研究报告: 电化学适配体传感器用于乳酸快速检测的研究

目的 构建用于检测L-乳酸的新型电化学适配体传感器。方法 基于金钯-掺氮多壁碳纳米管纳米复合材料（Au/Pd-N-MWCNTs）修饰的玻碳电极，通过三螺旋分子开关（triple-helix molecular switch，THMS）触发具有RNA剪切活性的Pb²⁺辅助的脱氧核酶（DNAzyme）对电极表面固定化信号探针的循环剪切效应，实现L-乳酸的超灵敏电化学检测。采用差分脉冲伏安法（DPV）记录电流信号变化。结果 信号探针浓度4 μmol/L、Pb²⁺浓度4 μmol/L、DNAzyme剪切孵育时间60 min为传感器最优测试条件。在最优实验条件下，该L-乳酸传感器线性范围为1~20 mmol/L，检出限为0.51 mmol/L。此外，该适配体传感器具有优异的稳定性（RSD=4.56%）、重现性（RSD=2.80%）和选择性。在人血清样本中检测L-乳酸时回收率为105.60%~110.80%，RSD为2.35%~4.56%，与传统方法具有较好的一致性。结论 该适配体传感器能实现L-乳酸的超灵敏检测，在生物医学诊断、食品工业和环境监测等领域具有广泛的应用前景。     

太赫兹刺激听神经的测试平台搭建

目的 近年来，用于脑功能调控的神经调控技术蓬勃发展，很多方法已在临床上被推广应用，主要包括电极深部脑刺激、经颅磁刺激、光遗传技术、超声深脑刺激等。但是这些调控技术存在刺激靶点改变灵活性差、空间分辨率不足、需要注射病毒转染等问题。与这些技术相比，太赫兹波调控则能以较高的时空分辨率、无需引入外源基因的方式对神经活动进行干预。激光神经刺激是一种具有较明确靶向性的刺激方法，可以通过调整不同激光参数（激光波长、脉冲能量等）控制引起神经兴奋或者抑制。但是由于该研究方向的实验手段和实验平台的缺乏，相关研究开展较少。方法 针对这个问题，从听觉神经入手，在分子、细胞和在体不同层面为相关领域的研究搭建了不同的测试平台。结果 实验结果表明，这些系统在时间和空间上具有良好的耦合性和靶向性，测得的信号受噪音干扰小。结论 这些系统可以有效测试神经系统对太赫兹刺激的响应并精确控制刺激时间和位置。     

新型锌多糖抗凝血涂层修饰聚氯乙烯导管

目的：提高体外循环聚氯乙烯导管的血液相容性。方法：采用层层自组装的方法在PVC表面形成锌离子和多糖（肝素或硫酸葡聚糖）的复合涂层来提高PVC的血液相容性。结果：傅立叶红外光谱表明锌多糖复合物成功的沉积到PVC管表面,与未修饰的PVC管相比,修饰后的PVC管具有较长的部分活化的凝血酶原时间和很少数量的血小板黏附。体系中引入硫酸葡聚糖后,表面涂层具有更好的稳定性。结论：锌多糖抗凝血涂层很好的提高了聚氯乙烯导管的血液相容性。     

用钴卟啉修饰电极催化测定多巴胺等神经递质

将5, 10, 15, 20－四－（3－甲氧基－4－羟基苯基）卟啉钴（CoTMHPP）修饰在玻碳电极表面,制备成对多巴胶等神经递质有高灵敏度响应的CoTMHPP修饰电极．电极具有灵敏度高、响应快、稳定性好等特点．电极响应时间小于10s,儿茶酚类化合物的检测浓度为10^-6mol／L．     

IV型限制酶ScoMcrA中SRA结构域介导的二聚体化对硫结合结构域功能的影响机制

[背景] 部分细菌的DNA骨架会发生磷硫酰化修饰,硫结合结构域（Sulfur Binding Domain,SBD）可以特异性识别这种生理修饰。与绝大多数SBD-HNH双结构域核酸酶不同,ScoMcrA的SBD和HNH结构域中间插入了一个特异性识别5-甲基胞嘧啶（5mC）修饰DNA的SET and RING-Associated （SRA）结构域。晶体结构显示,单独的SBD是单体,而SBD-SRA是双体。[目的] 探究ScoMcrA中SRA结构域的存在对SBD识别硫修饰DNA的影响及影响方式。[方法] 凝胶迁移实验（Electrophoresis Mobility Shift Assay,EMSA）比较SBD、SBD-SRA对硫修饰DNA结合力的差异;对参与SBD-SRA二聚体化的关键氨基酸残基突变,并检测点突变对SBD-SRA蛋白二聚体化及结合硫修饰DNA的影响。[结果] 相较于SBD结构域,SBD-SRA双结构域对磷硫酰化修饰DNA的结合能力明显增强。对SBD-SRA双体互作界面进行单点突变基本不影响其对硫修饰DNA的结合,当二聚体化界面连续的L₂₆₁LGET₂₆₅突变成A₂₆₁AAAA₂₆₅时,突变体对硫修饰DNA的结合力下降到与SBD相似的水平。[结论] 根据EMSA实验结果可以初步判断,SRA结构域介导的SBD-SRA双体化能增强SBD对硫修饰DNA的结合力;L₂₆₁LGET₂₆₅是SRA结构域上影响SBD对硫修饰DNA结合力的关键氨基酸位点。     

基于表面质子化聚多巴胺修饰电极用于拟南芥原生质体的黏附与测定

采用自氧化方法将多巴胺(DA)聚合修饰到光透ITO(或金)电极表面上,通过缓冲溶液(pH 3)处理后形成带正电、可与带负电荷原生质体静电相互作用的表面膜。经循环伏安、电化学阻抗方法证明修饰薄膜对拟南芥原生质体黏附的有效性,在一定原生质体数目范围(1000~30 000),质子化聚多巴胺膜界面电荷转移电阻(R_(ct))随原生质体数目(N_(cells))增加而增加,1/R_(ct)与1/N_(cells)呈线性关系。此外,石英晶体微天平动态测试结果亦证明,本方法制备的修饰薄膜对原生质体具良好的黏附效果。本研究提供了一种用于原生质体固定与传感的有效方法,为在细胞层次研究植物结构、功能与行为及植物生命多样性提供参考。     

血清铁蛋白（SF）及sod水平与2型糖尿病患者小纤维神经病变关系的研究

摘要 目的：探血清铁蛋白（SF）及超氧化物歧化酶（sod）水平与2型糖尿病患者小纤维神经病变的关系。方法：选择2017年6月至2019年12月我院接诊的120例2型糖尿病患者,根据病变发生情况分为病变组67例,未发生病变53例作为对照组,分析血清铁蛋白（SF）及sod在其中的表达及其预测小纤维神经病变的价值。结果：病变组铁蛋白（SF）水平显著高于对照组,sod水平显著低于对照组,差异显著（P＜0.05）;ROC结果显示,铁蛋白（SF）预测2型糖尿病患者小纤维神经病变的AUC为0.924,95%CI为0.892~0.957,截断值为223.407 ng/mL ;sod预测2型糖尿病患者小纤维神经病变的AUC为0.96,95%CI为0.944~0.987,截断值为126.862 U/mL;联合预测2型糖尿病患者小纤维神经病变的AUC为0.993,95%CI为0.986~1.000,单独检测分别和联合检测曲线下面积比较均具有显著差异,联合检测的特异度、准确度分别为94.26%、95.16%。结论：在2型糖尿病小纤维神经病变患者中血清铁蛋白（SF）及sod的表达异常,对于疾病的进展有诊断意义,临床应给与关注。     

双模态纳米诊疗一体药物在肝癌动物模型中的应用

摘要 目的：构建一种肿瘤诊断和治疗一体化药物，并利用肝癌动物模型开展诊疗效能评价。方法：利用多孔金属有机骨架材料ZIF-8，通过配位作用同时对化疗药物阿霉素（DOX）和近红外荧光染料IR-820进行负载。而后，利用超声的方法在ZIF-8-IR-820-DOX表面修饰了红细胞膜以提高载药体系的生物安全性和稳定性，得到具有生物伪装特性的pH响应型ZIF-8-IR-820-DOX@RM纳米颗粒。最后，通过对该药物体系的粒径、表面电位、形貌等理化性质进行表征，并利用肝癌动物模型验证该药物的诊断和治疗效能。结果：成功构建了一款红细胞伪装的金属框架纳米诊疗一体试剂，该试剂具有较好的pH相应性，在肿瘤pH 5.5 条件下，药物的释放率达到98.4 %，而在机体正常pH 7.4条件下，释放率仅为15.3 %。在小鼠肝癌动物模型的诊疗过程中，能通过近红外荧光较好的识别肿瘤的位置和大小，且对小鼠肿瘤具有较好的治疗效果。结论：本研究所构建的ZIF-8-IR-820-DOX @RM能通过肿瘤处增强的渗透性和保留（EPR）效应，精准的到达肿瘤部位，并利用pH响应性，在肿瘤酸性环境中精准释放携带的抗肿瘤药物和近红外荧光成像试剂，实现对肿瘤的诊断和治疗一体化设计。为肿瘤治疗的相关研究提供了一种思路和借鉴。     

基于生物阻抗谱的生物细胞活性免标记检测方法

目的 基于生物阻抗谱（bioelectrical impedance spectroscopy,BIS）技术,提出一种免标记的生物细胞活性实时检测方法。该方法依据不同浓度、生理、病理状态下细胞组织的电学特性差异来判断细胞是否具有活性,以协助医师在临床手术中快速精准定位患者烫伤组织并实现有效切除。方法 使用具有活性的斑马鱼胚胎干细胞来模拟人体烫伤组织,采用生物阻抗谱技术来鉴别细胞组织的生理状态。结果 在不同状态下,细胞的阻抗幅值变化有显著的差异,可以从中发现同等浓度下活性细胞的阻抗幅值比死亡细胞平均高出17.25%,活性细胞发生弛豫频率的时间也比死亡细胞早25%。结论 实验数据表明,生物阻抗谱法能有效区分胚胎干细胞的两类生理状态;从聚类区域中可以看出,BIS检测法具有明显的细胞活性及浓度区分能力,理论上能够快速地协助医师完成对患者烫伤组织检测。     

叶酸受体靶向的金纳米棒用于细胞内光声成像

目的 分子成像技术具有“早期检测”的特点,由于分子水平上的畸变早于解剖水平上的变化。本研究采用细胞内光声分子成像（PMI）方法,对靶向到癌细胞上的叶酸-金纳米棒（FA-AuNRs）精确定位成像。方法 本文合成了FA-AuNRs,并对其性质包括形貌、吸收光谱和生物相容性进行了研究。修饰叶酸赋予FA-AuNRs特异性靶向到叶酸受体高表达癌细胞的能力。然后,通过PMI实验研究FA-AuNRs对癌细胞的靶向特异性。结果 FA-AuNRs呈棒状,在~800 nm处有一近红外吸收峰。在癌细胞的细胞质中观察到强光声信号,而在正常细胞中只有弱光声信号,表明FA-AuNRs通过叶酸受体介导的内吞作用被癌细胞选择性摄取。这项研究证明了PMI能够实现对靶向到癌细胞上的FA-AuNRs精确定位成像。结论 借助特异性靶向作用,可以通过PMI获得癌细胞表面分子信息。该方法有望实现在细胞和分子水平上对生物过程进行可视化、表征和量化。     

Carbon nanotube-based biosensors for DNA structure characterization

The possibility of DNA detection using electrodes modified with carbon nanotubes (CNTs) was studied. CNTs facilitate the electrochemical oxidation of DNA guanine nucleotide, which allows direct detection of DNA on a modified electrode. Electrochemical properties of DNA depend on its secondary structure and molecular weight. Denaturation of native DNA improves the adsorption of biopolymer on CNTs and results in an increase in DNA oxidation current on the modified electrode. A similar effect is observed after ultrasonic shearing of DNA or its treatment with Fenton’s reagent due to the fragmentation of biopolymer. Our results demonstrate the feasibility of biosensors based on CNT-modified electrodes for the direct detection and characterization of DNA and DNA damaging factors.     

Intracellular Neural Recording with Pure Carbon Nanotube Probes

The computational complexity of the brain depends in part on a neuron’s capacity to integrate electrochemical information from vast numbers of synaptic inputs. The measurements of synaptic activity that are crucial for mechanistic understanding of brain function are also challenging, because they require intracellular recording methods to detect and resolve millivolt- scale synaptic potentials. Although glass electrodes are widely used for intracellular recordings, novel electrodes with superior mechanical and electrical properties are desirable, because they could extend intracellular recording methods to challenging environments, including long term recordings in freely behaving animals. Carbon nanotubes (CNTs) can theoretically deliver this advance, but the difficulty of assembling CNTs has limited their application to a coating layer or assembly on a planar substrate, resulting in electrodes that are more suitable for in vivo extracellular recording or extracellular recording from isolated cells. Here we show that a novel, yet remarkably simple, millimeter-long electrode with a sub-micron tip, fabricated from self-entangled pure CNTs can be used to obtain intracellular and extracellular recordings from vertebrate neurons in vitro and in vivo. This fabrication technology provides a new method for assembling intracellular electrodes from CNTs, affording a promising opportunity to harness nanotechnology for neuroscience applications.     

Monitoring of dopamine release in single cell using ultrasensitive ITO microsensors modified with carbon nanotubes

The study of single cell dynamics has been greatly adapted in biological and medical research and applications. In this work a novel microfluidic electrochemical sensor with carbon nanotubes (CNTs) modified indium tin oxide (ITO) microelectrode was developed for single cells release monitoring. The sensitivity of the electrochemical sensor after CNTs surface modification was improved by 2.5-3 orders of magnitude. The developed CNTs modified ITO sensor was successfully employed to monitor the dopamine release from single living rat pheochromocytoma (PC 12) cells. Its ultrahigh sensitivity, transparency and need for fewer agents enable this smart electrochemical sensor to become a powerful tool in recording dynamic release from various living tissues and organs optically and electrically.     

A sensitive nonenzymatic glucose sensor in alkaline media with a copper nanocluster/multiwall carbon nanotube-modified glassy carbon electrode

Copper (Cu) nanoclusters were electrochemically deposited on the film of a Nafion-solubilized multiwall carbon nanotube (CNTs)-modified glassy carbon electrode (CNTs-GCE), which fabricated a Cu-CNTs composite sensor (Cu-CNTs-GCE) to detect glucose with nonenzyme. Transmission electron microscopy (TEM) and X-ray diffraction (XRD) were used for the characterization of the distribution of the Cu nanoclusters on the CNTs matrix. The composite of the Cu-CNTs was investigated by the electrochemical characterization of cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The preliminary study shows that the nonenzymatic sensor has synergistic electrocatalytic activity to the oxidation of glucose in alkaline media. A well applicable sensor was constructed to use for the analysis of the glucose in real blood serum samples due to the large number of electrons taking part in the oxidation process, the high apparent kinetic rate constant, and the stable operation of the electrode. The linear range for the detection of the glucose is 7.0 x 10(-7) to 3.5 x 10(-3) M with a high sensitivity of 17.76 microA mM(-1), a low detection limit of 2.1 x 10(-7) M, and a fast response time of within 5s. Experiment results also showed that the sensor has good reproducibility and long-term stability and is interference free.     

Rapid Prototyping of a High Sensitivity Graphene Based Glucose Sensor Strip

A rapid prototyping of an inexpensive, disposable graphene and copper nanocomposite sensor strip using polymeric flexible substrate for highly sensitive and selective nonenzymatic glucose detection has been developed and tested for direct oxidization of glucose. The CuNPs were electrochemically deposited on to the graphene sheets to improve electron transfer rates and to enhance electrocatalytic activity toward glucose. The graphene based electrode with CuNPs demonstrated a high degree of sensitivity (1101.3±56 μA/mM.cm²), excellent selectivity (without an interference with Ascorbic Acid, Uric Acid, Dopamine, and Acetaminophen), good stability with a linear response to glucose ranging from 0.1 mM to 0.6 mM concentration, and detection limits of 0.025 mM to 0.9 mM. Characterization of the electrodes was performed by scanning electron microscopy (FESEM and SEM). The electrochemical properties of the modified graphene electrodes were inspected by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and amperometry.     

Nucleic acid sensor for insecticide detection

Nucleic acid sensor based on polyaniline (PANI) has been fabricated by covalently immobilizing double stranded calf thymus (dsCT) DNA onto perchlorate (ClO(-) (4))-doped PANI film deposited onto indium-tin-oxide (ITO) glass plate using 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC)/N-hydroxysuccinimide (NHS) chemistry. These dsCT-DNA-PANI-ClO(4)/ITO and PANI-ClO(4)/ITO electrodes have been characterized using square wave voltammetry, electrochemical impedance, scanning electron microscopy (SEM) and Fourier-transform-infrared (FTIR) measurements. This disposable dsCT-DNA-PANI-ClO(4)/ITO bioelectrode, stable for about 4 months, can be used to detect cypermethrin (0.005 ppm) and trichlorfon (0.01 ppm) in 30 and 60 s, respectively.     

Electrochemical immunosensor based on Pd–Au nanoparticles supported on functionalized PDDA-MWCNT nanocomposites for aflatoxin B1 detection

This paper reports a label-free electrochemical immunosensor for the determination of aflatoxin B1 (AFB1), which is based on a gold electrode modified by a biocompatible film of carbon nanotubes/poly(diallyldimethylammoniumchloride)/Pd–Au nanoparticles (CNTs/PDDA/Pd–Au). The nanocomposite was characterized by transmission electron microscopy and the electrochemical behavior of modified electrodes was investigated by cyclic voltammetry. The CNTs/PDDA/Pd–Au nanocomposites film showed good electron transfer ability, which ensured high sensitivity to detect AFB1 in a range from 0.05 to 25 ng mL⁻¹ with a detection limit of 0.03 ng mL⁻¹ obtained at 3σ (where σ is the standard deviation of the blank solution, n = 10). The proposed immunosensor provides a simple tool for AFB1 detection. This strategy can be extended to any other antigen detection by using the corresponding antibodies.     

Efficient Polymer Solar Cells on Opaque Substrates with a Laminated PEDOT:PSS Top Electrode

Solution processed polymer:fullerene solar cells on opaque substrates have been fabricated in conventional and inverted device configurations. Opaque substrates, such as insulated steel and metal covered glass, require a transparent conducting top electrode. We demonstrate that a high conducting (900 S cm^?1) PEDOT:PSS layer, deposited by a stamp‐transfer lamination technique using a PDMS stamp, in combination with an Ag grid electrode provides a proficient and versatile transparent top contact. Lamination of large size PEDOT:PSS films has been achieved on variety of surfaces resulting in ITO‐free solar cells. Power conversion efficiencies of 2.1% and 3.1% have been achieved for P3HT:PCBM layers in inverted and conventional polarity configurations, respectively. The power conversion efficiency is similar to conventional glass/ITO‐based solar cells. The high fill factor (65%) and the unaffected open‐circuit voltage that are consistently obtained in thick active layer inverted geometry devices, demonstrate that the laminated PEDOT:PSS top electrodes provide no significant potential or resistive losses.     

Electrochemical behavior of L-cysteine and its detection at carbon nanotube electrode modified with platinum

The electrochemical behavior of L-cysteine (CySH) on platinum (Pt)/carbon nanotube (CNT) electrode was investigated by cyclic voltammetry. CNTs used in this study were grown directly on graphite disk by chemical vapor deposition. Pt was electrochemically deposited on the activated CNT/graphite electrode by electroreduction of Pt(IV) complex ion on the surface of CNTs. Among graphite, CNT/graphite, and Pt/CNT electrodes, improved electrochemical behavior of CySH oxidation was found with Pt/CNT electrode. On the other hand, a sensitive CySH sensor was developed based on Pt/CNT/graphite electrode. A linear calibration curve can be observed in the range of 0.5 microM-0.1 mM. The detection limit of the Pt/CNT electrode is 0.3 microM (signal/nose=3). Effects of pH, scan rate, and interference of other oxidizable amino acids were also investigated and discussed. Additionally, the reproducibility, stability, and applicability of the Pt/CNT electrode were evaluated.     

Direct electrochemistry and electrocatalytic activity of catalase immobilized onto electrodeposited nano-scale islands of nickel oxide

Cyclic voltammetry was used for simultaneous formation and immobilization of nickel oxide nano-scale islands and catalase on glassy carbon electrode. Electrodeposited nickel oxide may be a promising material for enzyme immobilization owing to its high biocompatibility and large surface. The catalase films assembled on nickel oxide exhibited a pair of well defined, stable and nearly reversible CV peaks at about -0.05 V vs. SCE at pH 7, characteristic of the heme Fe (III)/Fe (II) redox couple. The formal potential of catalase in nickel oxide film were linearly varied in the range 1-12 with slope of 58.426 mV/pH, indicating that the electron transfer is accompanied by single proton transportation. The electron transfer between catalase and electrode surface, (k(s)) of 3.7(+/-0.1) s(-1) was greatly facilitated in the microenvironment of nickel oxide film. The electrocatalytic reduction of hydrogen peroxide at glassy carbon electrode modified with nickel oxide nano-scale islands and catalase enzyme has been studied. The embedded catalase in NiO nanoparticles showed excellent electrocatalytic activity toward hydrogen peroxide reduction. Also the modified rotating disk electrode shows good analytical performance for amperometric determination of hydrogen peroxide. The resultant catalase/nickel oxide modified glassy carbon electrodes exhibited fast amperometric response (within 2 s) to hydrogen peroxide reduction (with a linear range from 1 microM to 1 mM), excellent stability, long term life and good reproducibility. The apparent Michaelis-Menten constant is calculated to be 0.96(+/-0.05)mM, which shows a large catalytic activity of catalase in the nickel oxide film toward hydrogen peroxide. The excellent electrochemical reversibility of redox couple, high stability, technical simplicity, lake of need for mediators and short preparations times are advantages of this electrode. Finally the activity of biosensor for nitrite reduction was also investigated.