An electrochemical DNA biosensor for detecting HPV-16

Abstract

The application provides an electrochemical DNA biosensor for detecting human papilloma virus 16 subtype (HPV-16), which utilizes base complementary pairing to specifically combine different single-stranded DNAs, and when HPV-16 is added, a DNA strand displacement reaction can be triggered to increase the electrochemical impedance (EIS) response. Within a certain range, as the concentration of HPV-16 increases, the EIS response signal also increases. The electrochemical DNA biosensor provided by the application has the advantages of simple preparation process, convenient operation, low detection limit of 33.8 pM and fast detection speed.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological analysis detection, and particularly relates to an electrochemical DNA biosensor for detecting human papillomavirus 16 subtype (HPV-16). BACKGROUND

[0002] Cervical cancer is the second most common malignant tumor in women, and its main pathogenic cause is persistent infection of high-risk human papillomavirus (HPV). The high-risk HPV mainly includes 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73, 82 and other subtypes. Among them, the carcinogenicity of high-risk HPV-16 subtype is much higher than that of other types in the occurrence of cervical cancer. Therefore, sensitive detection of HPV-16 is crucial for early discovery, early diagnosis and early treatment of cervical cancer. The recognized HPV-16 detection method is hybrid capture II generation technology, but the biggest disadvantage of this technology is that it cannot be specifically typed. Polymerase chain reaction (PCR) has high sensitivity and is the main strategy for HPV gene detection and typing at present, but it is not conducive to popularization and application due to its high requirement for experimental conditions. The later developed microarray technology greatly depends on precision equipment and is not suitable for on-site rapid diagnosis and detection. Therefore, it is necessary to develop a sensitive, simple and economical method for detecting HPV-16. SUMMARY

[0003] In order to solve the problems of the prior art, the present application provides an electrochemical DNA biosensor for detecting HPV-16, which is simple to operate and can sensitively detect HPV-16.

[0004] To achieve the above purpose, the present application adopts the following technical solutions:

[0005] The first object of the present application is to provide a preparation method of an electrochemical DNA biosensor, as follows:

[0006] An electrochemical DNA biosensor for detecting HPV-16, characterized in that a glassy carbon electrode is used as a base electrode, various modifiers are sequentially modified on the surface of the glassy carbon electrode, including nano-gold (Au), deoxyribonucleic acid 1 (DNA1) with a mercapto group, 6-mercaptohexanol (MCH), and silver nanoclusters (AgNCs) synthesized with deoxyribonucleic acid 2 (DNA2) as a template. Further, the preparation steps of the electrochemical DNA biosensor are as follows:

[0007] (1) taking glassy carbon electrode (GCE) as a working electrode, saturated calomel electrode as a reference electrode, platinum electrode as a counter electrode, and then inserting the three-electrode system into a chloroauric acid solution (HAuCl4) with a concentration of 3 mM, and adopting constant potential polarization method to deposit nano gold (Au) on the surface of the glassy carbon electrode, so that a modified glassy carbon electrode GCE / Au is obtained;

[0008] (2) 8 μL of DNA1 solution is dropped on the surface of the GCE / Au, and after reaction for 12 hours at room temperature in dark, the excessive DNA1 is washed away by ultrapure water, so that a modified electrode GCE / Au / DNA1 is obtained;

[0009] (3) 8 μL of 6-mercaptohexanol (MCH) is dropped on the surface of the GCE / Au / DNA1 electrode, and after reaction for 30 minutes, the excessive MCH is washed away by ultrapure water after non-specific sites are blocked, so that a modified electrode GCE / Au / DNA1 / MCH is obtained;

[0010] (4) 10 μL of DNA2 is taken in a centrifuge tube, and 10 μL of 3-(N-morpholine)-propanesulfonic acid (10 mM, PH = 7.8), 2.5 μL of silver nitrate solution are added in the centrifuge tube, and then mixed uniformly, and after reaction for 30 minutes, 2.5 μL of sodium borohydride solution is added, and after reaction for 30 minutes, silver nanoclusters synthesized by taking DNA2 as a template, namely DNA2-AgNCs, are obtained;

[0011] (5) 8 μL of the DNA2-AgNCs solution obtained in step (4) is dropped on the surface of the GCE / Au / DNA1 / MCH electrode obtained in step (3), since DNA1 and DNA2 can occur hybridization reaction, DNA2-AgNCs can be combined with DNA1 together, and after 1 hour, the surface of the electrode is washed by ultrapure water, so that a modified electrode GCE / Au / DNA1 / MCH / DNA2-AgNCs is obtained.

[0012] Further, in step (1), the parameter setting of the constant potential polarization method is as follows: polarization potential: -0.2 V, and polarization time: 500 s. Further, in step (2), the sequence of the DNA1 is as follows: 5'-GCAAAGGCATTTTTTGCAATGTTAGCA-(CH2)6-SH-3'.

[0013] Further, in step (4), the sequence of the DNA2 is as follows: 5'-ACATTGCCCCCCCTGCCT-3'.

[0014] The second object of the present application is to provide an electrochemical DNA biosensor prepared by the above method.

[0015] The third object of the present application is to provide an electrochemical DNA biosensor for detecting human papillomavirus 16 subtype (HPV-16), and the detection procedure is as follows:

[0016] (I) 8 μL of HPV-16 solution with different concentrations is added dropwise to the surface of the GCE / Au / DNA1 / MCH / DNA2-AgNCs electrode, and the reaction is carried out for 20 min, and then the electrode surface is washed with ultrapure water;

[0017] (II) the glassy carbon electrode treated in step (I) is used as a working electrode, a mercury-tellurium electrode is used as a reference electrode, and a platinum electrode is used as a counter electrode to form a three-electrode system inserted into a potassium ferricyanide / potassium ferrocyanide solution with a concentration of 5 mM, and then electrochemical detection is carried out by using an electrochemical impedance method (EIS), and the concentration of HPV-16 is detected by measuring the impedance value.

[0018] Further, in step (I), the HPV-16 sequence is 5'-TGCTAACATTGCTGCCTTTGC-3'.

[0019] Further, in step (II), the frequency range of the electrochemical impedance method is 0.1-10 5 Hz.

[0020] The principle of detecting HPV-16 in the present application is as shown in the accompanying drawings. Figure 1 As shown in the accompanying drawings, the electrochemical DNA biosensor of the present application first connects the DNA1 with a mercapto group to the glassy carbon electrode modified with nano-gold through a gold-sulfur bond, then seals with MCH, and then synthesizes silver nanoclusters (DNA2-AgNCs) by using DNA2 as a template. Since DNA1 and DNA2 are base complementary pairs, DNA1 and DNA2-AgNCs are specifically combined, and finally, when HPV-16 is added, since the number of base complementary pairs between HPV-16 and DNA1 is more than that between DNA1 and DNA2-AgNCs, a DNA strand displacement reaction is triggered to displace DNA2-AgNCs. As shown in the accompanying drawings, when DNA2-AgNCs is combined on DNA1, since silver nanoclusters can promote electron transfer, the electrochemical impedance (EIS) response is obviously reduced, and when HPV-16 displaces DNA2-AgNCs, the EIS response signal is increased. Therefore, the change of the EIS signal is used to detect HPV-16. Figure 2

[0021] The electrochemical DNA biosensor of the present application has a simple preparation process, is easy to operate, has a low detection limit, and can be used for detecting HPV-16 genes. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 ​The principle diagram of the electrochemical DNA biosensor for detecting HPV-16 in the embodiment of the present application;

[0023] Figure 2 The electrochemical characterization diagram of the construction process of the electrochemical DNA biosensor in the embodiment of the present application, and the inserted partial enlarged view is shown in the figure;

[0024] Figure 3 The feasibility of the electrochemical DNA biosensor for detecting HPV-16 in the present application;

[0025] Figure 4 The EIS response diagram of the electrochemical DNA biosensor for detecting HPV-16 in the present application;

[0026] Figure 5 The standard curve diagram of the electrochemical DNA biosensor for detecting HPV-16 in the present application;

[0027] Figure 6 The equivalent circuit model corresponding to EIS. DETAILED DESCRIPTION

[0028] In order to further describe the technical means adopted by the present application and its performance, the present application is further described below in combination with the embodiments and the drawings. The specific embodiments described herein are only used to explain the present application, and not to limit the present application.

[0029] Embodiment 1

[0030] The present embodiment provides a preparation method of an electrochemical DNA biosensor, which specifically comprises the following steps:

[0031] 1. Pretreatment of the glassy carbon electrode: a little of Al2O3 (particle size of 0.05 μm) suspension liquid is dropped on the chamois leather, so that the surface of the glassy carbon electrode contacts with the suspension liquid and is perpendicular to the chamois leather, and then the chamois leather is moved in the shape of "8" to polish and grind, after 2 minutes of polishing and grinding, the electrode surface is washed with ultrapure water, and then the polished glassy carbon electrode is placed in a beaker containing ultrapure water, the beaker is placed in an ultrasonic water bath instrument, and ultrasonic cleaning is performed for 1 minute, and then ultrapure water is used for washing, so as to ensure that the electrode surface is free of any dirt;

[0032] 2. The pretreated glassy carbon electrode (GCE) is used as a working electrode, a saturated calomel electrode is used as a reference electrode, and a platinum electrode is used as a counter electrode to form a three-electrode system, which is inserted into a chloroauric acid solution (HAuCl4) with a concentration of 3 mM, and a constant potential polarization method is used to deposit nano-gold (Au) on the surface of the glassy carbon electrode, so as to obtain a modified glassy carbon electrode GCE / Au;

[0033] 3. Take 8 μL of DNA1 solution (sequence: 5'-GCAAAGGCATTTTTTGCAATGTTAGCA-(CH2)6-SH-3') and add it to the GCE / Au surface. After reacting at room temperature in the dark for 12 h, wash away the excess DNA1 with ultrapure water to obtain the modified electrode as GCE / Au / DNA1.

[0034] 4. Take 8 μL of 6-mercaptohexanol (MCH) and add it to the surface of GCE / Au / DNA1 electrode. After reacting for 30 min and blocking non-specific sites, wash away excess MCH with ultrapure water to obtain the modified electrode GCE / Au / DNA1 / MCH.

[0035] 5. Take 10 μL of DNA2 (sequence: 5'-ACATTGCCCCCCCTGCCT-3') into a centrifuge tube, add 10 μL of 3-(N-morpholino)-propanesulfonic acid (10 mM, pH=7.8) and 2.5 μL of silver nitrate solution to the centrifuge tube, mix well, react for 30 min, then add 2.5 μL of sodium borohydride solution, react for 30 min, to obtain silver nanoclusters synthesized with DNA2 as template, namely DNA2-AgNCs;

[0036] 6. Take 8 μL of the DNA2-AgNCs solution obtained in step 5 and drop it onto the surface of the GCE / Au / DNA1 / MCH electrode obtained in step 4. Since DNA1 and DNA2 can undergo hybridization, DNA2-AgNCs can bind to DNA1. After 1 hour, rinse the electrode surface with ultrapure water to obtain the modified electrode as GCE / Au / DNA1 / MCH / DNA2-AgNCs.

[0037] Example 2

[0038] In this embodiment, to verify the success of the electrochemical DNA biosensor construction, electrochemical impedance spectroscopy was used to electrochemically characterize the construction process of the electrochemical DNA biosensor. For example... Figure 2 As shown, the EIS response curves for each preparation step are displayed. Letters a to f in the figure represent: GCE, GCE / Au, GCE / Au / DNA1, GCE / Au / DNA1 / MCH, GCE / Au / DNA1 / MCH / DNA2-AgNCs, and GCE / Au / DNA1 / MCH / DNA2-AgNCs / HPV-16, respectively. As shown, the semicircle diameter is equal to the charge transfer resistance (Rct). In ZView software, the equivalent circuit diagram (e.g., ...) is used... Figure 6As shown in the figure, fitting the measured curves revealed that the Rct for unmodified GCE was approximately 44.7 Ω (curve a). The GCE / Au ratio was almost a straight line (curve b), indicating that Au exhibits good conductivity and thus promotes electron transfer. With DNA1 (curve c) and MCH (curve d) immobilized on the electrode, the Rct values ​​gradually increased to 1013 Ω and 1664 Ω, respectively. With DNA2-AgNCs (curve e) immobilized on the electrode, the Rct was 406.2 Ω, indicating that AgNCs also promote electron transfer, significantly decreasing the Rct. Finally, when the constructed electrochemical DNA biosensor was used to detect HPV-16 (curve f), the Rct was 1234 Ω, indicating that HPV-16 successfully replaced DNA2-AgNCs, increasing the Rct.

[0039] Example 3

[0040] In this embodiment, to verify the feasibility of the electrochemical DNA biosensor constructed in Example 1 for detecting HPV-16, 8 μL of HPV-16 solution and 8 μL of HPV-16-free solution were respectively added to the surface of the modified GCE / Au / DNA1 / MCH / DNA2-AgNCs electrode. After reacting for 20 min, a three-electrode system was constructed using the above electrode as the working electrode, a saturated calomel electrode as the reference electrode, and a platinum electrode as the counter electrode. This system was then inserted into a 5 mM potassium ferricyanide / potassium ferrocyanide solution. Electrochemical detection was performed using electrochemical impedance spectroscopy (EIS) at a frequency range of 0.1–10. 5 Hz, such as Figure 3 As shown. Similarly, in ZView software, the equivalent circuit diagram (such as...) is used... Figure 6 As shown in the figure, the measured curve was fitted, and it can be seen that the Rct (1234Ω) in the presence of HPV-16 is significantly higher than that in the absence of HPV-16 (434.2Ω). Therefore, this electrochemical DNA biosensor can be used to detect HPV-16.

[0041] Example 4

[0042] In this embodiment, an electrochemical DNA biosensor was used to detect different concentrations of HPV-16, specifically electrochemical impedance spectroscopy (EIS) was employed for electrochemical detection, with a frequency range of 0.1–10. 5 Hz, to obtain the EIS signal response at different concentrations. For example Figure 4 As shown, the concentrations of HPV-16 from a to e are 1.0 × 10⁻⁶. -10 M, 1.0×10 -9 M, 1.0×10 -8 M, 1.0×10 -7 M, 1.0×10 -6 M. ByFigure 4 It can be seen that the electrochemical response signal increases with the increase of the concentration of HPV-16. The measured curves were fitted in ZView software using equivalent circuit diagram (as shown in Figure 6 Figure 5 The error bars in the figure represent three parallel experiments), and the Rct value is linearly related to the logarithm of the concentration of HPV-16 in the range of 1.0 x 10 -10 ~ 1.0 x 10 -6 M, and the detection limit is determined according to the 3σ method (σ represents the standard deviation of the blank sample based on 10 repeated experiments). First, the blank response and its 3 times standard deviation are calculated. Then, it is fitted with the linear standard curve, and the estimated concentration value of the minimum detection limit of the method is 33.8 pM.

[0043] The technical scheme of the present application is not limited to the above specific embodiments, and any technical modification made according to the technical scheme of the present application falls within the protection scope of the present application.​

Claims

1. A method for preparing an electrochemical DNA biosensor for detecting HPV-16, characterized in that, The preparation method of this electrochemical DNA biosensor is as follows: (1) A glassy carbon electrode (GCE) was used as the working electrode, a saturated calomel electrode as the reference electrode, and a platinum electrode as the counter electrode to form a three-electrode system. The system was inserted into a chloroauric acid solution (HAuCl4) with a concentration of 3 mM. Using the constant potential polarization method, nano-gold (Au) was deposited on the surface of the glassy carbon electrode to obtain the modified glassy carbon electrode as GCE / Au. (2) Take 8 μL of DNA1 solution and add it to the surface of GCE / Au. After reacting at room temperature in the dark for 12 h, wash away the excess DNA1 with ultrapure water to obtain the modified electrode as GCE / Au / DNA1. (3) Take 8 μL of 6-mercaptohexanol (MCH) and add it to the surface of GCE / Au / DNA1 electrode. After reacting for 30 min, block the non-specific sites and wash away the excess MCH with ultrapure water to obtain the modified electrode GCE / Au / DNA1 / MCH. (4) Take 10 μL of DNA2 into a centrifuge tube, and add 10 μL of 3-(N-morpholino)-propanesulfonic acid (10 mM, pH=7.8) and 2.5 μL of silver nitrate solution to the centrifuge tube. Mix well and react for 30 min. Then add 2.5 μL of sodium borohydride solution and react for 30 min to obtain silver nanoclusters synthesized with DNA2 as template, namely DNA2-AgNCs. (5) Take 8 μL of the DNA2-AgNCs solution obtained in step (4) and drop it onto the surface of the GCE / Au / DNA1 / MCH electrode obtained in step (3). Since DNA2-AgNCs can hybridize with DNA1 through base complementary pairing, after reacting for 1 h, rinse the electrode surface with ultrapure water to obtain the modified electrode as GCE / Au / DNA1 / MCH / DNA2-AgNCs.

2. The method for preparing an electrochemical DNA biosensor for detecting HPV-16 according to claim 1, characterized in that, In step (1), the parameters of the constant potential polarization method are set as follows: polarization potential: -0.2V, polarization time: 500s.

3. The method for preparing an electrochemical DNA biosensor for detecting HPV-16 according to claim 1, characterized in that, In step (2), the DNA1 sequence is: 5,-GCAAAGGCATTTTTTGCAATGT TAGCA-(CH2)6-SH-3,.

4. The method for preparing an electrochemical DNA biosensor for detecting HPV-16 according to claim 1, characterized in that, In step (4), the DNA2 sequence is: 5,-ACATTGCCCCCCCTGCCT-3,.

5. An electrochemical DNA biosensor for detecting HPV-16, characterized in that, Prepared by the method described in claim 1.

6. An application of the electrochemical DNA biosensor for detecting HPV-16 as described in claim 5, characterized in that, The electrochemical DNA biosensor is used to detect human papillomavirus subtype 16 (HPV-16).

7. The application of the electrochemical DNA biosensor for detecting HPV-16 according to claim 6, characterized in that, The steps for testing for HPV-16 are as follows: (i) Take 8 μL of HPV-16 solution of different concentrations and drop it onto the surface of GCE / Au / DNA1 / MCH / DNA2-AgNCs electrode obtained in step (5) of the preparation method of electrochemical DNA biosensor as described in claim 2. React for 20 min and then rinse the electrode surface with ultrapure water. (II) Using the glassy carbon electrode processed in step (I) as the working electrode, the saturated calomel electrode as the reference electrode, and the platinum electrode as the counter electrode, a three-electrode system is formed and inserted into a 5 mM potassium ferrocyanide / potassium ferrocyanide solution. Electrochemical impedance spectroscopy (EIS) is then used for electrochemical detection, and the concentration of HPV-16 is detected by measuring the impedance value.

8. The application of the electrochemical DNA biosensor for detecting HPV-16 according to claim 7, characterized in that, In step (i), the HPV-16 sequence is: 5,-TGCTAACATTGCTGCCTTTGC-3,.

9. The application of the electrochemical DNA biosensor for detecting HPV-16 according to claim 7, characterized in that, In step (ii), the frequency range for the electrochemical impedance spectroscopy is 0.1–10 Hz. 5 Hz.

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