适用于发酵生产过程的谷氨酸微生物传感器的研制

采用夹层法将内含L-谷氨酸脱羧酶的大肠杆菌固定于聚脂(PET)膜与硅橡胶膜之间,并将此与CO_2电极复合构成L-谷氨酸微生物传感器。电极响应时间为3分钟,电极电位mv值与谷氨酸浓度对数的关系曲线在50～6000mg/L成线性关系。传感器的最低检测限为10mg/L,使用寿命可达两周以上。该电极的贮存性能良好,4℃贮存近两个月,未见其响应值下降。电极对浓度为800mg/L试样重复测定11次,其变异系数为0.13％。     

超高效液相色谱-串联质谱法检测河鲀毒素

目的建立准确、快速检测河鲀毒素(tetrodotoxin,TTX)的超高效液相色谱-串联质谱(ultra performance liquid chromatography-tandem mass spectrometry,UPLC-MS/MS)的分析方法,了解市售烤鱼片、鱿鱼干制品和鱼丸中河鲀毒素污染状况。方法样品经2%乙酸/甲醇溶液超声离心提取后,通过MCX固相萃取柱进行净化处理,采用液相色谱串联质谱法进行检测。结果市售的107份样品中,鱿鱼干制品和鱼丸中没有检出TTX;64份烤鱼片样品中检出16份含有TTX,检出率为25.0%。结论部分烤鱼片中TTX呈阳性,建议生产厂家严把原料关,监管部门应加强对市售烤鱼片标签标识的监管。     

固定化乳酸菌制备生物传感器测定抗生素

用海藻酸钠-淀粉凝胶作固定剂,将乳酸菌固定到两片核微孔膜中间制成“三明治”式传感膜,然后将其固定到玻碳电极上制成生物传感电极。通过电化学工作站检测3 种抗生素分别在不同质量浓度条件下的响应电流,结果表明：该传感器固定瑞士乳杆菌的最适量为0.05 g,此时对青霉素、链霉素、四环素的最低检测限分别为1×10－10、1×10－9、1×10－9 g/mL,检出时间为4 min,明显优于国内外对抗生素残留量的要求。而且经测定表明该电极在37 ℃的MRS培养基中至少可以稳定保存7 d,低温保藏应该可以保存更长时间,说明电极性能比较稳定。总之,固定化乳酸菌制备的生物传感器提供了一种新的定量测定抗生素的方法,其检测灵敏度高、成本低、简单、快速,不仅适用于乳制品中抗生素残留的定量化快速检测,而且可以实现对多种类型的抗生素进行检测。     

生物传感器法测定花生中黄曲霉毒素B_1

建立了生物传感器法测定花生中黄曲霉毒素B1含量的方法。黄曲霉毒素氧化酶经固定化后与过氧化氢电极构成电流型酶电极。花生经粉碎、过筛、提取、超声波萃取后利用电流型酶电极构成的生物传感器对黄曲霉毒素B1进行测定,与薄层色谱法、酶联免疫吸附法(ELISA法)有较好的一致性。黄曲霉毒素生物传感器测定时间为20 s,相对偏差2%,标样线性良好;进行加标回收试验,回收率为98%~106%。结果表明,该方法具有较高的灵敏度,操作简便,可用于花生中黄曲霉毒素的测定。     

冷却肉中乳酸含量的酶电极生物传感器测定

为测定冷却肉中乳酸含量,建立了冷却肉中乳酸提取及酶电极生物传感器的测定方法。冷却猪肉用1 mol/L高氯酸水溶液匀浆去除蛋白质及脂肪后,以乳酸氧化酶生物传感器检测。检测结果显示,该测定方法线性范围0～0.50 mg/mL乳酸(R=0.999 7),检测限5.0×10-5mg/mL。回收率为97.5%~102.3%,相对标准偏差为2.6%~3.1%,日内测定变异系数(CV)为1.08%,日间测定变异系数为1.26%。该方法应用于冷却猪肉乳酸含量测定获得较好的效果。     

鲀毒鱼毒素检测及物种鉴定技术研究进展

鲀毒鱼引起的河鲀毒素(tetrodotoxin,TTX)中毒是中国沿海地区食物中毒致死的主要原因之一.河鲀毒素中毒发病迅速,至今尚无特效药,因此,通过检测TTX含量或鉴定携带TTX的物种可以更好地进行TTX中毒的风险分析、管理和控制.本文综述了鲀毒鱼TTX的检测技术以及鲀毒鱼物种鉴定的方法,以期能预警并减少鲀毒鱼引起的...     

小麦粉中过氧化苯甲酰现场测定的肌红蛋白凝胶膜生物传感器研究

基于肌红蛋白-硅酸铝(Mb-AlSiO4)凝胶膜修饰电极,制备了一种非媒介体型的过氧化苯甲酰(BPO)生物传感器,并用于小麦粉中BPO的直接快速检测.用扫描电子显微镜(SEM)、X-光电子能谱(XPS)和原子力显微镜(AFM)对AlSiO4凝胶修饰电极进行了表征.电极表面的Mb-AlSiO4凝胶膜有均匀的多孔结构,能长时间保持肌红蛋白活性.该传感器对BPO的电化学还原表现出良好的催化活性,BOP还原峰电流与其浓度在1×10-6～4×10-4mol/L的范围内有良好的线性关系,相关系数为0.997,检测限是5×10-7mol/L.具有灵敏、稳定、易于制备的优点,适合于样品的现场快速测定,此技术平台可推广到粮油食品中其它添加剂的检测.     

固定化大鼠小肠组织制备生物传感器对白藜芦醇的检测

以海藻酸钠-淀粉凝胶作固定剂,将Sprague-Dawley（SD）大鼠的小肠回肠组织固定到两片核微孔膜中间制成“三明治”式传感膜,然后将其固定到玻碳电极上制成生物传感电极,通过电化学工作站测定出不同浓度白藜芦醇刺激其相应受体后的响应电流。结果表明：该传感器对白藜芦醇的最低检测限为1×10－13 mol/L,在其浓度为8.5×10－12 mol/L时电流变化率达到最大值,说明此时白藜芦醇的受体已经被饱和。用Origin软件对白藜芦醇与其受体的作用曲线进行双曲线拟合（R2＝0.988 7）,然后用双倒数法作图求出白藜芦醇与其受体的结合常数和解离常数分别为1.207×10－11和1.118×10－12。由此可以估测出,平均每个细胞的受体数约为85 个。通过将白藜芦醇固定化小肠上皮组织所制成的生物传感器的响应值和白藜芦醇与裸电极作用的响应值比较可知,小肠组织细胞对白藜芦醇的响应值显然得到了细胞内信号传递系统的放大作用,其放大倍数为100 倍。与此同时,细胞受体明显赋予该生物传感器的可饱和性特征,表现为类似于酶促反应动力学-米氏方程的典型特征。通过该传感器第一次定量化测定了白藜芦醇与受体互作并向机体内传递信号的动力学规律,这将为白藜芦醇受体分析、分离与纯化、信号传递及其生物功能评价与研究提供新的方法。     

重金属离子的聚苯胺修饰印刷电极的脲酶生物传感器检测

利用丝网印刷电极,构建了一种基于聚苯胺膜和壳聚糖的生物传感器,实现了重金属离子的快速检测。聚苯胺膜可通过电聚合修饰在丝网印刷电极表面,对电位变化有明显的响应,可以利用酶抑制作用,检测重金属离子。壳聚糖具有良好的生物兼容性,可以保持脲酶的活性。在最优条件下,用生物传感器检测重金属离子Hg~(2+)和Cd~(2+),重金属离子浓度的对数与抑制率呈良好的线性关系,Hg~(2+)的检出限为3.89μg/L,Cd~(2+)的检出限为5.41μg/L。该传感器用于纺织品样品的检测,加标回收率在88%~111%,可以满足实际检测需求。     

利用生物传感器测定氨基酸

生物传感器由酶和微生物等的生体触酶和物理化学装置构成。物理化学装置用各种离子选择性电极、半导体元件、热敏电阻、光子计算机等。根据用物理化学装置测定与生体有关的生物化学反应生成或消耗的化学物质、热、光等这一原理。生物传感器采用共有结合法把生物体触媒固定在合成高分子膜上或天然高分子膜上的方法和生物体触媒包括埋在高分子凝胶膜中的方法。     

Biosensors fob food industry

Novel biosensor systems were constructed for food industry. In order to determine fish freshness, a multifunctional enzyme sensor system was developed by combining a double membrane consisting of a 5'‐nucleotidase membrane and a nucleoside phosphorylase‐xanthine oxidase membrane with an oxygen electrode. Each nucleotide concentration was determined as the current decreased. One assay was completed within 20 min. Good comparative results were observed between the KI values determined by the sensor proposed and by the conventional method. An enzyme sensor for meat freshness consisted of a monoamine oxidase‐collagen membrane and an oxygen electrode. The response time of the electrode was 4 min. A linear relationship was observed between the amine (tyramine) concentration in the range 50–200 μM and the difference in current. Monoamine in meat extract was determined by the enzyme sensor. Micro‐glutamate sensor was constructed using silicon fabrication technology. Calibration curve for glutamate sensor was obtained in a glutamate concentration range between 5–50 mM. Moreover, an immuno sensor based on piezoelectric crystal was applied to the determination of toxic bacterium C. albicans. The frequency shift is correlated with C. albicans concentration in the range 10⁶ 5×10⁸ cell/ml.     

A pyruvate dehydrogenase-based amperometric biosensor for assessing pungency in onions (Allium cepa L.)

A disposable prototype electrochemical biosensor was constructed using pyruvate dehydrogenase immobilised on mediated Meldolas Blue electrodes to determine pungency in onions (Allium cepa L.). The optimum operating potential was +50 mV vs. Ag/AgCl reference/counter electrode. The biosensor was able to differentiate between mild and pungent bulbs with pyruvate concentrations ranging between ≈4 and 8 mM in freshly extracted juices. Resolution was 0.5 mM and thus was comparable to the standard Schwimmer and Weston-based colorimetric assay currently employed by industry for determining pungency in macerated onion tissue.     

Determination of carbamate pesticides by a cholinesterase-based flow injection biosensor

A potentiometric flow injection-type biosensor developed in our laboratory was used for the determination of carbamate pesticides. The principle of the biosensor is based on the inhibition of enzyme by pesticides, which is dependent on the concentration of the pesticides present. The sensor system consisted of a reactor with acetylcholinesterase immobilized on controlled pore glass and a detector with tubular H⁺-selective membrane electrode. The sensor protocol established was applied to the analysis of nine carbamate pesticides. All the pesticides at 10⁻⁶ M inhibited the sensor enzyme, demonstrating the utility of the developed method. Treatment of the inhibited enzyme with pyridine-2-aldoxime restored nearly all of the enzyme activity allowing repeated use of the sensor. Calibration curves for carbaryl and carbofuran over the concentration range 10⁻¹¹–10⁻⁴ M were obtained. The lower detection limits were 10⁻¹⁰ M and 10⁻¹¹ M, respectively.     

Construction of an amperometric polyphenol biosensor based on PVA membrane

A method is described for construction of an amperometric biosensor for determination of polyphenolic content using polyvinyl alcohol membrane bound polyphenol oxidase mounted on a gold coated Ag wire as a working electrode, a silver/silver chloride (Ag/AgCl) reference electrode and Pt wire as auxiliary electrode. The biosensor showed optimum response within 30 s, when operated at 30 °C. A linear relationship was obtained between L-DOPA concentration (0.5–20 μM) and current (μA). Limit of detection of the method was 0.5 μM. The biosensor measured polyphenols in tea leaves, alcoholic beverages and water. The enzyme electrode was used 280 times over 6 months, when stored at 4 °C. The biosensor is better than earlier membrane based biosensors in terms of detection limit (0.5 μM) and stability (6 months).     

Amperometric determination of tyramine in sauce and beer by epoxy resin biocomposite membrane bound tyramine oxidase

A method is described for construction of an amperometric biosensor for specific determination of tyramine, using black gram tyramine oxidase immobilized covalently on an epoxy resin membrane. The biosensor had optimum response within 10 s at pH 8.5 and 35 °C. A linear relationship was observed between tyramine concentrations and current (mA) in the range of 0.24 to 3.47 mg/dL. The biosensor was employed for determination of tyramine in beer and sauce. The detection limit of sensor was 0.24 mg/dL. The mean analytical recovery of added tyramine (0.5 and 1.0 mg/dL) was 97.3 ± 2.3 and 95.9 ± 3.4%. Within and between batch coefficient of variation were 5.1 and 5.34%, respectively. Enzyme electrode showed 35% loss in its initial activity after its regular use over a period of 2 months. The biosensor has the advantage that it does not suffer from leaching of enzyme and measures tyramine specifically.     

Isocitrate analysis using a potentiometric biosensor with immobilized enzyme in a FIA system

A potentiometric biosensor for the analysis of isocitrate was developed by using a CO₃²⁻-selective electrode and enzyme immobilization in flow injection analysis (FIA). The biosensor consisted of a peristaltic pump, injector, enzyme reactor, reference electrode, working electrode, pH/mV meter and record. The experimental parameters that influenced the sensitivity and selectivity of the biosensors were optimized in the FIA system. A linear correlation between the potential difference and logarithmic isocitrate concentration was obtained in the range of 10⁻³–10⁻¹ M isocitrate under optimal conditions. The interference effect of major sugars and organic acids on the sensor system was less than 5%. Isocitrate concentrations of some fruit juices analyzed by the isocitrate sensor system were compared with those analyzed by gas chromatography (GC). There was no significant difference between the two analytical methods in any of the fruit juices. This suggests that the isocitrate sensor system is reliable in determining the isocitrate concentrations of foods.     

一种新型过氧化氢传感器的构建及其在牛奶中的应用

为构建一种新型过氧化氢生物传感器,将血红蛋白吸附在聚吡咯膜为基底的金纳米颗粒上,通过SEM、AFM、XPS、CV和EIS表征修饰电极并将该传感器用于牛奶中过氧化氢的检测。结果表明各材料成功的修饰到电极表面,且纳米金颗粒的粒径约为15 nm,Hb/AuNPs/Ppy/GCE的最佳制备条件为吡咯聚合电量3.0×10-3 C,PBS溶液pH6.5,支持电解质溶液pH7.0。该传感器对过氧化氢的检出限为0.2 μmol/L(S/N=3),检测时间12 s。此外,所构建的传感器具有良好的稳定性和选择性,金纳米颗粒大,比表面积提供更多的位点固载血红蛋白,同时血红蛋白和纳米金颗粒对过氧化氢具有良好的协同催化效果。该传感器监测牛奶中过氧化氢的加标回收率为92.7%~116.0%。结果表明,Hb/AuNPs/Ppy/GCE是一种有前途的电化学生物传感器。     

L-抗坏血酸传感器在果汁中Vc含量的测定

本文利用花椰菜组织通过交联制备L-抗坏血酸传感器,该传感器在pH6.0、0.1mol/L的KH_2PO_4-Na_2HPO_4真缓冲溶液中对Vc标准溶液的线性范围为5.0×10~(-5)×1.8×10~(-3)mol/L,响应时间1min。电极使用寿命在1个月以上。利用该传感器测定了部分水果和果汁中Vc的含量,所得结果与分光光度法(2,4-二硝基苯肼法)测得结果一致。     

基于聚硫菫的一次性过氧化氢生物传感器的研究

用循环伏安法将硫堇电聚合在丝网印刷碳电极上,再用壳聚糖-二氧化硅溶胶凝胶将辣根过氧化物酶固定于聚硫堇电极表面,利用聚硫堇作为电子传递介体,用壳聚糖-二氧化硅溶胶凝胶固定辣根过氧化物酶并保持酶的生物活性,制成了新型过氧化氢生物传感器。实验发现,该传感器响应快、灵敏度高、稳定性好,对H2O2表现出良好的响应特性;检测线性范围为7.5×10-6～3.12×10-3mol/L,检出限为1.22×10-6mol/L,并具有抗葡萄糖、抗坏血酸等干扰的特点,有望用于食品中过氧化氢残留的检测。     

亚甲基蓝为介体的丝网印刷双酶修饰电极测定葡萄糖

以亚甲基蓝作为电子媒介,通过壳聚糖固定葡萄糖氧化酶和辣根过氧化酶在丝网印刷电极上,制备成葡萄糖生物传感器。循环伏安法和恒电位法用于研究修饰电极的电化学特性,在优化的实验条件下,获得传感器对葡萄糖响应的线性范围为8．0×10-5～3．5×10-3mol/L。此方法测定葡萄糖抗干扰能力强、重现性好、准确度高,电极制作简单,使用方便。