WO2022120923A1 - 一种基于功能化纳米探针的单细胞电化学传感器及其应用 - Google Patents

一种基于功能化纳米探针的单细胞电化学传感器及其应用 Download PDF

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WO2022120923A1
WO2022120923A1 PCT/CN2020/137569 CN2020137569W WO2022120923A1 WO 2022120923 A1 WO2022120923 A1 WO 2022120923A1 CN 2020137569 W CN2020137569 W CN 2020137569W WO 2022120923 A1 WO2022120923 A1 WO 2022120923A1
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cell
electrochemical
toxin
functionalized
nanoprobe
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孙秀兰
孙嘉笛
王利平
纪剑
张银志
高璐
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江南大学
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
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    • G01N27/28—Electrolytic cell components
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    • G01N27/28—Electrolytic cell components
    • G01N27/30—Electrodes, e.g. test electrodes; Half-cells

Definitions

  • the invention relates to a single-cell electrochemical sensor based on functionalized nano-probe and its application, belonging to the technical field of electrochemical sensor and toxin detection.
  • Cellular sensors can be used to qualitatively or quantitatively detect unknown toxic substances and determine the presence and content of such substances based on the specific properties of excitatory action potentials and cellular mechanisms to detect and evaluate harmful substances.
  • Research at the level of individual cells can obtain more accurate and comprehensive information reflecting the physiological state and process of cells, better understand some special cell functions in cell populations, and have a deeper understanding of cell-to-cell differences and cell-to-cell interactions. Information and deeper information such as neurotransmitters, physiological effects of drug stimulation, etc.
  • Nanoelectrochemistry plays a key role in a wide range of interdisciplinary research in biochemistry, neuroscience, catalysis, molecular electronics, nanoscience (such as nanopores, nanobubbles and nanoparticles), polymer science, electrodeposition and renewable technologies effect.
  • nanoelectrodes minimize damage during penetrating living cells and are particularly beneficial for intracellular measurements of these species.
  • chemical measurements in solution have nanometer spatial resolution, high temporal resolution, and ultra-high sensitivity and selectivity.
  • T-2 toxin is a fungal toxin produced by the genus Fusarium, belonging to the class A trichothecenes toxins, and is also the most toxic one. Because T-2 toxin is widely distributed in nature, T-2 toxin may exist in crops grown in the field such as barley, wheat, oats, rye and corn, as well as stored grains, and T-2 toxin may exist It can be produced between -2 and 35 °C, and the yield will increase with the increase of ambient humidity. T-2 toxin is difficult to degrade, and ordinary cooking methods will not reduce its toxicity, so it is a serious threat to human and animal health.
  • T-2 toxin As one of the most dangerous natural food contamination toxins. T-2 toxin should not exceed 0.5mg/kg in feed. T-2 toxin can induce oxidative stress in a variety of cells in vivo and in vitro. Many researchers also explain the various toxic effects caused by T-2 toxins from the perspective of oxidative stress, such as cytotoxicity, immunotoxicity, genotoxicity, reproductive toxicity and neurotoxicity. Hydrogen peroxide is the most representative free radical of intracellular reactive oxygen species (ROS). The level of intracellular reactive oxygen species and hydrogen peroxide is closely related to the physiology and pathology of organisms. However, overproduction of ROS can overwhelm cellular free radical scavenging and repair systems, leading to tissue dysfunction and oxidative stress. T-2 toxin can activate the ROS-dependent mitochondrial apoptosis pathway, thereby causing mitochondrial dysfunction.
  • ROS reactive oxygen species
  • toxicity evaluation methods mainly rely on multi-cell experiments and animal experiments.
  • Multi-cell experimental methods have low cost, short cycle and certain homology with the body.
  • multi-cell culture has low sensitivity and cannot realize real-time monitoring.
  • the results of animal toxicology experiments Although it can truly and comprehensively reflect the impact of drugs on the body, it has disadvantages such as high cost, long cycle, and unsatisfactory repeatability.
  • the patent discloses a graphene-based single-cell sensing method. Specifically, graphene is transferred to a transparent substrate, and an appropriate microfluidic channel is selected according to the size of the cell to be tested and pasted on the graphene. Focusing on the graphene covered with the microfluidic channel, the outgoing light is divided into s and p polarizations and irradiated to the two probes of the balanced detector respectively. The voltage signal is collected, and the cell signal is analyzed and processed to obtain the characteristic information of the cell.
  • this method can only distinguish single cell morphology, and further research is needed for drug detection.
  • the present invention provides a single-cell electrochemical sensor based on functionalized nanoprobes and its application on the mycotoxin T-2 toxin.
  • the invention utilizes the combination of nano-probes and electrochemical cell sensors to construct a reliable, easy-to-operate and highly repeatable liver cancer single-cell system, and the electrochemical chronoamperometry is used to determine the current value of the single cell to determine whether a single cell is stimulated by toxins. damage conditions for a rapid and efficient assessment of mycotoxin cytotoxicity.
  • the first object of the present invention is to provide a functionalized nano-probe for single-cell electrochemical sensing.
  • the construction method of the functionalized nano-probe includes the following process: drawing a capillary into a nano-microneedle, Gold nanoparticles are deposited on the tips of microneedles to make nanoprobes; then Prussian blue is deposited on the obtained nanoprobes to obtain functionalized nanoprobes.
  • the process of depositing gold nanoparticles is to immerse the microneedle tips in a sulfuric acid solution containing chloroauric acid, at an initial potential of -0.25V, and deposit for 15-20s.
  • the concentration of chloroauric acid in the sulfuric acid solution is 1 mmol ⁇ L -1 ; the concentration of sulfuric acid is 0.5 mol ⁇ L -1 .
  • the process of Prussian blue deposition is: electrochemical deposition in a plating solution containing 0.1M HCl, 2mM FeCl 3 , 0.1M KCl and 2mM K 3 [Fe(CN) 6 ]; potential Cycle between 0.2 and -0.6V for 50 cycles.
  • the tip radius of the functionalized nanoprobe is 200-400 nm.
  • the specific process of the nanoprobe includes: the initial drawing tip is 200nm; the gold nanoparticles are electroplated at 50-100nm, the initial point is -0.25V, and the time is 20s; the probe tip is A 1-2 cm gold layer was exposed as the electrochemical sensing part.
  • the preparation method of the functionalized nanoprobe specifically includes:
  • the nano-microneedle drawn by the glass capillary using a needle-pulling instrument is coated with gold nanoparticles on the tip of the needle to be characterized by the electrodeposition method, PDMS is used to insulate the outer layer of the electrode, and the nano-probe is wrapped with Apiezon wax. On the surface, the gold layer is exposed at the tip as the electrochemical sensing part;
  • nanoprobes were further modified by electrochemical deposition of Prussian blue, the potential was cycled for 50 cycles, and the Prussian blue-modified nanoprobes were rinsed with deionized water and dried at room temperature.
  • the second object of the present invention is to provide a single-cell electrochemical sensor, wherein the working electrode of the single-cell electrochemical sensor is the above-mentioned functionalized nanoprobe.
  • the third object of the present invention is to provide a single-cell toxicity detection method using the above-mentioned functionalized nanoprobes
  • the above-mentioned functionalized nano-probes are clamped on a micro-operating system for automatic control, and are directly pierced into cells for electrochemical detection.
  • the single-cell electrochemical sensor is a direct electrochemical detection of a single cell.
  • the cell is a human hepatoma cell HepG2.
  • the detection method is to localize the functionalized nanoprobes on a cell of a single cell, at a distance of up to 500 ⁇ m from other cells.
  • the fourth object of the present invention is to provide a method for detecting the toxicity of class A trichothecenes T-2 toxins by using the above single-cell electrochemical sensor. It was diluted into a gradient concentration solution, added to a cell culture dish, and electrochemically detected after 5 minutes, and the cytotoxicity of the toxin was analyzed by electrochemical chronoamperometry IT.
  • the process of analyzing the cytotoxicity of the toxin by the electrochemical chronoamperometry IT comprises:
  • the standard curve A was constructed by using the concentration values of H 2 O 2 standard samples with different concentrations and the current value output by the single-cell electrochemical sensor ; B; By detecting the current value of the sample to be tested, and based on the standard curves A and B, the concentration of the toxin in the sample to be tested is measured.
  • the single-cell sensor needs to be cultured before application, and the specific operation is as follows: cells in the logarithmic growth phase are passaged at 1:5, placed in 37°C, and the concentration of carbon dioxide is 5%. 2. Incubate in a 95% humidity incubator for 6-12 hours; dilute the toxin standard substance with MEM cell culture medium to form a gradient concentration solution, add it to the culture dish respectively, and conduct electrochemical detection after 5 minutes.
  • the current signal is measured in an Autolab PGSTAT302N electrochemical workstation, and a working signal of 600 mV is collected.
  • a standard curve of the concentration and the current value needs to be drawn for the hydrogen peroxide solution with a determined concentration.
  • the single-cell detection is performed under an inverted microscope using a micro-operating system SenSapex UMP.
  • the T-2 toxin evaluation is the detection of reactive oxygen species, especially hydrogen peroxide, produced in cells.
  • the fifth object of the present invention is the application of the single-cell electrochemical sensor in the fields of non-disease diagnosis and treatment of drug development, toxicology testing, and nano-environment real-time monitoring.
  • the present invention has the following advantages:
  • the present invention adopts the modified functionalized nano-probe, which can specifically detect hydrogen peroxide in cells, so that the prepared sensor has higher sensitivity and lower detection limit for toxin detection.
  • the nano-electrode used in the present invention can minimize the damage in the process of penetrating living cells due to its small size, perform intracellular measurement, and perform real-time signal detection of toxins.
  • the single-cell sensor of the present invention can evaluate the degree of toxicity of mycotoxin T-2 toxin. For a long time, food and feed in my country have been seriously polluted by mycotoxins.
  • the present invention can evaluate the cytotoxicity of a single toxin, and can further determine its mechanism type, which can provide a reference for the determination of relevant detection standards.
  • the present invention constructs a single-cell electrochemical detection system by rationally combining functionalized nanoprobes with single-cell electrochemical sensing. It is a new method and new idea, which is expected to be applied in the fields of food safety, biomedicine and so on.
  • Figure 1 Schematic diagram of the single-cell electrochemical sensor based on functionalized nanoprobes.
  • Figure 2 Characterization diagram of functionalized nanoelectrode construction. Among them, A is the electron microscope characterization image of the nano-electrode; B is the electrochemical characterization before and after Prussian blue modification.
  • Figure 3 Calibration plot of steady - state current versus H2O2 concentration. The inset shows the linear relationship of 1 nM - 100 nM H2O2 with peak current.
  • FIG. 4 Single-cell electrochemical sensor evaluation of T-2 toxin detection results.
  • A is a.1ppb, b.10ppb, c.100ppb, d.1ppm, e.0ppb (control group) real-time chronoamperometry of cells stimulated by T-2 toxin (the probe penetrated into a single cell at 35s)
  • inset is the light micrograph of nanoprobe penetration into single HepG2 cells
  • C is T-2 toxin stimulation detected by single-cell electrochemical sensing
  • the peak current value of the cells corresponds to the H2O2 concentration and a linear fit was performed. .
  • Figure 5 Experimental results of evaluating cell proliferation activity by CCK8 method.
  • Figure 6 The experimental results of DCFH-DA fluorescence method to evaluate intracellular reactive oxygen species.
  • A is the fluorescence intensity obtained by the determination of reactive oxygen species in HepG2 cells;
  • B is the fluorescence image of reactive oxygen species in HepG2 cells.
  • FIG. 7 Real-time chronoamperometry of HepG2 single cells stimulated by 1 ppb T-2 toxin (nanoprobe penetrates single cell at 0 s, T-2 toxin is added to the dish at 60 s).
  • Figure 8 A. DPV curves of HepG2 cells stimulated by T-2 toxin detected by electrochemical sensing of GelMA/AuNPs/GCE cells, the concentration of T-2 toxin is 0ppb, 1ppb, 2ppb, 5ppb, 10ppb from bottom to top , 20ppb, 100ppb, 200ppb, 500ppb, 1pppm, 2pppm; B. Linear fitting of T-2 toxin-stimulated cell peak current detected by GelMA/AuNPs/GCE multicellular electrochemical sensor.
  • Figure 9 DVP curves of the effect of different gold plating times on the nanoprobe signal.
  • Figure 10 Chronocurrent curves of the effect of modified Prussian blue on nanoprobes with different cycle periods.
  • a method for constructing a single-cell electrochemical sensor based on functionalized nanoprobes including the following steps:
  • HepG2 human hepatoma cells were cultured in a MEM medium containing 10% fetal bovine serum and 1% 100 ⁇ g/mL penicillin-streptomycin in a 37°C incubator with a saturated humidity of 5% CO 2 . nourish. The cells grow adherently, and the culture medium is changed every 3 days. When the cells cover 90% of the bottom area of the flask, they can be subcultured.
  • nano-probes The glass capillary was used to pull out nano-micro needles with a tip opening of about 200 nm, and gold nanoparticles of about 50-100 nm were plated on the tip of the needle to be characterized by the electrodeposition method (the The nanoprobe was immersed in a 0.5 mol ⁇ L -1 sulfuric acid solution containing 1 mmol ⁇ L -1 chloroauric acid, with an initial potential of -0.25 V for 20 seconds), and the outer layer of the electrode was insulated with PDMS, and the nanoprobe was wrapped with Apiezon wax. Surface, the tip exposed 1-2 cm Au layer as the electrochemical sensing part.
  • Prussian blue (PB) was used for electrochemical deposition of Prussian blue (PB) in a deposition solution containing 0.1 M HCl, 2 mM FeCl 3 , 0.1 M KCl and 2 mM K 3 [Fe(CN) 6 ].
  • the probe is further modified.
  • the potential was cycled between 0.2 and -0.6V for 50 cycles.
  • the PB-modified nanoprobes were then rinsed with deionized water and dried at room temperature.
  • the prepared functionalized nanoprobes were characterized by scanning electron microscopy (Fig. 2A).
  • the nanoprobe tip diameter is in the range of 200-400 nm.
  • Cyclic voltammogram characterization was tested using a CHI660e electrochemical workstation.
  • the probe tip was performed in 2.5mM Fe(CN) 6 3-/4- and 1.0M KCl electrolytes.
  • the reference electrode and auxiliary electrode were Ag electrode and Pt electrode, respectively.
  • the cycle voltage is -0.1 ⁇ 0.6V
  • the scanning speed is 0.1V/s.
  • Figure 2B shows that a characteristic signal peak appears at ⁇ -0.1 V after PB modification, and the reduction peak responds to the conversion of PB to Prussian white (PW ) , which is necessary for the electrocatalysis of H2O2 , PW has the function of reducing H2O2 as an electron transport medium, indicating that PB is successfully deposited on the nanoprobes .
  • PW Prussian white
  • the nanoprobes were used to collect current signals of H 2 O 2 solutions with different concentrations at a voltage of 0.6V.
  • Figure 3 is the corresponding calibration curve.
  • Example 1 The single-cell electrochemical sensor obtained in Example 1 was used to evaluate the single-cell toxicity of mycotoxins T-2 toxin, as follows:
  • Drug stimulation remove the original culture solution in the culture dish, dilute the toxin standard substance with MEM cell culture solution to a gradient concentration solution, and then add 0ppb, 1ppb, 10ppb, 100ppb, 1ppm T-2 toxin to the cell culture dish After 5 min, single-cell electrochemical detection was performed.
  • SD is the standard deviation of the lowest concentration
  • slope is the fitting slope of the curve.
  • T-2 toxin was added at the following concentrations: 0, 1, 10, 100, 1000 ppb (Table 1).
  • the average spike recovery of samples based on single-cell electrochemical sensing was 81.19%-130.17%, indicating that the method has high accuracy and detection efficiency, and can be used for the detection of T-2 toxin in real samples.
  • Detection of cytotoxicity after T-2 toxin effect by CCK8 method Human hepatoma cells HepG2 with a density of 5 ⁇ 10 4 cells/mL were inoculated into a 96-well plate, and the culture medium was removed after culturing for 24 hours. the same dose of toxin solution. After 24 hours of toxin stimulation, aspirate the supernatant and add 100 ⁇ L of culture medium containing 10% CCK8 to each well, incubate at 37°C for 2 hours, and then measure the absorbance value at 450 nm using a microplate reader, and calculate the cell activity inhibition rate.
  • the calculation method is as follows:
  • OD dosing absorbance value after 24h of toxin stimulation
  • OD 0 dosing absorbance value after 24h stimulation without toxin
  • OD blank absorbance value of pure cell culture solution.
  • DCFH-DA fluorescent probe was used to detect the levels of reactive oxygen species in vivo after cells were stimulated by mycotoxins. HepG2 cells were inoculated into six-well plates. After the cells adhered and entered the logarithmic growth phase, complete culture medium containing different concentrations of T-2 toxin was added and incubated in a carbon dioxide incubator for 24 hours. The culture medium was discarded and washed with PBS by centrifugation. And suspend by blowing, add DCFH-DA with a final concentration of 10 ⁇ mol/L, mix well, and incubate at 37 °C for 30 min in the dark to promote the full combination of probe and cells.
  • ROS reactive oxygen species
  • the cells were washed twice with serum-free MEM medium, and the average fluorescence intensity (excitation wavelength 488 nm, emission wavelength 530 nm) was measured by a microplate reader, and fluorescence pictures were taken by an inverted fluorescence microscope.
  • Electrochemical sensors can easily quantify targets and further analyze real-time data for key parameters of biochemical processes.
  • the nanoprobes were brought into contact with the cytoplasm, and 1 ppb of T-2 toxin was added to the dish.
  • Figure 7 demonstrates that real - time current traces of H2O2 in single HepG2 cells were detected by single-cell electrochemical sensing after T- 2 toxin stimulation.
  • T-2 toxin was added to the culture dish for about 60 s for stimulation, the current value increased 20 s after stimulation.
  • the experimental group showed a clear peak current at about 70s after stimulation. When the peak current is reached, the current is stable for 1-5min, and then gradually decreases. Controls the cellular redox balance by balancing ROS production and eliminating ROS through ROS scavenging systems.
  • the cleaned and polished glassy carbon electrode (GCE) was immersed in a 0.5M H2SO4 solution containing 1 mM HAuCl4 and electrodeposited using a potential - controlled coulomb method (potential of ⁇ 0.25 V for 100 s).
  • the modified electrodes were placed in the electrolyte for CV scanning.
  • the cycling voltage was -0.6–0.6 V, and the scan speed was 0.1 V/s.
  • the digested cell suspension was mixed with gelatin-methacryloyl (GelMA) hydrogel to ensure a concentration of 10 6 cells/mL. 6 ⁇ L of the mixture was then added to the electrode surface.
  • the gold deposition process was replaced by: optimizing the gold deposition time, soaking the nanoprobes in 1 mmol ⁇ L-1 chloroauric acid and 0.5 mol ⁇ L-1 sulfuric acid solution, the initial potential -0.25, and electrodepositing for 5s respectively , 10s, 15s, 20s, 25s, 30s, the electrical signals of the gold-plated electrodes were detected by DPV, and the changes of cell morphology were observed by penetrating the cells with nanoprobes.
  • the longer the gold plating time the greater the peak current displayed by the DPV.
  • the nanoprobes with the gold-plating time of 25s and 30s had obvious damage to the cells, and the cells had obvious depressions after penetrating, indicating that the gold-plated time was too long and the diameter of the probe was too large, so gold-plated nanoprobes were selected. Time 20s.
  • the alternative Prussian blue deposition process is: optimizing the Prussian blue deposition cycle, by electrochemical deposition of Prussian blue in a plating solution containing 0.1M HCl, 2mM FeCl 3 , 0.1M KCl and 2mM K 3 [Fe(CN) 6 ] Blue (PB) was used to further modify the nanoprobes.
  • the potential was cycled between 0.2 and -0.6V for 10, 20, 50, 100, and 150 cycles, respectively. 20 ⁇ M H2O2 was detected by chronoamperometry .

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Abstract

一种基于功能化纳米探针的单细胞电化学传感器及其应用，属于电化学传感器和毒素检测技术领域。单细胞电化学传感器是利用纳米探针和电化学细胞传感器结合，使用普鲁士蓝对纳米探针进行功能化修饰，通过显微操作平台对单个细胞进行电流信号分析。构建了一种可靠、操作简便、可重复性强的单细胞电化学检测平台，通过电化学计时电流法测定其电流值判断单个细胞受毒素刺激后的受损情况，从而快速、有效地评价真菌毒素细胞毒性，进一步使真菌毒素毒性在活细胞中实时监测和纳米环境检测中得到应用。

Description

一种基于功能化纳米探针的单细胞电化学传感器及其应用 技术领域
本发明涉及一种基于功能化纳米探针的单细胞电化学传感器及其应用，属于电化学传感器和毒素检测技术领域。
背景技术
细胞传感器可用于定性或定量地检测未知有毒物质，并基于兴奋作用电位和细胞机制的特定特性确定这种物质的存在和含量，以检测和评估有害物质。在单个细胞水平上进行研究，可以获得反映细胞生理状态和过程更为准确、全面的信息，更好地了解细胞群体中某些特殊的细胞功能，更加深入的认识细胞间差异、细胞间相互作用信息和神经递质、药物刺激的生理影响等更深层次的信息。纳米电化学在生物化学、神经科学、催化、分子电子学、纳米科学(如纳米孔、纳米气泡和纳米颗粒)、聚合物科学、电沉积和可再生技术等广泛的跨学科研究中发挥着关键作用。纳米电极由于其体积小，可将穿透活细胞过程中的损伤降至最低，特别有利于对这些物种的细胞内测量。近年来，随着纳米电化学的发展，在溶液中进行化学测量具有纳米的空间分辨率、高的时间分辨率和超高的灵敏度和选择性。
T-2毒素是由镰刀菌属产生的一种真菌毒素，属于A类单端孢霉烯族毒素，也是毒性最强的一种。因为T-2毒素在自然界中分布极其广泛，在田间生长的作物如大麦、小麦、燕麦、黑麦和玉米等，以及库存的谷物中都可能有T-2毒素的存在，而且T-2毒素在-2～35℃之间都可以产生，产量会随着环境湿度的升高而增加。T-2毒素难以降解，普通的烹饪方式不会降低它的毒性，因此对人类和动物的健康都是一个严重威胁。1973年，世界粮食组织/卫生组织(JECFA)将T-2毒素认定为最危险的天然食品污染毒素之一，2017年中国发布了关于植物性饲料原料及猪、禽配合饲料国家标准，规定了T-2毒素在饲料中不能超过0.5mg/kg。T-2毒素能够引起动物体内和体外多种细胞中发生氧化应激反应。许多研究者也从氧化应激角度解释T-2毒素引起的多种毒性作用，如细胞毒性、免疫毒性、遗传毒性、生殖毒性和神经毒性等。过氧化氢是细胞内活性氧(ROS)最具有代表性的自由基，细胞内活性氧过氧化氢水平与生物的生理、病理密切相关。然而，ROS的过度生成会压倒细胞自由基清除和修复系统，导致组织功能障碍和氧化应激。T-2毒素能够激活ROS依赖的线粒体凋亡通路，从而引起线粒体功能损伤。
目前毒性评价方法主要依赖于多细胞实验及动物实验，多细胞实验方法成本低、周期短、 与机体具有一定同源性，但是多细胞培养灵敏度低，无法实现实时监测，动物毒理实验的结果虽能够真实全面系统地反映药物对机体的影响，但存在着成本高、周期长、重复性不够理想等缺点。
随着科技发展与进步，传统细胞和传感器技术相结合的多种新技术、新方法为毒性机制研究提供更多新的手段。细胞传感器的构建中，细胞作为感受器被固定到界面上，当受到外界药物刺激后会引起细胞生理活性改变，这些变化可被转化为光电信号，信号变化的大小可对细胞受到的药物刺激进行定性定量分析。目前专利(CN201610231154.0)提供了一种细胞检测石房蛤毒素的方法，检测线性范围1-10nM，即2.99-29.9ppb，检测限较高，灵敏度较低，且该发明结合ELISA检测方法，并没有利用电化学传感方式。专利(CN201310511626.4)公开了一种基于石墨烯的单细胞传感方法，具体是将石墨烯转移到透明基底上，根据待测细胞大小选择合适的微流通道粘贴到石墨烯上，将光束聚焦照射到覆盖有微流通道的石墨烯位置，出射光分成s和p偏振分别照射到平衡探测器的两个探头，对电压信号进行采集，进行细胞信号分析处理，获得细胞的表征信息。但此方法只能区别单细胞形态，对药物检测还需进一步研究。
发明内容
为了解决上述至少一个问题，本发明提供了一种基于功能化纳米探针的单细胞电化学传感器及其在真菌毒素T-2毒素上的应用。本发明利用纳米探针和电化学细胞传感器结合，构建一种可靠、操作简便、可重复性强的肝癌单细胞体系，通过电化学计时电流法测定其电流值判断单个细胞受毒素刺激后的受损情况，从而快速、有效地评价真菌毒素细胞毒性。
本发明的第一个目的是提供一种用于单细胞电化学传感的功能化纳米探针，所述功能化纳米探针的构建方法包括如下过程：将毛细管拉制成纳米显微针，在显微针针尖上沉积金纳米粒子制成纳米探针；然后将普鲁士蓝沉积在所得纳米探针上，获得功能化纳米探针。
在本发明的一种实施方式中，所述沉积金纳米粒子的过程是将显微针针尖浸泡在含氯金酸的硫酸溶液中，初始电位-0.25V，沉积15-20s。
在本发明的一种实施方式中，所述硫酸溶液中氯金酸的浓度为1mmol·L -1；硫酸的浓度为0.5mol·L -1。
在本发明的一种实施方式中，普鲁士蓝沉积的过程为：在含有0.1M HCl，2mM FeCl 3，0.1M KCl和2mM K 3[Fe(CN) 6]的电镀液中电化学沉积；电位在0.2～-0.6V之间循环50个周期。
在本发明的一种实施方式中，所述功能化纳米探针的针尖半径为200-400nm。
在本发明的一种实施方式中，所述纳米探针的具体过程包括：初始拉制尖口开端为200nm；电镀金纳米粒子50-100nm，初始点位-0.25V，时间20s；探针尖端露出1-2cm金层作为电化学感应部分。
在本发明的一种实施方式中，所述功能化纳米探针的制备方法具体包括：
(1)将玻璃毛细管采用拉针仪拉制出的纳米显微针，通过电沉积法在待表征的针尖上镀约金纳米粒子，在电极外层采用PDMS绝缘，用Apiezon蜡包裹纳米探针表面，尖端露出金层作为电化学感应部分；
(2)通过电化学沉积普鲁士蓝对纳米探针进行进一步修饰，电位循环50个周期，用去离子水冲洗普鲁士蓝修饰的纳米探针，并在室温下干燥。
本发明的第二个目的是提供一种单细胞电化学传感器，所述单细胞电化学传感器的工作电极为上述的功能化纳米探针。
本发明的第三个目的是提供一种单细胞毒性检测方法，所述方法是利用上述功能化纳米探针
将上述功能化纳米探针夹持在显微操作系统上进行自动化操控，直接刺入细胞进行电化学检测。
在本发明的一种实施方式中，所述单细胞电化学传感器是对单个细胞进行直接电化学检测。
在本发明的一种实施方式中，所述细胞为人肝癌细胞HepG2。
在本发明的一种实施方式中，所述检测方法是将功能化纳米探针定位于一个单独细胞的细胞上，且距离其他细胞至500μm。
本发明的第四个目的是提供一种利用上述的单细胞电化学传感器检测A类单端孢霉烯族T-2毒素毒性的方法，所述方法为：将毒素标准物质用MEM细胞培养液稀释成梯度浓度溶液，加入细胞培养皿中，5min后进行电化学检测，利用电化学计时电流法IT分析毒素的细胞毒性。
本发明的一种实施方式中，所述电化学计时电流法IT分析毒素的细胞毒性的过程包括：
利用不同浓度的H 2O 2标准样品的浓度值与单细胞电化学传感器输出的电流值构建标准曲线A；然后在利用不同浓度的毒素标样的浓度值与H 2O 2浓度值构建标准曲线B；通过检测待测样的电流值，基于标准曲线A、B，测得待测样中毒素的浓度。
本发明的一种实施方式中，所述的单细胞传感器在应用之前需要先进行细胞培养，具体 操作为：将对数生长期的细胞进行1:5传代，放入37℃、二氧化碳浓度5％、湿度95％培养箱中孵育6～12h；将毒素标准物质用MEM细胞培养液稀释成梯度浓度溶液，分别加入培养皿中，5min后进行电化学检测。
在本发明的一种实施方式中，所述的电流信号是在Autolab PGSTAT302N电化学工作站测得，采集工作信号600mV。
在本发明的一种实施方式中，所述的过氧化氢检测之前需要对确定浓度的过氧化氢溶液进行浓度与电流值的标准曲线绘制。
在本发明的一种实施方式中，所述的单细胞检测采用微操作系统SenSapex UMP在倒置显微镜下进行。
在本发明的一种实施方式中，所述的T-2毒素评价是对细胞中产生的活性氧，特别是过氧化氢进行检测。
本发明的第五个目的是所述的单细胞电化学传感器在非疾病的诊断和治疗的药物开发、毒理学测试、纳米环境实时监测领域的应用。
与现有技术相比，本发明具有如下优势：
(1)本发明采用修饰功能化的纳米探针，可以特异性的检测细胞中过氧化氢，从而使所制备的传感器对毒素的检测具有更高的灵敏性和更低的检测限。
(2)本发明采用纳米电极由于其体积小，可将穿透活细胞过程中的损伤降至最低，进行单细胞内测量，可以对毒素进行实时信号检测。
(3)本发明的单细胞传感器可对真菌毒素T-2毒素毒性作用程度进行评价。一直以来，我国粮食、饲料受到真菌毒素的污染严重，本发明可以评价单一毒素的细胞毒性，还可以进一步判断其机理类型，可为相关检测标准确定提供参考依据。
(4)本发明通过功能化纳米探针与单细胞电化学传感领域合理结合，构建了单细胞电化学检测体系，该方法操作便捷、可靠、灵敏，为评价真菌毒素毒性纳米环境评价提供一种新方法、新思路，有望应用于食品安全、生物医药等领域。
附图说明
图1：基于功能化纳米探针的单细胞电化学传感器流程示意图。
图2：功能化纳米电极构建表征图。其中，A为纳米电极电镜表征图；B为普鲁士蓝修饰前后的电化学表征。
图3：稳态电流对H 2O 2浓度的校准图。插图显示了1nM-100nM H 2O 2与峰值电流的线性 关系。
图4：单细胞电化学传感器评价T-2毒素检测结果。其中，A为a.1ppb，b.10ppb，c.100ppb，d.1ppm，e.0ppb(对照组)T-2毒素刺激的细胞实时计时电流图(探针在35s时刺入单个细胞中)，插图是纳米探针渗透到单个HepG2细胞中的光学显微照片；B为HepG2单细胞传感器检测T-2毒素的峰值电流(n＝4)。p<0.05＝*，p<0.005＝**，p<0.001＝***，p<0.0001＝****，以下相同；C为通过单细胞电化学传感检测到的T-2毒素刺激细胞的峰值电流值对应H 2O 2浓度，并进行线性拟合。.
图5：CCK8法评价细胞增殖活性的实验结果。
图6：DCFH-DA荧光法评价细胞内活性氧的实验结果。A为HepG2细胞内活性氧测定获得的荧光强度；B为HepG2细胞中活性氧荧光图像。
图7：1ppbT-2毒素刺激HepG2单细胞的实时计时电流图(纳米探针在0s穿透单个细胞，T-2毒素在60秒时加入培养皿)。
图8：A.通过GelMA/AuNPs/GCE细胞电化学传感检测到T-2毒素刺激的HepG2细胞的DPV曲线，T-2毒素浓度从下到上依次为0ppb，1ppb，2ppb，5ppb，10ppb，20ppb，100ppb，200ppb，500ppb，1pppm，2pppm；B.GelMA/AuNPs/GCE多细胞电化学传感器检测的T-2毒素刺激细胞峰值电流的线性拟合。
图9：不同镀金时间对纳米探针信号影响的DVP曲线。
图10：修饰普鲁士蓝不同循环周期对纳米探针影响的计时电流曲线。
具体实施方式
以下对本发明的优选实施例进行说明，应当理解实施例是为了更好地解释本发明，不用于限制本发明。
实施例1单细胞电化学传感器的制备
一种基于功能化纳米探针构建单细胞电化学传感器的方法(图1)，包括以下步骤：
(1)细胞培养：将HepG2人肝癌细胞用含有10％胎牛血清和1％的100μg/mL青霉素-链霉素的MEM培养液，于饱和湿度5％CO 2的37℃恒温培养箱中进行培养。细胞为贴壁生长，每隔3天更换培养液一次，当细胞覆盖瓶底面积90％时，可传代培养。
(2)纳米探针的制备：将玻璃毛细管采用拉针仪拉制出尖端开口约200nm的纳米显微针，通过电沉积法在待表征的针尖上镀约50-100nm的金纳米粒子(将纳米探针浸泡在含1mmol·L -1氯金酸的0.5mol·L -1硫酸溶液中，初始电位-0.25V，20秒)，在电极外层采用PDMS 绝缘，用Apiezon蜡包裹纳米探针表面，尖端露出1-2cm Au层作为电化学感应部分。
(3)功能化纳米探针的修饰：在含有0.1M HCl，2mM FeCl 3，0.1M KCl和2mM K 3[Fe(CN) 6]的沉积液中通过电化学沉积普鲁士蓝(PB)对纳米探针进行进一步修饰。电位在0.2～-0.6V之间循环50个周期。然后用去离子水冲洗PB修饰的纳米探针，并在室温下干燥。
制备好的功能化纳米探针采用扫描电子显微镜表征(图2A)。纳米探针尖端直径在200-400nm范围内。
利用CHI660e电化学工作站测试循环伏安图表征，探针尖端在2.5mM Fe(CN) 6 3-/4-和1.0M KCl电解液中进行，参比电极和辅助电极分别是Ag电极和Pt电极，循环电压为-0.1～0.6V，扫描速度为0.1V/s。对比修饰前后氧化还原信号，图2B显示在PB修饰后在～-0.1V出现特征信号峰，还原峰响应为PB到普鲁士白(PW)的转化，这对于H 2O 2的电催化是必需的，PW作为电子传递介质具有还原H 2O 2的功能，表明PB成功沉积在纳米探针上。
用纳米探针对不同浓度的H 2O 2溶液在0.6V电压下进行电流信号采集，图3为对应的校准曲线，在0.1μM～100μM H 2O 2与电流值成线性，R 1 2＝0.98841，插图为1nM～100nM H 2O 2与电信号的线性关系，R 2 2＝0.97385。
实施例2基于功能化纳米探针的单细胞电化学传感器的应用
将实施例1得到的单细胞电化学传感器进行真菌毒素T-2毒素的单细胞毒性的评价，具体如下：
(1)药物刺激：移除培养皿中原有的培养液，将毒素标准物质用MEM细胞培养液稀释成梯度浓度溶液，然后将0ppb、1ppb、10ppb、100ppb、1ppmT-2毒素加在细胞培养皿中，5min后对其进行单细胞电化学检测。
(2)电化学信号值检测：电流信号是在Autolab PGSTAT302N电化学工作站上采用计时电流法于室温下测得，采集工作信号600mV。所有电化学实验均采用传统三电极体系，工作电极定位于一个单独细胞的细胞上，且距离其他细胞至少500μm。单细胞检测采用微操作系统SenSapex UMP在倒置显微镜下进行。PB修饰后的金纳米探针通过微操系统扎入HepG2细胞中。
以空气为空白对照，在600mV(vs-Ag/AgCl)的固定电位下记录了不同浓度T-2对细胞刺激的计时电流图。空白减法后，绘制电流与浓度的关系图，以获得线性图并得到检测限。检测限的计算方程如(1)所示：
Figure PCTCN2020137569-appb-000001
其中，SD为最低浓度标准差，slope为曲线的拟合斜率。
(3)结果判断
如图4A所示，在600mV时，纳米探针位于远离细胞的位置，然后逐渐接近并在约35秒时穿透细胞。不同浓度T-2毒素刺激的细胞中出现不同的阴极电流尖峰。对照组细胞的峰值电流为-0.14nA，1ppm T-2毒素刺激的细胞的峰值电流达到-0.24nA。图4B显示了每个峰值和控制组之间的显著差异。T-2毒素的浓度越高，峰值电流越高。此电流信号表明，在T-2毒素的刺激下，HepG2细胞不同程度地表现出氧化应激，产生H 2O 2并促使探针产生反应，导致电化学信号发生变化。将T-2毒素浓度与H 2O 2浓度对应并对其进行线性拟合图4C，由T-2毒素刺激的H 2O 2细胞在1ppb-1ppm下产生的H 2O 2浓度呈线性相关，R 2＝0.99055，检测限为0.13807ng/mL，检测浓度最低为1ng/mL。
(4)样品加标实验
在面粉上进行了样品添加实验，并以以下浓度添加了T-2毒素：0、1、10、100、1000ppb(表1)。基于单细胞电化学传感的样品的平均加标回收率为81.19％-130.17％，表明该方法具有较高的准确度和检测效率，可用于实际样品中T-2毒素的检测。
表1样品加标回收结果
Figure PCTCN2020137569-appb-000002
实施例3验证实验
CCK8法检测T-2毒素作用后的细胞毒性：将密度为5×10 4个/mL的人肝癌细胞HepG2贴壁接种到96孔板中，培养24h后去除培养液，加入100μL与实施例2中相同剂量的毒素溶液。毒素刺激24h后，吸出上清每孔加入100μL含10％CCK8的培养液，37℃孵育2h，然后在450nm下利用酶标仪测定吸光度值，并计算细胞活性抑制率，计算方法如下：
Figure PCTCN2020137569-appb-000003
其中，OD 加药：毒素刺激24h后的吸光度值，OD 0加药：无毒素刺激24h后的吸光度值，OD 空白：纯细胞培养液的吸光度值。
由图5可知，实施例1构建的单细胞电化学传感器评价T-2毒素细胞毒性的测定结构与传统细胞毒理学方法测定的结果一致性较好，能够有效判断毒素的细胞毒性。
细胞内活性氧(ROS)水平的测定：采用DCFH-DA荧光探针来检测细胞受真菌毒素刺激后体内活性氧的水平。将HepG2细胞接种到六孔板中，待细胞贴壁进入对数生长期后，加入含不同浓度T-2毒素的完全培养液，于二氧化碳培养箱中孵育24h，弃除培养液用PBS离心洗涤并吹悬，加入终浓度为l0μmol/L的DCFH-DA混匀，37℃避光孵育30min，以促使探针和细胞充分结合。最后用无血清MEM培养液洗涤细胞两次，酶标仪测定其平均荧光强度(激发波长488nm，发射波长530nm)，倒置荧光显微镜拍摄荧光图片。
由图6可知，实施例1构建的单细胞电化学传感器测量确定的剂量反应关系与ROS荧光测定值一致性较好，能够有效判断毒素的细胞毒性。
实施例4单细胞电化学传感实时监测
电化学传感器可以轻松地量化目标并进一步分析实时数据以获得关键参数的生化过程。为了实现对细胞生化过程的实时监控，将纳米探针与细胞质接触，并将1ppb T-2毒素添加到培养皿中。图7表明，在T-2毒素刺激后，通过单细胞电化学传感检测到单个HepG2细胞中H 2O 2的实时电流轨迹。当在大约60s的时间内将T-2毒素添加到培养皿中进行刺激时，刺激后20s电流值增加。与对照组相比，实验组在刺激后约70s出现了明显的峰值电流。当达到峰值电流时，电流稳定1-5min，然后逐渐减小。通过平衡ROS产生和通过ROS清除系统消除ROS来控制细胞的氧化还原平衡。
对照例1
将实施例1的单细胞电化学传感调整为多细胞电化学传感：
将经过清洗和抛光的玻碳电极(GCE)浸入含有1mM HAuCl 4的0.5M H 2SO 4溶液中，并使用电势控制库仑法(电势为-0.25V，100s)进行电沉积。将修饰的电极放置在电解质中以进行CV扫描。循环电压为-0.6–0.6V，扫描速度为0.1V/s。消化后的细胞悬液与明胶-甲基丙烯酰基(GelMA)水凝胶混合，以确保10 6细胞/mL的浓度。随后将6μL的混合物添加到电极表面。用光固定后，将不同浓度的T-2毒素刺激8小时，并进行GelMA/AuNP/GCE的电化学检测(图8A)。并线性拟合峰值电流(图8B)。在10ppb-1ppm范围内T-2毒素浓度与峰值电流具有良好的线性关系，R 2＝0.9776，最低检测浓度为10ng/mL。
结果表明，与传统的使用玻碳电极的多细胞电化学检测相比，单细胞电化学检测对T-2毒素检测操作更便捷、效率更快、更灵敏。
实施例5金沉积工艺对传感器的影响
参照实施例1，替换金沉积工艺为：优化金沉积时间，将纳米探针浸泡在1mmol·L-1氯金酸和0.5mol·L-1硫酸溶液中，初始电位-0.25，分别电沉积5s、10s、15s、20s、25s、30s，通过DPV检测镀金电极电信号，并通过纳米探针刺入细胞观察细胞形态的变化。
其他条件不变，制得相应的功能化纳米探针。
参照实施例2，如图9所示，镀金时间越长，DPV显示峰值电流越大。但是将镀金后的纳米探针刺入细胞，镀金时间25s、30s的纳米探针对细胞有明显损伤，细胞刺入后有明显凹陷，说明镀金时间过长使探针直径过大，所以选取镀金时间20s。
实施例6普鲁士蓝沉积工艺对传感器的影响
参照实施例1，替换普鲁士蓝沉积工艺为：优化普鲁士蓝沉积周期，含有0.1M HCl，2mM FeCl 3，0.1M KCl和2mM K 3[Fe(CN) 6]的电镀液中通过电化学沉积普鲁士蓝(PB)对纳米探针进行进一步修饰。电位在0.2～-0.6V之间分别循环10、20、50、100、150个周期。通过计时电流分析法检测20μM H 2O 2。
其他条件不变，制得相应的功能化纳米探针。
参照实施例2，如图10所示，循环周期越多，测得过氧化氢的电流值越大，到50周期以上时，电流值趋于稳定，因此选用循环50个周期作为普鲁士蓝修饰条件。

Claims (11)

  1. 一种单细胞电化学传感器检T-2毒素毒性的方法，其特征在于，所述方法是将毒素标准物质用培养液稀释成梯度浓度溶液，加入细胞培养皿中孵育，然后利用单细胞电化学传感器进行电化学检测，利用电化学计时电流法IT分析毒素的细胞毒性；
    所述电化学计时电流法IT分析毒素的细胞毒性的过程包括：
    利用不同浓度的H 2O 2标准样品的浓度值与单细胞电化学传感器输出的电流值构建标准曲线A；然后在利用不同浓度的毒素标样的浓度值与H 2O 2浓度值构建标准曲线B；通过检测待测样的电流值，基于标准曲线A、B，测得待测样中毒素的浓度；
    所述单细胞电化学传感器的工作电极为通过如下方法制备的功能化纳米探针：
    将毛细管拉制成纳米显微针，在显微针针尖上沉积金纳米粒子制成纳米探针；然后将普鲁士蓝沉积在所得纳米探针上，获得功能化纳米探针；其中，沉积金纳米粒子是将显微针针尖浸泡在含氯金酸的硫酸溶液中，初始电位-0.25V，沉积15-20s；普鲁士蓝沉积的过程为：在含有0.1M HCl，2mM FeCl 3，0.1M KCl和2mM K 3[Fe(CN) 6]的电镀液中电化学沉积；电位在0.2～-0.6V之间循环50个周期。
  2. 一种用于单细胞电化学传感的功能化纳米探针，其特征在于，所述功能化纳米探针的构建方法包括如下过程：将毛细管拉制成纳米显微针，在显微针针尖上沉积金纳米粒子制成纳米探针；然后将普鲁士蓝沉积在所得纳米探针上，获得功能化纳米探针。
  3. 根据权利要求2所述的功能化纳米探针，其特征在于，所述沉积金纳米粒子的过程包括：将显微针针尖浸泡在含氯金酸的硫酸溶液中，初始电位-0.25V，沉积15-20s。
  4. 根据权利要求2所述的功能化纳米探针，其特征在于，所述硫酸溶液中氯金酸的浓度为1mmol·L -1；硫酸的浓度为0.5mol·L -1。
  5. 根据权利要求2所述的功能化纳米探针，其特征在于，普鲁士蓝沉积的过程为：在含有0.1M HCl，2mM FeCl 3，0.1M KCl和2mM K 3[Fe(CN) 6]的电镀液中电化学沉积；电位在0.2～-0.6V之间循环50个周期。
  6. 根据权利要求2-5任一项所述的功能化纳米探针，其特征在于，所述功能化纳米探针的制备方法包括如下步骤：
    (1)将玻璃毛细管采用拉针仪拉制出的纳米显微针，通过电沉积法在待表征的针尖上镀约金纳米粒子，在电极外层采用PDMS绝缘，用Apiezon蜡包裹纳米探针表面，尖端露出金层作为电化学感应部分；
    (2)通过电化学沉积普鲁士蓝对纳米探针进行进一步修饰，电位循环50个周期，用去离子水冲洗普鲁士蓝修饰的纳米探针，并在室温下干燥。
  7. 一种单细胞电化学传感器，其特征在于，所述单细胞电化学传感器的工作电极为权利 要求2-6任一项所述的功能化纳米探针。
  8. 一种单细胞毒性检测方法，其特征在于，所述方法是利用权利要求7所述的单细胞电化学传感器进行检测，包括如下过程：将权利要求7所述的单细胞电化学传感器的功能化纳米探针夹持在显微操作系统上进行自动化操控，直接刺入细胞进行电化学检测。
  9. 一种利用权利要求7所述的单细胞电化学传感器检T-2毒素毒性的方法，其特征在于，所述方法是将毒素标准物质用培养液稀释成梯度浓度溶液，加入细胞培养皿中孵育，然后进行电化学检测，利用电化学计时电流法IT分析毒素的细胞毒性。
  10. 根据权利要求9所述的方法，其特征在于，所述电化学计时电流法IT分析毒素的细胞毒性的过程包括：
    利用不同浓度的H 2O 2标准样品的浓度值与单细胞电化学传感器输出的电流值构建标准曲线A；然后在利用不同浓度的毒素标样的浓度值与H 2O 2浓度值构建标准曲线B；通过检测待测样的电流值，基于标准曲线A、B，测得待测样中毒素的浓度。
  11. 权利要求7所述的单细胞电化学传感器在非疾病的诊断和治疗的药物开发、毒理学测试、纳米环境实时监测领域中的应用。
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