基于碳纳米管附载双酶体系的乳酸电化学发光生物传感器
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摘要
本文研究了在弱碱性体系中,应用鲁米诺的电化学发光(ECL)作为酶催化反应的信号输出研制ECL生物传感器。由于酶催化反应的产物为H2O2,因此,可以通过H2O2对鲁米诺电化学发光的增敏作用来制作不同的酶生物传感器,并将它用于实际样品的检测。由于电化学发光的高灵敏度以及酶的高选择性,因此制备的酶生物传感器能够很灵敏的检测底物的含量,检测的灵敏度高,检测限低。
第一部分研究了弱碱性体系中双酶电化学发光生物传感器的制备,采用的是铂盘电极。在双酶(乳酸脱氢酶、丙酮酸氧化酶)体系中加入乳酸时,在一定的范围内,随着乳酸浓度的增加,该体系发光强度不断增加。这是由于乳酸与乳酸脱氢酶在辅酶I的作用下,反应生成丙酮酸,丙酮酸同溶解氧在丙酮酸氧化酶的作用下反应产生的过氧化氢可以增敏鲁米诺的电化学发光。将双酶通过碳纳米管附载的方式修饰在电极表面上,修饰后的检出限为8.9×10-12Mol/L。将它应用于人体汗液中乳酸的测定及其运动员耐力训练水平的预测,得到的结果满意,重现性高,稳定性好,酶膜不容易脱落。
第二部分考察了pH=8的缓冲溶液体系中,以ITO玻璃为工作电极,研究了鲁米诺电化学发光的变化情况。鲁米诺在ITO玻璃电极上的电化学发光能在较低的电位下发生,且ITO玻璃电极便宜,方便,可一次性使用。采用壳聚糖固定酶技术,我们将双酶修饰在ITO玻璃电极上,得到的结果满意,重现性高,稳定性好。
This paper studied the application of luminol’s electrochemiluminescent (ECL) in enzymatic reaction in alkaline solution. Because of H2O2 produced by enzymatic reaction can increase the ECL of luminol, with this it can be used to fabricate different enzyme biosensor. It can detect the lower concentration of samples due to the highly sensitivity of ECL and highly selectivity of enzyme.
The first part studied the fabrication of Dual enzyme biosensor with Pt electrode. An ECL-based Lactic acid (LA) biosensor is developed which signaled with luminol. LA could be oxidized under the catalysis of immobilized lactic dehydrogenase (LDH) and pyruvate oxidase (PyOD) to produce hydrogen peroxide with nicotinamide adenine dinucleotide (NAD) as coenzyme. The enzymatic generated hydrogen peroxide can enhance the ECL intensity of luminol. The ECL-based biosensor showed excellent character for LA measurement. Its linear detection limit decreased to 8.9×10-12mol/L with relative standard deviation of 4.13% (CLA 1.34×10-10mol/l, n = 6) and average recovery of 101.3%. This ECL-based LA biosensor has reached a lower detection limit. It can be applied to detect the concentration of LA in the sweat of athletes during the exercitation for evaluation of exercise degree, and get a good result.
The second part studied that the ECL mechanism of luminol on ITO electrode in the alkaline solution.The results indicated that the phenomenon that ECL of luminol on the ITO electrode could occurre under a lower voltage, and ITO was cheap and convenient. We modified the Dual enzyme in the ITO electrode, obtained satisfactory results, high reproducibility, and good stability.
第一部分研究了弱碱性体系中双酶电化学发光生物传感器的制备,采用的是铂盘电极。在双酶(乳酸脱氢酶、丙酮酸氧化酶)体系中加入乳酸时,在一定的范围内,随着乳酸浓度的增加,该体系发光强度不断增加。这是由于乳酸与乳酸脱氢酶在辅酶I的作用下,反应生成丙酮酸,丙酮酸同溶解氧在丙酮酸氧化酶的作用下反应产生的过氧化氢可以增敏鲁米诺的电化学发光。将双酶通过碳纳米管附载的方式修饰在电极表面上,修饰后的检出限为8.9×10-12Mol/L。将它应用于人体汗液中乳酸的测定及其运动员耐力训练水平的预测,得到的结果满意,重现性高,稳定性好,酶膜不容易脱落。
第二部分考察了pH=8的缓冲溶液体系中,以ITO玻璃为工作电极,研究了鲁米诺电化学发光的变化情况。鲁米诺在ITO玻璃电极上的电化学发光能在较低的电位下发生,且ITO玻璃电极便宜,方便,可一次性使用。采用壳聚糖固定酶技术,我们将双酶修饰在ITO玻璃电极上,得到的结果满意,重现性高,稳定性好。
This paper studied the application of luminol’s electrochemiluminescent (ECL) in enzymatic reaction in alkaline solution. Because of H2O2 produced by enzymatic reaction can increase the ECL of luminol, with this it can be used to fabricate different enzyme biosensor. It can detect the lower concentration of samples due to the highly sensitivity of ECL and highly selectivity of enzyme.
The first part studied the fabrication of Dual enzyme biosensor with Pt electrode. An ECL-based Lactic acid (LA) biosensor is developed which signaled with luminol. LA could be oxidized under the catalysis of immobilized lactic dehydrogenase (LDH) and pyruvate oxidase (PyOD) to produce hydrogen peroxide with nicotinamide adenine dinucleotide (NAD) as coenzyme. The enzymatic generated hydrogen peroxide can enhance the ECL intensity of luminol. The ECL-based biosensor showed excellent character for LA measurement. Its linear detection limit decreased to 8.9×10-12mol/L with relative standard deviation of 4.13% (CLA 1.34×10-10mol/l, n = 6) and average recovery of 101.3%. This ECL-based LA biosensor has reached a lower detection limit. It can be applied to detect the concentration of LA in the sweat of athletes during the exercitation for evaluation of exercise degree, and get a good result.
The second part studied that the ECL mechanism of luminol on ITO electrode in the alkaline solution.The results indicated that the phenomenon that ECL of luminol on the ITO electrode could occurre under a lower voltage, and ITO was cheap and convenient. We modified the Dual enzyme in the ITO electrode, obtained satisfactory results, high reproducibility, and good stability.
引文
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