step method (i.e., the effect verification part) was used for testing, and the results are as follows: Figure 3 As shown in C.

[0083] Depend on Figure 3 As shown in C, the S / B ratio (signal-to-noise ratio) increases with the increase of the I / B ratio until I / B is 1:1.5. When I / B is greater than 1:1.5, the S / B ratio (signal-to-noise ratio) decreases with the increase of the I / B ratio. Therefore, in the subsequent experiment, the applicant chose an I / B ratio of 1:1.5.

[0084] ② Optimize the concentration of the reaction substrate

[0085] The optimal ratio of double-stranded promoters, i.e., I / B of 1:1.5, was selected to prepare double-stranded promoters of different concentrations, with substrate concentrations of 5 nM, 10 nM, 15 nM, 20 nM and 30 nM.

[0086] The above one-step method (i.e., the effect verification part) was used for testing, and the results are as follows: Figure 4 As shown in D.

[0087] Depend on Figure 3 As shown in D, the S / B ratio increases from I / B concentration to 20 nM, and decreases from I / B concentration to 30 nM. Therefore, the optimal I / B concentration is chosen to be 20 nM.

[0088] ③ Optimize reaction time

[0089] The optimal ratio and concentration of the double-stranded promoter and reaction substrate were selected for the reaction at 0.5h, 1h, 2h, 3h and 4h.

[0090] The above one-step method (i.e., the effect verification part) was used for testing, and the results are as follows: Figure 3 As shown in E.

[0091] Depend on Figure 3E indicates that the optimal reaction time is 3 hours.

[0092] 4. Detection performance of EDN biosensors

[0093] The sensitivity of the proposed biosensor was evaluated by measuring different concentrations of APE1 under optimal conditions. The specific steps are as follows:

[0094] (1) Linear detection:

[0095] ① Dilute the original concentration of APE1 with bovine serum albumin (BSA) to prepare final concentrations of 0 and 5 × 10⁻⁶. -6 5×10 -5 5×10 -4 5×10 -3 5×10 -2 5×10 -1 Targets of 5, 10, 20, and 30 U / mL;

[0096] ② Select the optimal ratio (i.e., I:B = 1:1.5) and optimal concentration (20 nM) of the double-stranded promoter, and choose final concentrations of 0 and 5 × 10⁻⁶ respectively. -6 5×10 -5 5×10 -4 5×10 -3 5×10 -2 5×10 -1 APE1 at concentrations of 5, 10, 20, and 30 U / mL was mixed by vortexing and then reacted at 25°C for 3 hours.

[0097] ③ Measure fluorescence intensity. The parameter settings during fluorescence intensity measurement are the same as above. Based on the detection results, calculate the relationship between the logarithm of fluorescence intensity and APE1 concentration. The resulting regression equation is: F = 47.92lg(C × 10⁻⁶). 6 -10.43, linear regression correlation coefficient R 2 =0.9885, F is the fluorescence intensity at 520 nm wavelength, and C is the concentration of APE1 (e.g., Figure 4 (As shown in A).

[0098] As can be seen from the linear regression equation above, the fluorescence intensity gradually increases with the increase of APE1 concentration from 0 to 30 U / mL.

[0099] Based on the fluorescence intensity of the blank test plus three times the standard deviation, the detection limit was determined to be 6.92 × 10⁻⁶. -6 U / mL. Clearly, the biosensor of this application exhibits a lower detection limit and a more considerable linear detection range.

[0100] To verify the universality and specificity of this protocol, this application introduces several functionally similar nucleases found in potential real-world clinical samples for detection. The specific steps are as follows:

[0101] KF enzyme, Nb.BbvC1 enzyme, DpnI enzyme, FspI enzyme, T4 PNK enzyme, and UDG enzyme, respectively, were selected at a final concentration of 2 U / mL to replace APE1 (final concentration of 0.25 U / mL) for one-step detection. Fluorescence intensity was measured, and the parameters were set as above during the fluorescence intensity measurement. The results are as follows. Figure 4 As shown in B.

[0102] Depend on Figure 4 As shown in Figure B, the highest fluorescence intensity was obtained in the presence of APE1. Conversely, there was a weak signal in the non-specific group, indicating that there was almost no interference or crosstalk in the non-specific group. This result demonstrates that the biological detector of this application has fairly high specificity for APE1.

[0103] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. A composition for a biosensor for detecting APE1, characterized in that, The composition includes a double-stranded promoter, an H1 hairpin, an H2 hairpin, and an FQ reporter molecule, wherein the double-stranded promoter includes promoter I and a blocking strand B, and the FQ reporter molecule includes a RepF strand and a RepQ strand; The nucleotide sequence of promoter I is shown in SEQ ID NO.1; The nucleotide sequence of the blocking chain B is shown in SEQ ID NO.2; The nucleotide sequence of the RepF chain is shown in SEQ ID NO.3; The nucleotide sequence of the RepQ chain is shown in SEQ ID NO.4; The nucleotide sequence of the H1 hairpin is shown in SEQ ID NO.5; The nucleotide sequence of the H2 hairpin is shown in SEQ ID NO.

6.

2. The use of the composition according to claim 1 in the working solution for preparing a biosensor or in a biosensor.

3. The working fluid of a biosensor, characterized in that, Includes the composition as described in claim 1 and acceptable excipients or additives.

4. The application of the working solution of the biosensor as described in claim 3 in the preparation of products for detecting APE1.

5. A product for detecting APE1, characterized in that, Includes the working fluid of the biosensor as described in claim 4.

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