Biosensor for L-phenylalanine based on the optical detection of NADH using a UV light emitting diode
Biosensor for L-phenylalanine based on the optical detection of NADH using a UV light emitting diode
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DOI：
10.1007/s00604-010-0536-5
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发表时间：
2011-04-01
影响因子：
5.7
通讯作者：
Mitsubayashi, Kohji
中科院分区：
文献类型：
--
作者：
Arakawa, Takahiro;Koshida, Tomoyuki;Mitsubayashi, Kohji
研制了检测L-苯丙氨酸的生物传感器，并用于苯丙酮尿症的诊断。它由固定在膜上的L-苯丙氨酸脱氢酶(L-苯丙氨酸脱氢酶)、紫外发光二极管激发系统和光电倍增管组成。用2-甲基丙烯酰氧乙基磷酰胆碱修饰的聚四氟乙烯膜固定L-PheDH，放置在光纤的远端。通过将传感器浸泡在也含有NAD(+)的样品溶液中并测量酶反应产生的NADH的荧光来测定Phe的浓度。从中间热放线菌和孢子链霉菌中分离到2个L-PheDHs对其进行了研究和比较。该传感器的荧光强度与L-苯丙氨酸浓度在10~10 mmoL L~(-1)~10 mmoL L~(-1)范围内呈良好的线性关系。传感器也在动力学模式下工作，通过在2分钟内对信号的斜率进行差示确定。该传感器的分析范围足以用于PKU的基因分型诊断(诊断价值为600mU·moL·L-1)。该生物传感系统具有灵敏度高、性价比高、功耗低等特点，可应用于新生儿常规筛查。
A biosensor was developed for the detection of L-phenylalanine (Phe) and demonstrated for use in the diagnosis of phenylketonuria (PKU). It consists of L-phenylalanine dehydrogenase (L-PheDH) immobilized on a membrane, an ultraviolet light-emitting diode excitation system, and a photo multiplier tube. The L-PheDH was immobilized on a teflon membrane modified with 2-methacryloyloxyethyl phosphorylcholine and placed at the distal and of an optical fiber. The concentration of Phe was determined by immersing the sensor into a sample solution that also contained NAD(+) and measurement of the fluorescence of the NADH produced by enzymatic reaction. Two L-PheDHs (from Thermoactinomyces intermedius and Sporosarcina sp.) were studied and compared. The fluorescence intensities of the biosensor are linearly related to the L-Phe concentrations in the range from 10 mu mol L-1 to 10 mmol L-1. The sensor also was operated in the kinetic mode by differential determination of the slope of the signal within 2 min. The analytical range of the sensor is adequate for application in the genotypic diagnosis of PKU (diagnostic value > 600 mu mol L-1). High sensitivity, good cost-benefit ratio, and low power consumption are typical features of this biosensing system that can can be applied to routine screening of newborn.