CN116183704A - Coaxial integrated implantable optical fuel sensor and method for detecting persistent organic pollutants - Google Patents
Coaxial integrated implantable optical fuel sensor and method for detecting persistent organic pollutantsDownload PDFInfo
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Abstract
Description
Translated fromChinese技术领域technical field
本发明属于光电化学传感领域，尤其是涉及一种同轴集成的植入式光燃料传感器及其制备方法和在持久性有机污染物方面的检测应用。The invention belongs to the field of photoelectrochemical sensing, and in particular relates to a coaxially integrated implanted photofuel sensor, its preparation method and its application in the detection of persistent organic pollutants.
背景技术Background technique
持久性有机污染物是指具有高毒性、持久性、生物蓄积性和长距离迁移性的人工合成化学品，其在环境中无处不在，已成为全球关注的主要环境污染问题。持久性有机污染物泄漏到环境中可通过食物链进行生物积累和生物放大，最终对人体健康造成严重的负面影响，如内分泌障碍、免疫和生殖系统功能障碍、发育性神经毒性以及某些癌症等。因此，迫切需要一种快速、灵敏、便携式和现场检测持久性有机污染物的分析方法，这是评估其环境风险的关键一步。鱼类作为人类膳食中的重要组成部分，能从饮食来源和鳃膜中积累持久性有机污染物，导致其组织中的污染物富集比周围水域高，是人类蓄积持久性有机污染物的主要来源。因此，鱼类中持久性有机污染物的原位定量检测对于全面评估持久性有机污染物对生态环境和人类健康的危害具有重要意义。Persistent organic pollutants refer to artificially synthesized chemicals with high toxicity, persistence, bioaccumulation and long-distance migration. They are ubiquitous in the environment and have become a major environmental pollution issue of global concern. Leakage of persistent organic pollutants into the environment can bioaccumulate and biomagnify through the food chain, and eventually cause serious negative effects on human health, such as endocrine disorders, immune and reproductive system dysfunction, developmental neurotoxicity, and certain cancers. Therefore, there is an urgent need for a rapid, sensitive, portable and on-site analytical method for the detection of persistent organic pollutants, which is a crucial step in assessing their environmental risks. As an important part of the human diet, fish can accumulate persistent organic pollutants from dietary sources and gill membranes, resulting in higher pollutant enrichment in its tissues than the surrounding waters, which is the main reason for human accumulation of persistent organic pollutants. source. Therefore, in situ quantitative detection of persistent organic pollutants in fish is of great significance for comprehensive assessment of the hazards of persistent organic pollutants to the ecological environment and human health.
传统的动物样品中污染物的分析技术通常需要破坏或杀死生物体，不仅对动物不道德，对濒危物种不利，而且不能反映持久性有机污染物在活体内的真实积累和变化状态。固相微萃取技术虽然可以实现活体内原位取样，但是后续需要复杂的洗脱步骤并且要用大型仪器如高效液相色谱进行样品分析，难以实现目标污染物现场监测和及时反馈。在各种体内和原位分析技术中，电化学传感以其高时空分辨率、设备简单等优点得到了广泛应用。光电化学传感器作为电化学分析的进化产物，不仅继承了上述优点，而且由于光激发信号与电检测信号的分离而具有更高的灵敏度。此外，由只有阳极和阴极的光燃料电池构成的自供电光电化学传感器可以在没有外接电源的情况下工作，更容易实现设备便携和低成本检测。但是，目前的光电化学活体传感器基本上都需要植入一个分离的三电极系统，包括光阳极、参比电极和对电极，这容易导致以下三个问题：1)复杂的活体环境中的生物大分子对三个电极都造成生物污染，体内的电活性小分子容易干扰光阳极，同时光阳极上的光活性材料也可能对活体造成生物毒性；2)无法精确控制在自由活动的动物体内植入电极之间的距离，从而影响检测精度；3)多个电极附加光源植入活体需要多次插入操作，会对生物体造成较大的机械损伤。Traditional analysis techniques for pollutants in animal samples usually need to destroy or kill organisms, which is not only unethical to animals and unfavorable to endangered species, but also cannot reflect the true accumulation and change status of persistent organic pollutants in vivo. Although solid-phase microextraction technology can achieve in situ sampling in vivo, it requires complicated elution steps and large-scale instruments such as high-performance liquid chromatography for sample analysis, making it difficult to achieve on-site monitoring and timely feedback of target pollutants. Among various in vivo and in situ analytical techniques, electrochemical sensing has been widely used due to its high spatiotemporal resolution and simple equipment. As an evolutionary product of electrochemical analysis, photoelectrochemical sensors not only inherit the above-mentioned advantages, but also have higher sensitivity due to the separation of the photoexcitation signal from the electrical detection signal. In addition, self-powered photoelectrochemical sensors consisting of photofuel cells with only anode and cathode can work without an external power source, making it easier to achieve device portability and low-cost detection. However, the current photoelectrochemical in vivo sensors basically need to be implanted with a separate three-electrode system, including a photoanode, a reference electrode and a counter electrode, which easily leads to the following three problems: 1) Biomass in a complex living environment Molecules cause biological contamination to the three electrodes, small electroactive molecules in the body can easily interfere with the photoanode, and the photoactive material on the photoanode may also cause biological toxicity to the living body; 2) Implantation in freely moving animals cannot be accurately controlled The distance between the electrodes affects the detection accuracy; 3) The implantation of multiple electrodes and additional light sources into the living body requires multiple insertion operations, which will cause greater mechanical damage to the living body.
发明内容Contents of the invention
本发明所要解决的技术问题是针对上述现有技术存在的不足而提供一种同轴集成的植入式光燃料传感器及其制备方法和在持久性有机污染物方面的检测应用，解决了单独植入多个电极以及光源到复杂环境造成的灵敏度较低、选择性较差、易被污染、难以实现原位检测等问题。The technical problem to be solved by the present invention is to provide a coaxial integrated implantable optical fuel sensor and its preparation method and its application in the detection of persistent organic pollutants in view of the above-mentioned deficiencies in the prior art. Introducing multiple electrodes and light sources into a complex environment causes problems such as low sensitivity, poor selectivity, easy contamination, and difficulty in in-situ detection.
本发明为解决上述提出的问题所采用的技术方案为：The technical scheme that the present invention adopts for solving the above-mentioned problem is:
一种同轴集成的植入式光燃料传感器，包括光阳极、生物阴极和燃料，所述的光阳极是以导光光纤为基底，在其一端依次修饰金层和碳纳米管以及Ag2S-Bi2S3异质结所得；所述生物阴极是以玻璃毛细管为基底，在其一端修饰金层后，再继续修饰用于特异性识别目标物的核酸适配体，然后在该端的端口处用全氟磺酸溶液固化形成质子交换膜所得；所述燃料是抗坏血酸溶液。A coaxially integrated implantable optical fuel sensor, including a photoanode, a biocathode and a fuel. The photoanode is based on a light-guiding optical fiber, and one end of the photoanode is sequentially modified with a gold layer, carbon nanotubes and Ag2 S - Obtained from a Bi2 S3 heterojunction; the biocathode is based on a glass capillary, after modifying a gold layer at one end, and then continuing to modify the nucleic acid aptamer for specific recognition of the target, and then at the end of the port It is obtained by solidifying a perfluorosulfonic acid solution to form a proton exchange membrane; the fuel is an ascorbic acid solution.
按上述方案，所述燃料注入生物阴极内，光阳极插入生物阴极内与燃料相接触，光阳极和生物阴极处于同轴。According to the above scheme, the fuel is injected into the biocathode, the photoanode is inserted into the biocathode to contact with the fuel, and the photoanode and the biocathode are coaxial.
按上述方案，所述碳纳米管可以为多壁碳纳米管或者单壁碳纳米管等。According to the above solution, the carbon nanotubes may be multi-walled carbon nanotubes or single-walled carbon nanotubes.
上述同轴集成的植入式光燃料传感器的制备方法，主要包括如下步骤：The preparation method of the coaxially integrated implantable optical fuel sensor mainly includes the following steps:
S1.选取玻璃毛细管和导光光纤，玻璃毛细管的内径大于导光光纤的直径；S1. select the glass capillary and the light-guiding fiber, the inner diameter of the glass capillary is greater than the diameter of the light-guiding fiber;
S21.在S1所述玻璃毛细管的一端采用种子介导生长法修饰金层，然后通过金硫键在金层表面修饰能特异性识别目标物的核酸适配体，并用6-巯基-1-己醇(MCH)溶液封闭活性位点，得到玻璃毛细管/金层/核酸适配体电极；S21. Use the seed-mediated growth method to modify the gold layer at one end of the glass capillary described in S1, and then modify the surface of the gold layer with a nucleic acid aptamer that can specifically recognize the target object through a gold-sulfur bond, and use 6-mercapto-1-hexyl Alcohol (MCH) solution seals the active site to obtain a glass capillary/gold layer/nucleic acid aptamer electrode;
S22.将全氟化树脂溶液注入玻璃毛细管/金层/核酸适配体电极的修饰端，干燥后在该电极修饰端的端口处形成一层质子交换膜，得到生物阴极；S22. Inject the perfluorinated resin solution into the modified end of the glass capillary/gold layer/nucleic acid aptamer electrode, and form a layer of proton exchange membrane at the port of the modified end of the electrode after drying to obtain a biocathode;
S31.在S1所述导光光纤的一端采用种子介导生长法修饰金层，然后在金层表面利用层层自组装法修饰多壁碳纳米管(MWNT)，得到光纤/金层/MWNT电极；S31. Use the seed-mediated growth method to modify the gold layer at one end of the light-guiding fiber described in S1, and then use the layer-by-layer self-assembly method to modify the multi-walled carbon nanotube (MWNT) on the surface of the gold layer to obtain an optical fiber/gold layer/MWNT electrode ;
S32.在光纤/金层/MWNT电极的修饰端利用连续离子层吸附修饰具有高光电转换效率的Ag2S-Bi2S3异质结，构建得到光阳极；S32. Using continuous ion layer adsorption to modify the Ag2 S-Bi2 S3 heterojunction with high photoelectric conversion efficiency at the modified end of the optical fiber/gold layer/MWNT electrode to construct a photoanode;
S4.将燃料抗坏血酸溶液灌入S22所得生物阴极内，并将S32所得光阳极的修饰端插入生物阴极内与燃料相接触，即得到同轴集成的植入式光燃料传感器。S4. Pour the fuel ascorbic acid solution into the biocathode obtained in S22, and insert the modified end of the photoanode obtained in S32 into the biocathode to contact with the fuel to obtain a coaxially integrated implantable photofuel sensor.
按上述方案，透明导光光纤的直径优选在100～200μm，修饰端的长度优选在2～3cm；所述玻璃毛细管的内径和外径分别优选在250～350μm和350～450μm，修饰端的长度优选在1.5～2.5cm。进一步地，所述玻璃毛细管修饰端包括直管段以及位于直管段一端的缩口段(用拉制仪拉制而成)，缩口段尖端直径优选在35～45μm。According to the above scheme, the diameter of the transparent light-guiding fiber is preferably 100-200 μm, and the length of the modified end is preferably 2-3 cm; the inner diameter and outer diameter of the glass capillary are preferably 250-350 μm and 350-450 μm respectively, and the length of the modified end is preferably 2-3 cm. 1.5～2.5cm. Further, the modified end of the glass capillary includes a straight pipe section and a necked section (drawn by a drawing machine) located at one end of the straight pipe section, and the diameter of the tip of the necked section is preferably 35-45 μm.
按上述方案，S21中生长金层的方法为：将玻璃毛细管的一端在多巴胺溶液浸泡0.5～1.5h，水洗干燥后在金种中浸泡10～13h，最后在盐酸羟胺和氯金酸的混合溶液中静置生长金层，水洗干燥即可。其中，多巴胺溶液浓度在1～2mg/mL范围内,金种是指含金纳米粒子的水溶液，浓度优选在50～100μg/mL范围内；盐酸羟胺和氯金酸的混合溶液中，盐酸羟胺和氯金酸各自浓度优选在0.5～1.5mM和0.03～0.07wt％范围内，静置生长3～5min。According to the above scheme, the method for growing the gold layer in S21 is: soak one end of the glass capillary in the dopamine solution for 0.5-1.5 hours, wash and dry it, soak it in the gold seed for 10-13 hours, and finally soak it in the mixed solution of hydroxylamine hydrochloride and chloroauric acid. Let it stand in the middle to grow the gold layer, then wash and dry. Wherein, the concentration of dopamine solution is in the range of 1-2 mg/mL, the gold species refers to the aqueous solution containing gold nanoparticles, and the concentration is preferably in the range of 50-100 μg/mL; in the mixed solution of hydroxylamine hydrochloride and chloroauric acid, hydroxylamine hydrochloride and The respective concentrations of the chloroauric acid are preferably in the ranges of 0.5-1.5 mM and 0.03-0.07 wt%, and the growth is allowed to stand for 3-5 minutes.
按上述方案，S21中通过金硫键修饰核酸适配体的方法为：首先，将巯基化核酸适配体溶于5～20mM PBS缓冲溶液(pH 7.0～7.5)中，浓度在6～10μM范围内,然后加入三(2-羧乙基)膦(TCEP)水溶液活化适配体1～3h(TCEP在适配体溶液中的浓度为0.2～1mM)，得到适配体溶液；然后，将玻璃毛细管生长有金层的一端置于上述适配体溶液中在4℃下孵育12～20h，取出后用PBS缓冲溶液清洗；最后，再将其置于0.5～2mM 6-巯基-1-己醇(MCH)水溶液中封闭1～3h，再次取出用PBS缓冲溶液清洗，得到玻璃毛细管/金层/核酸适配体电极。According to the above scheme, the method for modifying nucleic acid aptamers through gold-sulfur bonds in S21 is as follows: first, dissolve the thiolated nucleic acid aptamers in 5-20 mM PBS buffer solution (pH 7.0-7.5), and the concentration is in the range of 6-10 μM Inside, then add tris(2-carboxyethyl)phosphine (TCEP) aqueous solution to activate the aptamer for 1~3h (the concentration of TCEP in the aptamer solution is 0.2~1mM) to obtain the aptamer solution; then, the glass Place the end of the capillary with the gold layer in the aptamer solution and incubate at 4°C for 12-20 hours, take it out and wash it with PBS buffer solution; finally, place it in 0.5-2mM 6-mercapto-1-hexanol (MCH) aqueous solution was blocked for 1-3 hours, and then taken out and washed with PBS buffer solution to obtain a glass capillary/gold layer/nucleic acid aptamer electrode.
按上述方案，S22的具体操作方法为：将全氟磺酸树脂溶液(Nafion)注入到毛细玻璃管/金层/核酸适配体电极的修饰端端口，然后室温下固化在该端口形成质子交换膜。其中，Nafion溶液的浓度在0.4～0.6wt％范围，注入体积在0.04～0.06μL范围内，能实现在修饰端的端口固化形成质子交换膜即可。According to the above scheme, the specific operation method of S22 is: inject the perfluorosulfonic acid resin solution (Nafion) into the modified end port of the capillary glass tube/gold layer/nucleic acid aptamer electrode, and then solidify at the port at room temperature to form proton exchange membrane. Wherein, the concentration of the Nafion solution is in the range of 0.4-0.6 wt %, and the injection volume is in the range of 0.04-0.06 μL, so that the port of the modified end can be solidified to form a proton exchange membrane.
按上述方案，S31中生长金层的方法为：将干净的导光光纤在1～2mg/mL多巴胺溶液中浸泡1.5～2.5h后水洗干燥，然后采用丙酮浸泡处理2～4s去掉光纤前端0.7～0.9cm的聚多巴胺层和光纤包层使其能在前端透光，水洗干燥后，将该端1.5～2.5cm在金种中浸泡3～5h，再在盐酸羟胺和氯金酸的混合溶液中静置生长3～5min，水洗干燥即可(注：该光纤前端0.7～0.9cm这一段未生长金层)。金种是指含金纳米粒子的水溶液，浓度优选在50～100μg/mL范围内；盐酸羟胺和氯金酸的混合溶液中，盐酸羟胺和氯金酸各自浓度优选在0.5～1.5mM和0.03～0.07wt％范围内。According to the above scheme, the method for growing the gold layer in S31 is: soak the clean light-guiding fiber in 1-2 mg/mL dopamine solution for 1.5-2.5 hours, wash and dry it, and then soak it in acetone for 2-4 seconds to remove the 0.7- The 0.9cm polydopamine layer and the optical fiber cladding make it transparent at the front end. After washing and drying, soak the 1.5-2.5cm end in gold seeds for 3-5 hours, and then soak it in the mixed solution of hydroxylamine hydrochloride and chloroauric acid. Leave to grow for 3-5 minutes, then wash and dry (note: no gold layer grows in the 0.7-0.9 cm section of the front end of the optical fiber). The gold species refers to the aqueous solution containing gold nanoparticles, and the concentration is preferably in the range of 50-100 μg/mL; in the mixed solution of hydroxylamine hydrochloride and chloroauric acid, the respective concentrations of hydroxylamine hydrochloride and chloroauric acid are preferably 0.5-1.5mM and 0.03- 0.07wt% range.
进一步地，S31、S21中，导光光纤和玻璃毛细管生长金层后，电阻在10～20Ω。Further, in S31 and S21, after the gold layer is grown on the light-guiding optical fiber and the glass capillary, the resistance is 10-20Ω.
按上述方案，S31和S32中，金层与碳纳米管未完全重叠，而是在该修饰端部分重叠，碳纳米管的修饰区域相对金层的修饰区域更靠近(或者说是延伸至)光纤修饰端的端口方向，且碳纳米管的修饰区域大于金层的修饰区域，即金层修饰区域未延伸至修饰端端口，距离修饰端端口存在一小段距离(比如0.7～0.9cm)，而碳纳米管修饰区域延伸至修饰端端口，碳纳米管的修饰区域包括金层修饰区域以及光纤前端未修饰金层的区域；Ag2S-Bi2S3异质结的修饰区域与碳纳米管的修饰区域基本重叠。具体地，光纤修饰端最前端0.7～0.9cm部分没有修饰金层，但修饰了碳纳米管，碳纳米管的修饰区域包括金层修饰区域和光纤修饰前端没有修饰金层的0.7～0.9cm部分。According to the above scheme, in S31 and S32, the gold layer and the carbon nanotube do not completely overlap, but partially overlap at the modified end, and the modified region of the carbon nanotube is closer to (or extends to) the optical fiber than the modified region of the gold layer The port direction of the modified end, and the modified area of the carbon nanotube is larger than the modified area of the gold layer, that is, the modified area of the gold layer does not extend to the port of the modified end, and there is a small distance (such as 0.7 to 0.9 cm) from the port of the modified end, while the modified area of the carbon nanotube The modified region extends to the modified end port. The modified region of the carbon nanotube includes the modified region of the gold layer and the region of the unmodified gold layer at the front end of the fiber; the modified region of the Ag2 S-Bi2 S3 heterojunction and the modified region of the carbon nanotube basically overlap. Specifically, the 0.7-0.9 cm portion of the front end of the optical fiber modification end is not modified with the gold layer, but the carbon nanotubes are modified, and the modification area of the carbon nanotubes includes the gold layer modification area and the 0.7-0.9 cm portion of the fiber modification front end that is not modified with the gold layer .
按上述方案，S31中层层自组装法修饰碳纳米管的方法为：将生长金层后的光纤置于聚二烯丙基二甲基氯化铵(PDDA)水溶液中进行吸附，用水浸洗后再置于羧基化碳纳米管水溶液中进行吸附，此为S31中的一个循环；一个循环后，进行水洗干燥，然后进行下一个循环，实现碳纳米管修饰。其中，循环次数的范围是3～5次；PDDA的浓度0.5～1.5wt％，吸附时间在4～6min；羧基化碳纳米管水溶液的浓度在0.4～0.6mg/mL范围内，吸附时间各自4～6min。According to the above scheme, the method for modifying carbon nanotubes by the layer-by-layer self-assembly method in S31 is as follows: place the optical fiber after growing the gold layer in an aqueous solution of polydiallyldimethylammonium chloride (PDDA) for adsorption, and after rinsing with water, It is then placed in an aqueous solution of carboxylated carbon nanotubes for adsorption, which is a cycle in S31; after one cycle, it is washed and dried with water, and then the next cycle is performed to realize carbon nanotube modification. Among them, the range of cycle times is 3-5 times; the concentration of PDDA is 0.5-1.5wt%, and the adsorption time is 4-6min; the concentration of carboxylated carbon nanotube aqueous solution is in the range of 0.4-0.6mg/mL, and the adsorption time is 4 ~6min.
按上述方案，S32中在光纤/金层/MWNT电极上利用连续离子层吸附修饰Ag2S-Bi2S3异质结的方法为：在黑暗环境下，将光纤/金层/MWNT电极的修饰端置于0.08～0.12M AgNO3水溶液中浸泡25～35min，用水浸洗后，继续在0.1～0.14M Na2S溶液中浸泡25～35min，水洗干燥；然后，接着分别在8～12mM Bi(NO3)3溶液和8～12mM Na2S溶液中各浸泡8～12min，进行水洗干燥后，此为吸附Bi2S3的一个循环；吸附Bi2S3的循环次数为1～3次。According to the above scheme, the method of modifying the Ag2 S-Bi2 S3 heterojunction on the optical fiber/gold layer/MWNT electrode by continuous ion layer adsorption in S32 is: in a dark environment, the optical fiber/gold layer/MWNT electrode The modified end was soaked in 0.08-0.12M AgNO3 aqueous solution for 25-35min, after soaking in water, continued to soak in 0.1-0.14M Na2 S solution for 25-35min, washed and dried; then, followed by 8-12mM Bi Soak in (NO3 )3 solution and 8-12mM Na2 S solution for 8-12 minutes each, wash and dry with water, this is a cycle of adsorption of Bi2 S3 ; the number of cycles of adsorption of Bi2 S3 is 1-3 times .
按上述方案，S4中，燃料抗坏血酸溶液的浓度在20～100mM，在生物阴极内的灌注高度要高于光阳极修饰端。According to the above scheme, in S4, the concentration of the fuel ascorbic acid solution is 20-100 mM, and the perfusion height in the biocathode is higher than that in the modified photoanode.
按上述方案，所述目标物为持久性有机污染物，本发明以3,3’,4,4’-四氯联苯(PCB77)为例；能特异性识别3,3’,4,4’-四氯联苯(PCB77)的核酸适配体为SH-Aptamer，序列是5′-SH-(CH2)6-GGC-GGG-GCT-ACG-AAG-TAG-TGA-TTT-TTT-CCG-ATG-GCC-CGT-G-3′。若选择别的污染物作为待测目标物，则可以选择对应的能够特异性识别的分子，只需要识别分子含有氨基或者巯基等能修饰到金层的基团即可。According to the above scheme, the target object is a persistent organic pollutant. The present invention takes 3,3',4,4'-tetrachlorobiphenyl (PCB77) as an example; it can specifically recognize 3,3',4,4 The nucleic acid aptamer of '-tetrachlorobiphenyl (PCB77) is SH-Aptamer, the sequence is 5'-SH-(CH2 )6 -GGC-GGG-GCT-ACG-AAG-TAG-TGA-TTT-TTT- CCG-ATG-GCC-CGT-G-3'. If other pollutants are selected as the target to be detected, the corresponding molecules that can be specifically recognized can be selected, and the recognition molecules only need to contain groups such as amino groups or sulfhydryl groups that can be modified to the gold layer.
上述同轴集成的植入式光燃料传感器在检测3,3’,4,4’-四氯联苯(PCB77)方面的应用，具体应用方法为：将所述同轴集成的植入式光燃料传感器插入PCB77溶液中，并用激光源照射光阳极的非修饰端，光随着光纤传输到光阳极的修饰端激发上面修饰的Ag2S-Bi2S3产生光生电子，电子再通过外电路传输到生物阴极形成开路电压；目标物通过适配体识别到生物阴极上后会阻碍电子传输，减小开路电位；生物阴极和光阳极均通过表面修饰的金层与电位计的两端导线连接，然后测定开路电压随PCB77浓度的变化，采用标准曲线法测定待测物中PCB77的含量。The application of the above-mentioned coaxially integrated implantable optical fuel sensor in the detection of 3,3',4,4'-tetrachlorobiphenyl (PCB77), the specific application method is: the coaxially integrated implantable optical fuel sensor The fuel sensor is inserted into the PCB77 solution, and a laser source is used to irradiate the non-modified end of the photoanode. The light is transmitted to the modified end of the photoanode along with the optical fiber to excite the Ag2 S-Bi2 S3 modified above to generate photogenerated electrons, and the electrons pass through the external circuit. Transmit to the biocathode to form an open circuit voltage; after the target is recognized by the aptamer on the biocathode, it will hinder the electron transmission and reduce the open circuit potential; both the biocathode and the photoanode are connected to the two ends of the potentiometer through the surface-modified gold layer. Then measure the change of open circuit voltage with the concentration of PCB77, and use the standard curve method to determine the content of PCB77 in the test object.
按上述方案，所述待测样本可以是鱼、老鼠、植物等活体，也可以是血液、尿液等溶液。According to the above scheme, the sample to be tested can be a living body such as fish, mouse, plant, etc., or a solution such as blood or urine.
按上述方案，所述电位计可以是电化学工作站，也可以是便携式电位计。便携式电位计可以用能进行电位读数的便携式pH计代替。According to the above solution, the potentiometer can be an electrochemical workstation or a portable potentiometer. The portable potentiometer can be replaced by a portable pH meter capable of potentiometric readings.
本发明还提供一种同轴集成的植入式光燃料传感器系统，包括本发明上述同轴集成的植入式光燃料传感器和光源以及电位计；其中同轴集成的植入式光燃料传感器用于插入待测物中，光源与传感器的光阳极可以通过光阳极的基底光纤进行连接，传感器的生物阴极和光阳极的金层采用细铜丝分别与电位计的两端电连接。光源的波长为能够激发Ag2S-Bi2S3材料的可见～近红外波长范围内。The present invention also provides a coaxially integrated implantable optical fuel sensor system, including the above-mentioned coaxially integrated implantable optical fuel sensor, light source and potentiometer of the present invention; wherein the coaxially integrated implantable optical fuel sensor is used When inserted into the object to be measured, the light source and the photoanode of the sensor can be connected through the base optical fiber of the photoanode, and the biocathode of the sensor and the gold layer of the photoanode are electrically connected to the two ends of the potentiometer respectively with thin copper wires. The wavelength of the light source is within the visible to near-infrared wavelength range capable of exciting the Ag2 S-Bi2 S3 material.
本发明的检测原理为：光纤表面修饰的光活性材料Ag2S-Bi2S3被沿光纤传输的光(激光器与光纤未修饰的一端连接)所激发产生电子-空穴对。由于Ag2S和Bi2S3的能带匹配，Bi2S3中的激发电子被传输到具有相对负的价带能级的Ag2S上，然后转移到碳纳米管，再转移到光纤/金层上，最后通过外部电路到达玻璃毛细管/金层/适配体生物阴极上，从而产生了电能(开路电位)。玻璃毛细管内的抗坏血酸燃料用于填充Bi2S3的光生空穴从而促进电子-空穴对的分离。当目标PCB77通过与适配体作用识别到生物阴极表面形成导电性能差的适配体-PCB77复合物，阻碍了空穴-电子对的分离，降低了OCP信号，从而实现对PCB77的检测。其中，由于金层不透光，所以光纤最前端由于丙酮处理后没有生长金层，而是修饰了透光性较好的碳纳米管，保证光纤传输的光能够照到最外层的Ag2S-Bi2S3而实现材料激发。生物阴极尖端修饰了质子交换膜后不影响阴极、阳极之间的电子传递，同时由于质子膜只能通过质子、电子，所以藏在阴极内的光阳极可以避免与复杂的生物环境接触而影响光阳极的选择性。The detection principle of the present invention is: the photoactive material Ag2 S-Bi2 S3 modified on the surface of the optical fiber is excited by the light transmitted along the optical fiber (the laser is connected to the unmodified end of the optical fiber) to generate electron-hole pairs. Due to the energy band matching ofAg2S andBi2S3 , the excitedelectrons inBi2S3 are transportedtoAg2S with a relatively negative valence band energy level, then transferred to carbon nanotubes, and then transferred to optical fibers / gold layer, and finally reach the glass capillary / gold layer / aptamer biocathode through an external circuit, thereby generating electrical energy (open circuit potential). Ascorbic acid fuel inside the glass capillary is used to fill the photogenerated holes ofBi2S3 to promotethe separation of electron-hole pairs. When the target PCB77 is recognized by the aptamer to the surface of the biocathode to form an aptamer-PCB77 complex with poor conductivity, which hinders the separation of hole-electron pairs and reduces the OCP signal, thereby realizing the detection of PCB77. Among them, since the gold layer is opaque, the front end of the optical fiber does not grow a gold layer after being treated with acetone, but is modified with carbon nanotubes with better light transmission to ensure that the light transmitted by the optical fiber can reach the outermost Ag2 S-Bi2 S3 to achieve material excitation. The proton exchange membrane modified on the tip of the biocathode does not affect the electron transfer between the cathode and the anode. At the same time, because the proton membrane can only pass protons and electrons, the photoanode hidden in the cathode can avoid contact with the complex biological environment and affect the light. Anode selectivity.
与现有技术相比，本发明的有益成果是：Compared with the prior art, the beneficial results of the present invention are:
1、本发明制备的同轴集成的植入式光燃料传感器，光阳极藏在生物阴极内，固定并缩短了光阳极与生物阴极之间的距离，减少光阳极的生物污染，提高了传感灵敏度和准确性；1. In the coaxially integrated implantable photofuel sensor prepared by the present invention, the photoanode is hidden in the biocathode, which fixes and shortens the distance between the photoanode and the biocathode, reduces the biological pollution of the photoanode, and improves the sensing efficiency. Sensitivity and accuracy;
2、本发明制备的同轴集成的植入式光燃料传感器可以消除复杂活体环境或溶液环境中电活性小分子对光阳极的干扰以及克服光活性材料可能存在的生物毒性；2. The coaxially integrated implantable photofuel sensor prepared by the present invention can eliminate the interference of small electroactive molecules on the photoanode in complex living environment or solution environment and overcome the possible biological toxicity of photoactive materials;
3、本发明制备的同轴集成的植入式光燃料传感器减少了在活体中额外插入参比电极、对电极和光源所造成的损伤，极大程度上降低了对活体组织的机械损伤；3. The coaxial integrated implantable optical fuel sensor prepared by the present invention reduces the damage caused by the additional insertion of reference electrodes, counter electrodes and light sources in the living body, and greatly reduces the mechanical damage to living tissues;
4、本发明制备的同轴集成的植入式光燃料传感器能应用于鱼脑等活体中PCB77的生物积累水平的原位监测，体内PCB77的波动可以通过现场手持电位计直接反映，为活体原位监测技术提供了重要的技术突破。4. The coaxially integrated implantable optical fuel sensor prepared by the present invention can be applied to the in-situ monitoring of the bioaccumulation level of PCB77 in living bodies such as fish brains. The fluctuation of PCB77 in the body can be directly reflected by the on-site hand-held potentiometer, which is the source of the living body. Bit monitoring technology provides an important technological breakthrough.
综上所述，本发明所述同轴集成的植入式光燃料传感器实现了在现场采用便携式电位计进行快速原位检测活体内持久性有机污染物，兼具灵敏度高、抗污染性良好、生物相容性优良、生物毒性低、植入损伤小等优势；并且，成本便宜，可以在没有外部电压的情况下工作，具有便携化和低成本的性能，适用于多种目标物的现场检测。In summary, the coaxially integrated implantable optical fuel sensor of the present invention realizes rapid in-situ detection of persistent organic pollutants in vivo by using a portable potentiometer on site, and has the advantages of high sensitivity, good pollution resistance, It has the advantages of excellent biocompatibility, low biotoxicity, and small implant damage; and it is cheap, can work without external voltage, has portability and low-cost performance, and is suitable for on-site detection of various targets .
附图说明Description of drawings
图1为本发明光阳极的制备过程示意图；其中，Optical fiber(OF)表示光纤，PDA为聚多巴胺，Acetone为丙酮，Remove cladding表示去除包层，AuNPs seeds表示金种。1 is a schematic diagram of the preparation process of the photoanode of the present invention; wherein, Optical fiber (OF) means optical fiber, PDA means polydopamine, Acetone means acetone, Remove cladding means removing cladding, and AuNPs seeds means gold species.
图2为本发明生物阴极的制备过程示意图；其中，Glass capillary表示玻璃毛细管，Au-decorated表示修饰金层，PCB77 aptamer表示PCB77适配体，MCH为巯基己醇。2 is a schematic diagram of the preparation process of the biocathode of the present invention; wherein, Glass capillary means glass capillary, Au-decorated means modified gold layer, PCB77 aptamer means PCB77 aptamer, and MCH means mercaptohexanol.
图3为应用例1的线性关系图；Fig. 3 is the linear relationship diagram of application example 1;
图4为应用例2同轴集成的植入式光燃料传感器活体检测示意图；Fig. 4 is a schematic diagram of living body detection of the coaxially integrated implantable optical fuel sensor in application example 2;
图5为应用例2的鱼在污染物溶液中暴露不同浓度PCB77不同时间的鱼脑富集污染物浓度的测试结果图。Fig. 5 is a graph showing the test results of fish brain-enriched pollutant concentrations exposed to different concentrations of PCB77 for different times in application example 2.
图6为应用例2的用我们构建的传感器和气相色谱法测定不同浓度PCB77暴露6天后鱼脑中的PCB77浓度的测试结果对比图。Fig. 6 is a comparison chart of test results of PCB77 concentrations in fish brains after 6 days of exposure to different concentrations of PCB77 using our sensor and gas chromatography in Application Example 2.
具体实施方式Detailed ways
为了更好地理解本发明，下面结合实施例进一步阐明本发明的内容，但本发明不仅仅局限于下面的实施例。In order to better understand the present invention, the content of the present invention is further illustrated below in conjunction with the examples, but the present invention is not limited to the following examples.
下述实施例中，导光光纤和玻璃毛细管生长金层后，电阻在10～20Ω。In the following embodiments, after the gold layer is grown on the light-guiding optical fiber and the glass capillary, the resistance is 10-20Ω.
实施例Example
一种同轴集成的植入式光燃料传感器的制备方法，具体步骤如下：A method for preparing a coaxially integrated implantable optical fuel sensor, the specific steps are as follows:
S11.用拉制仪将内径300μm、外径400μm的毛细管在拉制仪下进行拉制，得到尖端端口直径为40μm的锥形玻璃毛细管；将能特异性识别目标物PCB77的核酸适配体SH-Aptamer溶于10mM PBS(pH＝7.4)溶液中，得到8μM的适配体溶液，并加入0.5mM TCEP活化适配体2h；S11. Pull a capillary with an inner diameter of 300 μm and an outer diameter of 400 μm under the drawing instrument to obtain a tapered glass capillary with a tip port diameter of 40 μm; the nucleic acid aptamer SH that can specifically recognize the target PCB77 - Aptamer was dissolved in 10 mM PBS (pH=7.4) solution to obtain an 8 μM aptamer solution, and 0.5 mM TCEP was added to activate the aptamer for 2 h;
S12.将毛细玻璃管的尖端(约2cm长)在1.5mg/mL的多巴胺溶液浸泡1h，水洗干燥后在金种中浸泡12h，最后在1mM盐酸羟胺和0.05wt％的氯金酸的混合溶液中静置生长4min，水洗干燥；然后接着置于S11所得适配体溶液中在4℃下孵育16h，取出后，用10mM PBS(pH＝7.4)缓冲溶液清洗后，再在1mM MCH溶液中封闭2h，取出后，再次用10mM PBS(pH＝7.4)缓冲溶液清洗后得到毛细玻璃管/金层/核酸适配体电极；S12. soak the tip (about 2cm long) of the capillary glass tube in the dopamine solution of 1.5mg/mL for 1h, wash and dry and soak in gold seeds for 12h, and finally in the mixed solution of 1mM hydroxylamine hydrochloride and 0.05wt% chloroauric acid and then placed in the aptamer solution obtained in S11 and incubated at 4°C for 16 hours. After taking it out, it was washed with 10mM PBS (pH=7.4) buffer solution, and then blocked in 1mM MCH solution. 2h, after taking it out, wash it again with 10mM PBS (pH=7.4) buffer solution to get the capillary glass tube/gold layer/nucleic acid aptamer electrode;
S13.取0.05μL 0.5％Nafion 117全氟化树脂溶液注入到毛细玻璃管/金层/核酸适配体电极(即缩口段)的尖端，在室温下固化尖端端口形成Nafion 117膜，由此得到生物阴极；S13. Get 0.05 μ L of 0.5% Nafion 117 perfluorinated resin solution and inject it into the tip of the capillary glass tube/gold layer/nucleic acid aptamer electrode (i.e. the shrinking section), and solidify the tip port at room temperature to form a Nafion 117 film, thereby get a biocathode;
S21.将干净的长度为7cm,直径为125μm的透明导光光纤的前端(前端约2cm长)在1.5mg/mL的多巴胺溶液浸泡2h，水洗干燥，采用丙酮处理3s去掉光纤前端(0.8cm)的聚多巴胺层和光纤包层使其能在前端透光，水洗干燥后在金种中浸泡4h，最后在1mM盐酸羟胺和0.05％的氯金酸的混合溶液中静置生长4min，水洗干燥，得到前端生长有金层的光纤，其中，光纤的金层修饰区域的纵向长度约1.7cm，该金层修饰区域距离该光纤修饰端的端口的距离约0.8cm；S21. Soak the front end of a clean transparent light-guiding optical fiber with a length of 7 cm and a diameter of 125 μm (the front end is about 2 cm long) in 1.5 mg/mL dopamine solution for 2 hours, wash and dry, and use acetone for 3 seconds to remove the front end of the optical fiber (0.8 cm). The polydopamine layer and the optical fiber cladding make it possible to transmit light at the front end. After washing and drying, soak in gold seeds for 4 hours, and finally grow in a mixed solution of 1mM hydroxylamine hydrochloride and 0.05% chloroauric acid for 4 minutes, wash and dry, Obtain an optical fiber with a gold layer grown on the front end, wherein the longitudinal length of the gold layer modified region of the optical fiber is about 1.7 cm, and the distance between the gold layer modified region and the port of the fiber modified end is about 0.8 cm;
S22.将S21所得光纤前端在含1wt％PDDA溶液中吸附5min，用水浸洗后在0.5mg/mL羧基化MWNT溶液中吸附5min，此为一个循环；共4个循环，水洗干燥，得到光纤/金层/MWNT电极；光纤修饰端最前端0.8cm部分没有修饰金层，但修饰了碳纳米管，碳纳米管修饰区域的纵向长度约2.5cm；S22. Adsorb the front end of the optical fiber obtained in S21 in a solution containing 1wt% PDDA for 5 minutes, soak it in water and then absorb it in a 0.5 mg/mL carboxylated MWNT solution for 5 minutes. This is a cycle; a total of 4 cycles are washed and dried to obtain an optical fiber/ Gold layer/MWNT electrode; the first 0.8cm part of the modified end of the optical fiber is not decorated with gold layer, but is decorated with carbon nanotubes, and the longitudinal length of the carbon nanotube modified area is about 2.5cm;
S23.将光纤/金层/MWNT电极的修饰端在黑暗环境下置于0.1M AgNO3溶液中浸泡30min，用水浸洗后继续在0.12M Na2S溶液中浸泡30min，水洗干燥；然后依次在10mM Bi(NO3)3溶液和10mM Na2S溶液中各浸泡10min，此为吸附Bi2S3一个循环，循环两次，水洗干燥，由此得到光阳极；S23. Soak the modified end of the optical fiber/gold layer/MWNT electrode in a 0.1MAgNO solution for 30 minutes in a dark environment, soak it in water for 30 minutes, then soak it in a 0.12 M Na2 S solution, wash and dry it; Soak in 10mM Bi(NO3 )3 solution and 10mM Na2 S solution for 10 min each, this is one cycle of adsorption of Bi2 S3 , cycle twice, wash and dry with water, thus obtain photoanode;
S3.将S13所得生物阴极内灌注“燃料”抗坏血酸(50mM)，将S23制备的光阳极修饰端插入生物阴极的修饰端，抗坏血酸溶液要高于光阳极修饰端的高度，即得到同轴集成的植入式光燃料传感器，可以用于活体内跟踪检测持久性有机污染物。S3. Pour "fuel" ascorbic acid (50mM) into the biocathode obtained in S13, and insert the modified end of the photoanode prepared in S23 into the modified end of the biocathode. The photofuel sensor can be used to track and detect persistent organic pollutants in vivo.
应用例1Application example 1
采用实施例制备的同轴集成的植入式光燃料传感器对缓冲溶液中的PCB77进行检测。以650nm的激光笔作为光源，用便携式pH计测量开路电位，将同轴集成的植入式光燃料传感器插入含有不同浓度PCB77的缓冲溶液中，并用激光笔与光阳极的非修饰端连接，生物阴极和光阳极分别与pH计的两端通过铜丝连接，然后测定开路电压，得到该同轴集成的植入式光燃料传感器对系列浓度的PCB77的响应信号如表1所示。The coaxially integrated implantable optical fuel sensor prepared in the embodiment is used to detect the PCB77 in the buffer solution. Using a 650nm laser pointer as the light source, the open circuit potential was measured with a portable pH meter, the coaxially integrated implantable photofuel sensor was inserted into the buffer solution containing different concentrations of PCB77, and the laser pointer was connected to the non-modified end of the photoanode. The cathode and the photoanode were respectively connected to the two ends of the pH meter through copper wires, and then the open circuit voltage was measured to obtain the response signals of the coaxial integrated implantable photofuel sensor to a series of concentrations of PCB77, as shown in Table 1.
表1PCB77测试结果Table 1 PCB77 test results
分析表1数据可知，该植入式光燃料传感器对PCB77系列浓度响应随着浓度的增加，产生的开路电位信号逐渐减小；线性拟合发现开路电位变化值ΔOCP(目标物的OCP与背景的OCP之差)与PCB77浓度的对数之间呈线性关系(附图3)，所得回归方程为：ΔOCP＝22.6+12.21lg C(R2＝0.998)(ΔOCP：mV；C：pg/mL)，线性范围为0～10000pg/mL，检测灵敏度为2.8fg/mL，能满足实际检测要求。Analysis of the data in Table 1 shows that the implantable photofuel sensor responds to the concentration of PCB77 series, and the open circuit potential signal gradually decreases with the increase of the concentration; linear fitting finds that the open circuit potential change value ΔOCP (the OCP of the target object and that of the background The difference between OCP) and the logarithm of PCB77 concentration showed a linear relationship (accompanying drawing 3), and the obtained regression equation was: ΔOCP=22.6+12.21lg C (R2 =0.998) (ΔOCP: mV; C: pg/mL) , the linear range is 0-10000pg/mL, and the detection sensitivity is 2.8fg/mL, which can meet the actual detection requirements.
应用例2Application example 2
采用实施例制备的同轴集成的植入式光燃料传感器对鱼脑内富集的PCB77进行原位监测，具体过程如下：The coaxial integrated implantable optical fuel sensor prepared in the embodiment is used to monitor the PCB77 enriched in the fish brain in situ, and the specific process is as follows:
在进行体内试验之前，将鱼放入含脱氯自来水的充气水族箱中两周，以适应饲料。将36尾草鱼分为4组(每50L水族箱(40L水)9尾鱼)，分别在0(对照组)、0.1、1、10ng/mLPCB77水中饲养3、6、9天。为了保持水中PCB77浓度稳定，每12小时更换三分之二的污染水，每天监测水质(pH 6.8±0.2，溶解氧7.0±0.3mg/L，温度25±0.7℃)。在每个暴露间隔结束时，每个水族箱取出3条鱼进行体内PCB77的光电化学检测。Fish were placed in aerated aquariums with dechlorinated tap water for two weeks to acclimate to the diet prior to in vivo trials. 36 grass carp were divided into 4 groups (9 fish per 50L aquarium (40L water)), and fed in 0 (control group), 0.1, 1, 10ng/mLPCB77 water for 3, 6, 9 days respectively. In order to keep the concentration of PCB77 in the water stable, two-thirds of the polluted water was replaced every 12 hours, and the water quality was monitored daily (pH 6.8±0.2, dissolved oxygen 7.0±0.3 mg/L, temperature 25±0.7°C). At the end of each exposure interval, three fish were removed from each aquarium for in vivo photoelectrochemical detection of PCB77.
体内分析首先用0.03％丁香酚麻醉鱼，直到失去垂直平衡。然后用一根26号针灸针沿着鱼身轴线在两只眼睛的中点刺入鱼头，插入约7mm，以穿透头骨；之后，将针取出，将集成传感器插入约孔中1.7cm，以穿透整个大脑。同时，以650nm的激光笔作为激发源，激光笔与光阳极的非修饰端连接，生物阴极和光阳极分别通过其表面的金层与pH计的两端以铜丝连接。最后，将鱼放入淡水中恢复垂直平衡，同时通过pH计检测OCP响应。In vivo analysis Fish were first anesthetized with 0.03% eugenol until vertical balance was lost. Then use a 26-gauge acupuncture needle to pierce the head of the fish along the axis of the fish body at the midpoint of the two eyes, and insert about 7mm to penetrate the skull; after that, take out the needle and insert the integrated sensor into the hole about 1.7cm, to penetrate the entire brain. At the same time, a 650nm laser pointer is used as the excitation source, and the laser pointer is connected to the non-modified end of the photoanode, and the biocathode and the photoanode are respectively connected to the two ends of the pH meter through the gold layer on its surface with copper wires. Finally, the fish were placed in fresh water to restore vertical balance while monitoring the OCP response by a pH meter.
对暴露于不同浓度的PCB77和不同时间的鱼的大脑中PCB77的浓度进行了监测(如图5)，结果显示对照组鱼脑中(无PCB77暴露)的传感器的OCP信号在整个实验过程中基本保持不变，说明传感器背景稳定，PCB77在鱼脑中的积累与暴露剂量和暴露时间呈正相关，证实构建的光燃料微传感器可以直接跟踪持久性有机污染物在活体中的生物积累水平。最后，采用气相色谱法检测了鱼脑中的PCB77浓度，所得结果与所制备的微传感器检测结果非常接近(如图6)，证明了本发明所述光燃料传感器具有较高的可靠性。The concentration of PCB77 in the brains of fish exposed to different concentrations of PCB77 and at different times was monitored (Fig. 5), and the results showed that the OCP signal of the sensor in the brains of control fish (without PCB77 exposure) remained basically the same throughout the experiment. It remained unchanged, indicating that the background of the sensor was stable, and the accumulation of PCB77 in the fish brain was positively correlated with the exposure dose and exposure time, confirming that the constructed photofuel microsensor could directly track the bioaccumulation level of persistent organic pollutants in vivo. Finally, the concentration of PCB77 in the fish brain was detected by gas chromatography, and the obtained result was very close to the detection result of the prepared microsensor (as shown in Figure 6), which proved that the optical fuel sensor of the present invention has high reliability.
根据上述说明书的揭示和教导，本发明所属领域的技术人员还可以对上述实施方式进行变更和修改。因此，本发明并不局限于上面揭示和描述的具体实施方式，对发明的一些修改和变更也应当落入本发明的权利要求的保护范围内。此外，尽管本说明书中使用了一些特定的术语，但这些术语只是为了方便说明，并不对本发明构成任何限制范围。According to the disclosure and teaching of the above-mentioned specification, those skilled in the art to which the present invention belongs can also make changes and modifications to the above-mentioned embodiment. Therefore, the present invention is not limited to the specific embodiments disclosed and described above, and some modifications and changes to the invention should also fall within the protection scope of the claims of the present invention. In addition, although some specific terms are used in this specification, these terms are only for convenience of description and do not constitute any limitation to the scope of the present invention.
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