阅读说明：本技术 一种核酸适配体光波导传感器及应用其的检测方法 (Nucleic acid adapter optical waveguide sensor and detection method using same ) 是由 娄新徽 赵家兴 陆张伟 王朔 于 2020-03-16 设计创作，主要内容包括：本发明涉及一种具有靶标原位富集和纯化功能的核酸适配体光波导传感器及基于小分子靶标和与核酸适配体互补短链DNA与偶联在光纤表面的核酸适配体竞争性结合,实现对小分子靶标的定量检测的方法。通过将光波导传感器的二氧化硅纤维进行脱氧核糖核酸适配体和固相微萃取层修饰,实现核酸适配体与靶标的特异性结合与靶标在原位高效富集纯化同步进行。无需任何基于酶的信号放大反应,对多种小分子靶标实现具有超高灵敏度和超高特异性的快速检测。检出限非常低,有优越普适性。可用于复杂样本中靶标直接检测,仅需对液体样品进行稀释,无需样品前处理,检测灵敏度均满足食品和环境中对各靶标的限量标准。检测时间短,传感器再生速度快,可重复使用。(The invention relates to a nucleic acid aptamer optical waveguide sensor with target in-situ enrichment and purification functions and a method for realizing quantitative detection of a small molecular target based on competitive combination of the small molecular target, short-chain DNA complementary with the nucleic acid aptamer and the nucleic acid aptamer coupled on the surface of an optical fiber. The specific combination of the aptamer and the target and the in-situ efficient enrichment and purification of the target are synchronously performed by modifying the deoxyribonucleic acid aptamer and the solid-phase microextraction layer of the silicon dioxide fiber of the optical waveguide sensor. The method does not need any enzyme-based signal amplification reaction, and realizes the rapid detection with ultrahigh sensitivity and ultrahigh specificity on various small molecular targets. The detection limit is very low, and the method has excellent universality. The method can be used for directly detecting the targets in the complex samples, only a liquid sample needs to be diluted, sample pretreatment is not needed, and the detection sensitivity meets the limit standards of each target in food and environment. The detection time is short, the regeneration speed of the sensor is high, and the sensor can be repeatedly used.)

The aptamer optical waveguide sensor is characterized by being the aptamer optical waveguide sensor with target in-situ enrichment and purification functions.

The aptamer optical waveguide sensor of claim 1, wherein an extraction layer SPME with high target extraction efficiency and a target-specific aptamer are co-assembled on the fiber sensing interface.

The aptamer optical waveguide sensor of claim 2, wherein the extraction layer SPME is bare fiber or tween 80.

An aptamer optical waveguide sensor according to claim 2 or 3, wherein the target-specific aptamer is

NH ₂-(EG) ₁₈TGGGGGTTGAGGCTAAGCCGAGTCACTAT, or

NH ₂-(EG) ₁₈GAGGGCAACGAGTG TTTATAGA, or

NH ₂-(EG) ₁₈CTTTCTGTCCTTCCGTCACATCCCACGCATTCTCCACAT, or

NH ₂AAAAAAAAAATAGCTTAACTAGTGTTCAAGCTG, the target-specific aptamer is attached to the surface of the optical fiber.

The method for detecting the small molecules is characterized in that the quantitative detection of the small molecule targets is realized based on the competitive combination of the small molecule targets, short-chain DNA complementary with nucleic acid aptamers and the nucleic acid aptamers coupled on the surface of an optical fiber.

The method of claim 5, wherein the SPME on the surface of the optical fiber efficiently concentrates the small molecules in solution near the surface of the optical fiber, facilitating binding between the aptamer and the small molecule coupled to the surface of the optical fiber.

The method of claim 6, comprising the steps of: step 1, hydroxylation of the surface of an optical fiber; step 2, silanization of the surface of the optical fiber; step 3, coupling the aptamer on the surface of the optical fiber; and step 4, reduction and sealing.

The method of claim 7, wherein step 1 hydroxylating the surface of the optical fiber is as follows: firstly, the optical fiber with a clean surface is treated by the following steps of mixing the optical fiber with concentrated sulfuric acid with the volume ratio of 3: 1: soaking the optical fiber in a 30% hydrogen peroxide mixed solution at the temperature of 100-120 ℃ for 1 hour, then taking out the optical fiber from the mixed solution, washing the optical fiber to be neutral by using ultrapure water, drying the optical fiber by using nitrogen, placing the optical fiber in a drying oven at the temperature of 70-90 ℃ for 4-6 hours, taking out the optical fiber, and cooling the optical fiber to room temperature in a dryer.

The method of claim 8, wherein step 2 silanization of the surface of the optical fiber is as follows: putting the optical fiber into an anhydrous toluene solution of 3-aminopropyltriethoxysilane, soaking and reacting for 1-2 hours at room temperature, taking out, respectively washing with anhydrous toluene, toluene-ethanol with a v/v ratio of 1:1, and ethanol for three times, drying by nitrogen, placing in a 180 ℃ oven for 1 hour, taking out, and cooling to room temperature in a dryer.

The method according to claim 9, wherein step 3 coupling of the aptamer to the surface of the optical fiber: placing the silanized optical fiber into 10 millimole per liter of phosphate buffer solution containing glutaraldehyde, and reacting for 4 hours at room temperature; after the reaction is finished, the fiber is washed with ultrapure water for three times, nitrogen is blown to dry, the fiber is put into the solution of the aptamer modified by the amino group, the soaking reaction is carried out for 6 to 8 hours at room temperature, and then the fiber is washed with ultrapure water for three times.

The method of claim 10, wherein step 4 reduction and blocking are as follows: and (3) putting the optical fiber into a sodium borohydride solution for soaking for 30 minutes, sealing an optical fiber interface by using an extracting agent with a certain concentration, then cleaning the optical fiber interface by using ultrapure water for three times, and storing the optical fiber interface in a refrigerator at 4 ℃.

The method of claim 7, further comprising the steps of (5) installing the optical fiber into a reaction cell of the waveguide sensor, pumping a mixed solution containing a certain concentration of the small molecule target and the complementary strand of the fluorescence-modified aptamer into the reaction cell after the baseline is stabilized, and testing the change of the fluorescence signal in real time; step 6, flushing the optical fiber with sodium dodecyl sulfate solution to regenerate the sensing interface; repeating the step 5; step 7, drawing working curves of the optical waveguide sensor for detecting different targets; step 8, selectivity experiment: the target in step 5 may be replaced by a selectively tested substance.