阅读说明：本技术 一种比率型荧光分子印迹纸芯片及其制备方法和应用 (A kind of Ratio-type fluorescent molecule trace paper chip and its preparation method and application ) 是由 张忠 秦潇潇 马昕 于 2019-09-29 设计创作，主要内容包括：本发明提供了一种比率型荧光分子印迹纸芯片及其制备方法和应用,属于材料科学与工程和微流控芯片技术领域,包括以下步骤：将CDs和APTES-NBD偶联物荧光材料依次修饰在纤维素纸表面,利用表面印迹技术在纤维素纸表面形成苯醚甲环唑印迹孔穴,制得荧光分子印迹纸,将荧光分子印迹纸固定在具有三维结构的微流控基底上,即得到比率型荧光分子印迹纸芯片；本发明实现对于农药苯醚甲环唑的快速检测,发展了一种具有低毒、便携、高效、经济等特性的微流控快速检测纸芯片。(The present invention provides a kind of Ratio-type fluorescent molecule trace paper chips and its preparation method and application, belong to Materials Science and Engineering and micro fluidic chip technical field, the following steps are included: successively modifying CDs and APTES-NBD conjugate fluorescent material on cellulose paper surface, difenoconazole trace hole is formed on cellulose paper surface using surface imprinted technology, fluorescent molecule trace paper is made, fluorescent molecule trace paper is fixed on the micro-fluidic substrate with three-dimensional structure to get Ratio-type fluorescent molecule trace paper chip is arrived；The present invention realizes the quick detection for pesticide difenoconazole, has developed a kind of micro-fluidic quick detection paper chip with the characteristics such as less toxic, portable, efficient, economic.)

1. a kind of preparation method of Ratio-type fluorescent molecule trace paper chip, which comprises the following steps:

CDs and APTES-NBD conjugate is successively modified on cellulose paper surface, later using surface imprinted technology in cellulose Paper surface forms difenoconazole trace hole, and fluorescent molecule trace paper is made, fluorescent molecule trace paper is fixed on three Tie up structure micro-fluidic substrate on to get arrive Ratio-type fluorescent molecule trace paper chip.

2. the preparation method of Ratio-type fluorescent molecule trace paper chip according to claim 1, which is characterized in that specific packet Include following steps:

S1: preparing CDs using one step hydro thermal method, and CDs carried out amino acid modification, and the CDs after modification is bonded by amide Branch is inlaid with the cellulose fluorescent paper of CDs in amination cellulose paper surface, synthesis；

S2: on the cellulose fluorescent paper that the modification of APTES-NBD conjugate is inlaid with CDs made from the S1, it is glimmering that cellulose is made Machine-glazed paper；Synthesis has the imprinted layer of difenoconazole on cellulose fluorescent paper, is formed after completely removing template difenoconazole Difenoconazole trace hole obtains fluorescent molecule trace paper；

S3: S2 fluorescent molecule trace paper is fixed on the micro-fluidic substrate with three-dimensional structure, forms Ratio-type fluorescent molecule Trace paper chip.

3. the preparation method of Ratio-type fluorescent molecule trace paper chip according to claim 2, which is characterized in that the S1 The cellulose fluorescent paper for being inlaid with CDs is made particular by following steps:

S11: folic acid powder being added in water, is stirred evenly, and 2-3h is reacted at 170-190 DEG C, and CDs crude product is made in natural cooling； The CDs crude product of synthesis through membrane filtration and is crossed into column purification, sterling CDs solution is made；The folic acid and water consumption ratio are 0.0025~0.00375g:1mL；

S12: L-cysteine being added in the sterling CDs solution of S11 synthesis, under the conditions of being protected from light, 36-38 DEG C of isothermal reaction 35- Amino acid modification CDs solution is made in 45min；The L-cysteine and sterling CDs solution usage ratio be 0.3564~ 0.7128g:1L；

S13: by cellulose paper immerse mass fraction be 0.2% hydrochloric acid in be acidified, be placed on mass fraction be 50% second In the mixed liquor that pure and mild APTES is constituted, 2-3h is vibrated, is cleaned, amination cellulose paper is made；Ethyl alcohol and APTES amount ratio are 1mL:10 μ L；

S14: the EDC/MES solution that EDC mass concentration is 20mg/mL is added in amino acid modification CDs solution made from S12, It is uniformly mixed, mixed solution is made；Amination cellulose paper made from S13 is immersed in mixed solution, and it is dense that NHS mass is added Degree is the NHS/MES solution of 20mg/mL, after standing 0.5-1.5h, is protected from light oscillation 12-14h, the cellulose for being inlaid with CDs is made Fluorescent paper；Amino acid modification CDs solution: EDC/MES solution: NHS/MES liquor capacity ratio is 1.3-1.5:1:1.

4. the preparation method of Ratio-type fluorescent molecule trace paper chip according to claim 2, which is characterized in that the S2 Middle fluorescence trace paper is made particular by following steps:

S21: mass fraction is added in NBD and is 50% ethyl alcohol, and APTES is added, 9-11h is reacted under the conditions of being stirred at room temperature, is made Obtain APTES-NBD conjugate；NBD: ethanol consumption ratio is 1g:10L, and ethyl alcohol: APTES amount ratio is 1mL:3.68-4.5 μ L；

S22: by be inlaid with made from S1 CDs cellulose fluorescent paper immerse mass fraction be 50% ethyl alcohol and TEOS constitute In mixture, it is protected from light oscillation 3-5h, APTES-NBD conjugate and difenoconazole made from S21 is added, is protected from light oscillation 25- After 35min, ammonium hydroxide and second batch TEOS is added, is protected from light oscillation 4-6h and fluorescence trace paper is made after elution, cleaning；

Ethyl alcohol in mixture: TEOS amount ratio is 1mL:2.4-2.67 μ L, and ethyl alcohol: APTES-NBD conjugate volume ratio is 18.78-20.83:1, difenoconazole and APTES-NBD conjugate amount ratio are 7.88-11.79g:1L, and ethyl alcohol and ammonium hydroxide are used Amount is than being 1mL:4.4-6 μ L；Ammonium hydroxide and second batch TEOS volume ratio are 2:1.

5. the preparation method of Ratio-type fluorescent molecule trace paper chip according to claim 2, which is characterized in that the S3 In the micro-fluidic substrate with three-dimensional structure be three layers of micro-fluidic paper chip folding of cellulose paper.

6. Ratio-type fluorescent molecule trace paper chip made from a kind of preparation method of any one according to claim 1~5.

7. Ratio-type fluorescent molecule trace paper chip answering in pesticide difenoconazole context of detection according to claim 6 With.

Technical field

The invention belongs to material science engineering and micro fluidic chip technical fields, and in particular to a kind of Ratio-type fluorescent molecule Trace paper chip and its preparation method and application.

Background technique

Carbon quantum dot (CDs) is a kind of carbon-based zero dimension material, by few molecule or former molecular nanocluster, approximate ball Type and diameter < 10nm, the structure and composition of carbon quantum dot determine their property qualitative diversities, and carbon quantum dot is in ultraviolet region There is stronger absorption peak, and have long streaking in visible light region, most of absorption peak bands concentrate on 260~320nm, usual table Reveal the optical signatures such as fluorescence maximum emission wavelength, excitation wavelength dependence；Carbon quantum dot with good aqueous solubility is in illumination Under, its own can issue bright fluorescence, and optical stability is fine, this also becomes the most widely used property of carbon quantum dot. As novel Preen nono material, carbon quantum dot has good fluorescent characteristic and quantum effect, compared to traditional semiconductor Quantum dot has many advantages, such as that toxicity is low, environmentally protective, and with up-conversion fluorescence characteristic, excitation wavelength and launch wavelength can Control, higher stability and the anti-light advantageous properties such as Bleachability.Although carbon quantum dot has preferable fluorescent characteristic, Merely itself with regard to carbon quantum dot, as fluorescence probe, maximum disadvantage is exactly that selectivity is poor.In addition, the prior art Middle fluorescence probe is general only to be detected according to a kind of variation of fluorescence signal, is easy to cause to exist between measured value and true value and be missed The problem of difference.

Summary of the invention

The present invention provides a kind of Ratio-type fluorescent molecule trace paper chips and its preparation method and application, solve above-mentioned Technical problem.

The first purpose of the invention is to provide a kind of preparation method of Ratio-type fluorescent molecule trace paper chip, features It is, comprising the following steps:

By CDs and APTES-NBD (3- aminopropyl triethoxysilane/- 1,3 diazole of nitro benzo -2- oxa-) conjugate Fluorescent material is successively modified on cellulose paper surface, forms phenyl ether methyl cyclic on cellulose paper surface using surface imprinted technology later Azoles trace hole is made fluorescent molecule trace paper, fluorescent molecule trace paper is fixed on the micro-fluidic substrate with three-dimensional structure On to get arrive Ratio-type fluorescent molecule trace paper chip.

Preferably, the preparation method of the Ratio-type fluorescent molecule trace paper chip, specifically includes the following steps:

S1: CDs is prepared using one step hydro thermal method, and CDs is subjected to amino acid modification, the CDs after modification is passed through into amide Key grafts on amination cellulose paper surface, and synthesis is inlaid with the cellulose fluorescent paper of CDs；

S2: on the cellulose fluorescent paper that the modification of APTES-NBD conjugate is inlaid with CDs made from the S1, fiber is made Plain fluorescent paper；Synthesis has the imprinted layer of difenoconazole on cellulose fluorescent paper, after completely removing template difenoconazole Difenoconazole trace hole is formed, fluorescent molecule trace paper is obtained；

S3: S2 fluorescent molecule trace paper is fixed on the micro-fluidic substrate with three-dimensional structure, forms Ratio-type fluorescence Molecular engram paper chip.

Preferably, the cellulose fluorescent paper that the S1 is inlaid with CDs is made particular by following steps:

S11: folic acid powder being added in water, is stirred evenly, and 2-3h is reacted at 170-190 DEG C, it is thick that CDs is made in natural cooling Product；The CDs crude product of synthesis through membrane filtration and is crossed into column purification, sterling CDs solution is made；The folic acid: water consumption is 0.0025~0.00375g:1mL；

S12: L-cysteine being added in the sterling CDs solution of S11 synthesis, under the conditions of being protected from light, 36-38 DEG C of isothermal reaction Amino acid modification CDs solution is made in 35-45min；The L-cysteine: sterling CDs solution usage ratio be 0.3564~ 0.7128g:1L；

S13: cellulose paper is immersed into the hydrochloric acid that mass fraction is 0.2% and is acidified, the second that mass fraction is 50% is placed in In the mixed liquor that pure and mild APTES is constituted, 2-3h is vibrated, is cleaned, amination cellulose paper is made；Ethyl alcohol and APTES amount ratio are 1mL:10 μ L；

S14: being 20mg/mL's by EDC (1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride) mass concentration EDC/MES solution adds in amino acid modification CDs solution made from S12, is uniformly mixed, and mixed solution is made；It will be made from S13 Amination cellulose paper immerses in mixed solution, and it is 20mg/mL's that NHS (n-hydroxysuccinimide) mass concentration, which is added, NHS/MES solution after standing 0.5-1.5h, is protected from light oscillation 12-14h, the cellulose fluorescent paper for being inlaid with CDs is made；Amino acid Modify CDs solution: EDC/MES solution: NHS/MES liquor capacity ratio is 1.3-1.5:1:1.

Preferably, fluorescence trace paper is made particular by following steps in the S2:

S21: mass fraction is added in NBD and is 50% ethyl alcohol, and APTES is added, 9-11h is reacted under stirring condition, is made Obtain APTES-NBD conjugate；NBD: ethanol consumption ratio is 1g:10L, and ethyl alcohol: APTES amount ratio is 1mL:3.68-4.5 μ L；

S22: the cellulose fluorescent paper that CDs is inlaid with made from S1 is immersed into ethyl alcohol and the TEOS that mass fraction is 50% In the mixture that (ethyl orthosilicate) is constituted, it is protected from light oscillation 3-5h, APTES-NBD conjugate and phenylate first made from S21 is added Ammonium hydroxide and second batch TEOS is added after being protected from light oscillation 25-35min in ring azoles, is protected from light oscillation 4-6h and is made glimmering after elution, cleaning Light trace paper；

Ethyl alcohol in mixture: TEOS amount ratio is 1mL:2.4-2.67 μ L, ethyl alcohol: APTES-NBD conjugate volume ratio are as follows: 18.78-20.83:1, difenoconazole and APTES-NBD conjugate amount ratio are 7.88-11.79g:1L, and ethyl alcohol and ammonium hydroxide are used Amount is than being 1mL:4.4-6 μ L；Ammonium hydroxide and second batch TEOS volume ratio are 2:1.

Preferably, the micro-fluidic substrate in the S3 with three-dimensional structure is that fold three layers of cellulose paper are micro-fluidic Paper chip.

A second object of the present invention is to provide one kind Ratio-type fluorescent molecule trace paper as made from above-mentioned preparation method Chip.

Third object of the present invention is to provide above-mentioned Ratio-type fluorescent molecule trace paper chips in pesticide difenoconazole The application of context of detection.

Compared with prior art, the present invention has the following advantages:

(1) incorporating quantum point technology, surface molecule print technology and core chip technology of the present invention, are prepared for Ratio-type fluorescence Molecular engram paper chip improves the selectivity of CDs by the above method, can be used for difenoconazole efficient identification and quickly Detection；In addition, being different from other core flake products, which is grafted with two different colors of fluorescent material, respectively blue CDs and green APTES-NBD conjugate, by under a kind of excitation of wavelength, while the fluorescence intensity of two emission peaks of measurement, Target molecule is detected according to the ratio of two fluorescence signals, solves easily to go out when only being detected according to a kind of variation of fluorescence signal There are problems that error between existing measured value and true value, realize inherent self-correction, even is eliminated some difficulties to reduce With the influence of the other factors of control, to obtain more accurate result；

(2) in two kinds of fluorescent materials being grafted in paper chip, one of which is carbon quantum dot, test proves that carbon quantum Point is different from the quantum dots such as CdSe and CdTe, and for CDs to cell almost without toxicity, the present invention has developed a kind of hypotoxicity, environment The paper chip of friendly；

(3) in addition to this, micro-fluidic paper chip has the characteristics such as portable, efficient and economic, realizes p-phenylene's methyl cyclic-azole Quickly detection, has greatly met the needs that present food security field is quickly detected.

Detailed description of the invention

Fig. 1 is the Ratio-type fluorescent molecule trace paper chip preparation process that the embodiment of the present invention 1 provides；

Fig. 2 is MTT toxic effect of the CDs to HMC cell for the various concentration that the embodiment of the present invention 1 provides；

Fig. 3 is MTT toxic effect of the CDs-Cys to HMC cell for the various concentration that the embodiment of the present invention 1 provides；

Fig. 4 is the cell quantity detection containing fluorescent material that the embodiment of the present invention 1 provides；A:PBS control group；B:CDs Group；C:PBS control group；D:CDs-Cys；

Fig. 5 is the infrared spectroscopy that the embodiment of the present invention 1 provides；Wherein a: general fibre element paper；B: cellulose fluorescent paper；C: Fluorescent molecule trace paper；D:NIP；

Fig. 6 is the SEM figure in the Ratio-type fluorescent molecule trace paper chip preparation process that the embodiment of the present invention 1 provides；Its Middle A: general fibre element paper；B:CDs fluorescent paper；C:CDs and NBD-APTESC fluorescent paper: D: fluorescent molecule trace paper；

Fig. 7 is the Ratio-type fluorescent molecule print under the difenoconazole effect for the various concentration that the embodiment of the present invention 1 provides The fluorescence intensity change of mark paper chip.

Specific embodiment

In order to enable those skilled in the art to more fully understand, technical solution of the present invention is practiced, below with reference to specific The invention will be further described for embodiment and attached drawing, but illustrated embodiment is not as a limitation of the invention.