Preparation method of glove type flexible aptamer sensor based on DNA hydrogel

Abstract

The invention discloses a preparation method of a glove type flexible aptamer sensor based on DNA hydrogel, and relates to a preparation method of a novel wearable biosensor. The aptamer sensor for rapidly detecting organophosphorus pesticide residues in vegetables is constructed by utilizing the specific response of DNA aptamer hydrogel to organophosphorus pesticides and the flexible integration of a screen-printed electrode. The specific preparation process is as follows: a DNA hydrogel embedded with methylene blue is prepared by a rolling ring amplification method, a flexible three-electrode system is constructed on a butyronitrile glove by using a screen printing technology, and an electrochemical detection mechanism that organophosphorus pesticide and an aptamer are combined to trigger the hydrogel to disintegrate and release methylene blue is realized. The aptamer sensor disclosed by the invention is simple in preparation method, high in detection speed, good in stability and high in selectivity, and can be used for high-sensitivity detection of residual organophosphorus pesticides in vegetables.

Description

TECHNICAL FIELD

[0001] The application relates to a glove-type flexible aptamer sensor preparation method based on a DNA hydrogel, and belongs to the field of electrochemical biosensors. TECHNICAL BACKGROUND

[0002] Organophosphorus pesticides are organic compounds containing phosphorus elements, which are widely used in agriculture for insect killing, accounting for about 40% of commercial pesticides. Common examples include malathion, phorate, isocarbophos and omethoate. In China, organophosphorus pesticides have replaced organochlorine pesticides and become the dominant pesticide. However, organophosphorus pesticides have adverse effects on human health, can irreversibly inhibit acetylcholinesterase, destroy nerve signals, cause muscle spasms, pupil contraction and respiratory distress, and can even cause seizures or death in severe cases. Therefore, the residues of organophosphorus pesticides in agricultural products and the environment are key issues in food safety and ecological protection research.

[0003] Rolling circle amplification is an advanced isothermal nucleic acid amplification technique that has been selected for its unique advantages. It allows efficient amplification at constant temperature without the need for complex thermal cycling equipment, thereby reducing experimental costs and technical barriers. In addition, rolling circle amplification provides strong signal enhancement for the detection of low concentrations of targets. Rolling circle amplification uses circular DNA templates and specific polymerases, as well as short DNA or RNA primers, to produce long single-stranded DNA or RNA products under isothermal conditions. These products contain hundreds to thousands of repeated units. This mechanism rapidly generates numerous repeated units, significantly enhancing signal strength. In recent years, the combination of rolling circle amplification with electrochemical sensors has enabled ultra-sensitive detection through a dual amplification strategy, enhancing system signal response. In addition, the combination of rolling circle amplification with hydrogels can create programmable responsive materials for biosensing, improving synthesis efficiency and tunable targeted release characteristics. The simplicity, mild reaction conditions and adaptability of rolling circle amplification make it suitable for laboratory and field use, providing high specificity and flexibility. Its applications include medical diagnosis, biosensor development and food safety monitoring, making rolling circle amplification an important tool for nucleic acid analysis and signal amplification.

[0004] Nucleic acid aptamers are single-stranded DNA or RNA molecules that fold into specific three-dimensional structures, capable of binding targets with high affinity and specificity. They offer a number of advantages, including rapid screening, simple synthesis, consistent quality and economic feasibility. Aptamers are the core of DNA hydrogel design. By embedding aptamers and signal markers, these hydrogels can achieve specific responses to targets, the presence of which triggers competitive binding with aptamers, disrupting the hydrogel and releasing catalytic color-developing or conductive substances for signal-based detection.

[0005] Screen-printing technology was chosen for the fabrication of screen-printed electrodes to enable controlled construction and patterning of electrodes on flexible or irregular substrates. Screen-printed electrodes are low-cost, simple to use, and do not require complex equipment. They are compatible with a wide range of conductive materials, providing extensive material flexibility. In addition, screen-printed electrodes support the patterning of prints on diverse substrates such as plastics, paper, and fabrics, meeting the needs of a wide range of applications. By customizing the printing stencil, the shape, size, and layout of the electrode can be customized, enhancing the design of miniaturized and integrated electrochemical sensors. After decades of improvement, screen-printed electrodes have become a cornerstone of electrochemical sensor technology. They are widely used in environmental monitoring, biological analysis, and interdisciplinary research. Continued advances in materials science and printing technology are expected to further enhance the performance of screen-printed electrodes and expand their scope. SUMMARY

[0006] The present application integrates pesticide-specific aptamer DNA hydrogel with screen-printed electrodes to achieve rapid detection of organophosphorus pesticides in vegetables. A broad-spectrum organophosphorus aptamer is used as a rolling circle amplification primer to synthesize a methylene blue-embedded DNA hydrogel. A flexible nitrile glove is used as the substrate, and a screen-printed three-electrode interface with high ductility and low resistivity is prepared. This study elucidates the hydrogel-pesticide response mechanism and the detection mechanism of the flexible aptamer sensor, and establishes a sensor construction and detection protocol to achieve rapid and reliable analysis of organophosphorus residues in vegetables.

[0007] The technical solution of the present application is to integrate DNA hydrogel and screen-printed electrodes on a glove substrate through layer-by-layer assembly technology. The specific preparation process is as follows: (1) preparing DNA hydrogel; (2) constructing a screen-printed electrode on a glove substrate; (3) assembling the hydrogel and electrode system; (4) optimizing the detection conditions; (5) completing the preparation of the aptamer sensor; (6) detecting organophosphorus pesticides.

[0008] Preferably, the glove substrate is a flexible nitrile glove, and the three-electrode system is prepared by screen-printing technology, including mixing of carbon paste and silver paste, using a 10% silver-carbon paste mixture, and curing at 120°C for 30 min to achieve high ductility and low resistivity.

[0009] Preferably, the DNA hydrogel preparation method is as follows: mix 50 μL of 50 μM primer with 50 μL of 50 μM linear DNA, heat to 95°C for 5 min, cool to room temperature, add 50 μL of 500 U / μL T4 DNA ligase, incubate at 25°C for 16 h to form circular DNA; then take 20 μL of circular DNA, add 6.5 μL of 13 U / μL phi29 DNA polymerase, 2.5 μL of dNTPs, 21 μL of TE buffer, and 1 μL of 15 mM methylene blue, and react at 37°C for 24 h to prepare methylene blue-embedded DNA hydrogel.

[0010] Preferably, the detection mechanism is that the hydrogel is placed on the thumb, the organophosphorus pesticide binds with the aptamer to cause the hydrogel to disintegrate, and methylene blue is released; the silk screen printed electrode on the index finger is in contact with the thumb by gently pressing, and differential pulse voltammetry electrochemical analysis is performed.

[0011] Preferably, the aptamer is a broad-spectrum organophosphorus pesticide aptamer, which can recognize phorate, isocarbophos and omethoate as a rolling circle amplification primer, the amount of the hydrogel is 10 μL, and the reaction time is 15 min.

[0012] Compared with the prior art, the present application has the beneficial effects that the present application innovatively combines silk screen printing and rolling circle amplification technology, fully utilizes the stimulus response characteristics of DNA hydrogel and the wearable nature of flexible gloves, enhances signal amplification and on-site detection capability, and improves the detection sensitivity and practicability of the aptamer sensor for organophosphorus pesticide residues in vegetables. BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1 Preparation process of the sensor.

[0014] Figure 2 Material characterization (SEM, Mapping, AFM images).

[0015] Figure 3 Optimization conditions of the hydrogel and the electrode.

[0016] Figure 4 DPV curve and standard curve of the sensor for three kinds of organophosphorus pesticides.

[0017] Figure 5 Actual sample spiking recovery rate graph.

[0018] Figure 6 Repeatability, stability and specificity of the sensor. DETAILED DESCRIPTION

[0019] The present application will be further described in detail below in combination with the drawings and examples, but the examples do not limit the present application in any form.

[0020] Example 1: Preparation of the aptamer sensor Figure 1 is the preparation process of the sensor, first, a three-electrode system (working electrode, reference electrode, counter electrode) is constructed on a butyronitrile glove substrate using silk screen printing technology, and a mixture of carbon paste and silver paste is used, and after curing, it is dried. Next, the DNA hydrogel is prepared by rolling circle amplification method: mixing primers and linear DNA, incubating with ligase to form circular DNA, and embedding methylene blue in polymerase reaction. Subsequently, the hydrogel is placed on the surface of the thumb electrode.

[0021] Example 2: Characterization of materials (1) SEM and Mapping representation The surface morphology of the screen-printed electrode and the DNA hydrogel was characterized using scanning electron microscopy. The screen-printed electrode featured tightly packed nanoparticles and a uniformly distributed carbon-silver pattern. Figure 2 AB), the hydrogel has a porous structure ( Figure 2 DF). (2) AFM characterization Atomic force microscopy characterization demonstrated that the electrode surface had low roughness (Ra = 0.128 μm), reflecting a smooth and defect-free surface at the microscale. Figure 2 C).

[0022] Example 3: Condition Optimization of Aptamer Sensors Methylene blue concentration, PHI29 polymerase concentration, rolling circle amplification reaction time, electrode morphology, materials, screen mesh count, pH value, hydrogel dosage, and reaction time are key factors affecting sensor performance. For example... Figure 3 As shown in Figure A, the electrochemical signal intensity increases with increasing methylene blue concentration. The signal intensity reaches its peak when the methylene blue concentration is at a moderate value, and then begins to decrease with further increases in concentration. Therefore, a moderate methylene blue concentration was chosen for subsequent experiments. The effect of phi29 polymerase concentration on the aptamer sensor signal intensity is shown in Figure A. Figure 3 B. The electrochemical signal intensity gradually increases with increasing polymerase concentration, but the change becomes less pronounced after 13 U / μL, indicating that approximately 13 U / μL is required for complete polymerase-substrate reaction. The rolling circle amplification reaction time is also a key factor affecting the electrochemical signal intensity. Figure 3 As shown in Figure C, the optimal response was obtained at a reaction time of 24 h. During hydrogel formation, reaction times were tested between 6, 12, 18, 24, and 30 h. The absorbance peaks were lowest at 24 and 30 h, indicating minimal methylene blue release and optimal hydrogel formation. However, since 24 h was more efficient than 30 h, 24 h was chosen as the optimal duration. The combination of 13 U / μL polymerase with a 24-h gelation time produced the lowest absorbance and lightest solution color, confirming complete encapsulation of methylene blue and optimal hydrogel formation. The effect of electrode morphology on sensor performance is as follows: Figure 3 D. The electrochemical signal intensity significantly improves with the optimization of electrode morphology (such as a wavy design). The signal intensity reaches its maximum when a wavy morphology is used, followed by lower signal intensities with other morphologies. Therefore, a wavy electrode morphology was selected for further experiments. The influence of electrode materials is as follows: Figure 3E. The electrochemical signal intensity varies with the material ratio (e.g., 10% silver-carbon paste mixture). The signal intensity is highest when the material is a 10% silver-carbon paste mixture, indicating that this ratio provides optimal conductivity and ductility. The effect of screen-printed mesh size on sensor performance is as follows: Figure 3 F, the signal strength increases as the grid size decreases. An optimal balance is achieved when the grid size is moderate, avoiding the drawbacks of being too large or too small; therefore, this grid size is selected. The effect of pH value on the signal strength of the aptamer sensor is as follows: Figure 3 G. The electrochemical signal intensity changes with increasing pH, reaching its maximum at pH 7.4. Thereafter, the signal intensity decreases with further increases or decreases in pH; therefore, pH 7.4 was chosen as the optimal value. The amount of hydrogel used is a key component of the detection system, and its dosage significantly affects the electrochemical signal intensity. As the amount of hydrogel increases, the signal intensity increases significantly. Figure 3 When the amount of hydrogel (H) is increased to 10 μL, the signal intensity reaches its maximum and then remains constant; therefore, a hydrogel amount of 10 μL is chosen. The effect of reaction time on sensor performance is as follows: Figure 3 The signal intensity gradually increases with increasing reaction time, but the change becomes less pronounced after 15 minutes, indicating that complete binding of the organophosphorus pesticide to the aptamer requires approximately 15 minutes. From 5 to 15 minutes, the electrical signal steadily increases, representing sustained target-hydrogel interaction and methylene blue release. At 15 minutes, the signal reaches 15 μA, accounting for 93.75% of the maximum signal (16 μA), reflecting high reaction efficiency. Extending the reaction time to 20, 25, or 30 minutes only increases the signal by 6.7%, while the time cost increases by 33.3% to 100%, reducing efficiency. Therefore, a duration of 15 minutes optimally balances responsiveness and time efficiency.

[0023] Example 4: Detection of organophosphorus pesticides using aptamer sensors Under optimal experimental conditions, the prepared sensor was used to detect three organophosphorus pesticides: phorate, methamidophos, and dimethoate. Figure 4 AC represents the relationship between the concentrations of three pesticides and the intensity of the differential pulse voltammetric signal. Figure 4 A: Phosphorus phosphate; Figure 4 B: Ammonium thion; Figure 4 C: Omethoate), with increasing pesticide concentration, the electrochemical signal intensity gradually increases. Figure 4 DF display ( Figure 4 D: Phosphorus phosphate; Figure 4 E: Ammonium thion; Figure 4F: oxydemeton-methyl), the electrochemical signal intensity showed a good linear relationship with the logarithm of the pesticide concentration in a certain concentration range, and had a low detection limit (S / N = 3) of about 6.8 x 10 -4 ng / mL, and the linear range was 0.001 ng / mL to 100 ng / mL. The sensor platform was used to detect organophosphorus pesticides in vegetables, including unknown concentration samples and spiked sample tests, and was analyzed in parallel with liquid chromatography as a reference method. The sensor and liquid chromatography showed excellent performance in detecting pesticide residues in spinach, celery and eggplant. For unspiked samples, both methods detected no residues (0 ng / mL), confirming that there were no organophosphorus pesticides in the purchased vegetables. For spiked samples, the sensor recovery rate ranged from 96.80% to 102.37%, indicating high accuracy, and the relative standard deviation ranged from 2.08% to 4.14%. The LC-MS recovery rate ranged from 96.43% to 101.49%, and the relative standard deviation was 1.45% to 2.72%, showing strong consistency between the two methods, verifying the reliability of the sensor. These results confirm the reliability of the sensor in complex matrices, making it suitable for food safety monitoring Figure 5 ).

[0024] Example 5: Performance analysis of aptamer sensor (1) Reproducibility test The results of the sensor reproducibility test are shown in Figure 6 A, five sensors were prepared under the same conditions to detect the same batch of RCA hydrogel, and five identical detection experiments were performed under constant conditions. The results showed that the signal intensities of the five experiments were highly consistent, with a relative standard deviation of 3.48%, indicating low variability and excellent reproducibility, thus confirming its suitability for batch applications. (2) Stability test Figure 6 B is the stability test results of the sensor. Multiple sensors were prepared under the same conditions, and a concentration of 10 ng / mL of organophosphorus pesticides was immediately incubated on the surface of some of the sensors, and their electrochemical signal intensity was detected. The remaining sensors were stored in a 4°C refrigerator, and every 5 days some of the sensors were taken out for detection. It was found that on the 1st, 5th, 10th, 15th and 20th day, the hydrogel appearance was basically unchanged, and on the 20th day, the hydrogel showed consistent signal output intensity, indicating that even after prolonged storage, it had robust detection performance. (3) Specificity test Four pesticides, deltamethrin, imidacloprid, glyphosate and chlorothalonil, were selected to analyze the specificity of the sensor, as shown in Figure 6As shown in Fig. C, at the same concentration, the four non-target pesticides, either in single system or mixed system, cannot effectively trigger signal change, and the signal intensity is very low, while once there is an organophosphorus pesticide component in the system, the electrochemical signal intensity will be significantly enhanced (for example, the signals of phorate, isocarbophos and oximepoxide are much higher than the control group), confirming the specificity of the platform.

Claims

1. A method for preparing a DNA hydrogel-based glove-type flexible aptasensor, characterized by, DNA hydrogel embedded with methylene blue was prepared by rolling circle amplification method, and the release of methylene blue signal was realized by the response mechanism of hydrogel to organophosphorus pesticides. A three-electrode system was constructed on a flexible glove substrate by screen printing technology to realize efficient electrochemical detection. The specific preparation process is as follows: (1) Preparation of DNA hydrogel; (2) Construction of screen-printed electrode on glove substrate; (3) Assembly of hydrogel and electrode system; (4) Optimization of detection conditions; (5) Preparation of aptamer sensor is completed.

2. The method for fabricating a glove-shaped flexible aptamer sensor based on DNA hydrogel according to claim 1, characterized in that, The glove substrate is a flexible nitrile glove, and the three-electrode system is prepared by screen printing technology, including mixing of carbon paste and silver paste. A 10% silver-carbon paste mixture is used, and the mixture is cured at 120°C for 30 min to achieve high ductility and low resistivity.

3. The method for preparing a glove-shaped flexible aptamer sensor based on DNA hydrogel according to claim 1, characterized in that, DNA hydrogel preparation method: mix 50 μL of 50 μM primer with 50 μL of 50 μM linear DNA, heat to 95°C for 5 min, cool to room temperature, add 50 μL of 500 U / μL T4 DNA ligase, incubate at 25°C for 16 h to form circular DNA; then take 20 μL of circular DNA, add 6.5 μL of 13 U / μL phi29 DNA polymerase, 2.5 μL of dNTPs, 21 μL of TE buffer and 1 μL of 15 mM methylene blue, and react at 37°C for 24 h to prepare DNA hydrogel embedded with methylene blue.

4. The method of claim 1, wherein the DNA hydrogel-based glove-type flexible aptasensor is prepared by the steps of: The detection method is to place the hydrogel on the thumb, and the organophosphorus pesticide binds with the aptamer to cause the hydrogel to disintegrate and release methylene blue; the screen-printed electrode on the index finger is gently pressed into contact with the thumb, and differential pulse voltammetry electrochemical analysis is performed.

5. The method for fabricating a glove-shaped flexible aptamer sensor based on DNA hydrogel according to claim 1, characterized in that, The aptamer is a broad-spectrum organophosphorus aptamer, which can recognize phorate, isocarbophos and oxon.

Images