组成图示
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传感器类型
电化学生物传感器
检测对象
重组质粒pEThIL-2（recombinant plasmid pEThIL-2，含人IL-2编码DNA）；选择性对照：非互补质粒pET21a(+)；样品基质：质粒溶液/Tris–HCl缓冲液（pH 7.0，含20 mM NaCl）
检测原理
将chIL-2探针电化学固定于活化PGE表面。当含IL-2互补序列的pEThIL-2质粒与探针杂交时，电极表面形成双链DNA，使更多鸟嘌呤（G）碱基靠近电极并参与阳极氧化。ADPV在0.50–1.15 V扫描，G氧化产生阳极电流；杂交后电流高于仅探针背景，差值ΔI随质粒浓度增加而增大，约5000 pg/µL饱和。非互补pET21a(+)主要发生非特异吸附或缺陷杂交，经300 s洗涤可解吸或降低信号，因此选择性提高。无外源标记或酶放大，信号直接来自DNA中G的氧化。
检测灵敏度
LOD: 10.31 pg/µL；线性范围: 0–500 pg/µL；R^2 = 0.9992；信号饱和: ca. 5000 pg/µL
效应效果
在优化条件下（PGE活化300 s、洗涤300 s），互补pEThIL-2使鸟嘌呤氧化电流由54.19±2.26 nA增至192.03±11.12 nA；非互补pET21a(+)仅由54.19±2.26 nA增至65.8±4.38 nA，c/n=2.92、n/p=1.21，表明对与骨架93%同源的质粒具有选择性。三个独立修饰电极在200 pg/µL下RSD为4.2%，重现性良好。检测限10.31 pg/µL，信号约5000 pg/µL饱和。未报告实际样品加标回收率或与ELISA/HPLC/qPCR的对比；作者认为该无标记PGE传感器可低成本、直接检测重组IL-2质粒并鉴别其表达载体。
传感器的构成
- 工作电极基底：铅笔石墨电极（PGE），直径2.0 mm，横截面积0.037 cm²，作为导电基底与电化学换能器。
- 电极封装：聚四氟乙烯（Teflon）包覆铅笔芯，铜线焊接金属夹，仅横截面接触溶液，防止侧向导电。
- 电极活化处理：0.50 M醋酸缓冲液（pH 4.80）含20 mM NaCl中1.80 V活化300 s，形成适合探针固定的活性表面并降低非特异吸附。
- 识别元件：20-mer反义单链寡核苷酸探针chIL-2（5′-CTAAATTTAGCACCTCCTCC-3′），在0.50 V下电化学固定于PGE，与pEThIL-2中IL-2互补序列杂交。
- 信号标记物：无外源标记，依赖杂交双链DNA中鸟嘌呤（G）的阳极氧化电流作为信号。
- 测量介质：20 mM Tris–HCl（pH 7.0）含20 mM NaCl，用于杂交后洗涤和ADPV测量。
- 参比/辅助电极：饱和甘汞电极（SCE）与铂丝，构成三电极系统用于ADPV测量。
中文摘要
本文报道了重组质粒pEThIL-2的构建及其基于白细胞介素-2（IL-2）DNA插入片段的电化学传感与鉴别。pEThIL-2通过PCR扩增IL-2编码DNA并克隆至pET21a(+)载体构建，总长5839 bp。采用无标记DNA杂交生物传感器检测该质粒：将与人IL-2基因相关的20碱基反义单链寡核苷酸探针chIL-2固定于铅笔石墨电极（PGE）表面，随后以阳极差分脉冲伏安法（ADPV）监测鸟嘌呤氧化信号变化。以不含IL-2编码DNA的非互补质粒pET21a(+)（5443 bp）评估选择性。通过优化电极活化条件与洗涤策略，消除非特异性吸附；发现PGE活化300 s后，在含20 mM NaCl的20 mM Tris–HCl（pH 7.0）中洗涤300 s可使非特异性吸附质粒解吸。该传感器检测限为10.31 pg/µL。
英文摘要
Construction, electrochemically biosensing and discrimination of recombinant pEThIL-2 plasmid, with 5839bp size, on the basis of interleukine-2 (IL-2) DNA insert are described. Plasmid pEThIL-2 was constructed by PCR amplification of IL-2 encoding DNA and subcloning into pET21a(+) vector using BamHI and SacI sites. The recombinant pEThIL-2 plasmid was detected with a label-free DNA hybridization biosensor using a non-inosine substituted probe. The proposed sensor was made up by immobilization of a 20-mer antisense single strand oligonucleotide (chIL-2) related to the human interleukine-2 gene on the pencil graphite electrode (PGE) as a probe and then the sensing of recombinant pEThIL-2 plasmid was conducted by anodic differential pulse voltammetry (ADPV) based on guanine oxidation signal. Selectivity of the detection was assessed with pET21a(+) non-complementary plasmid, with 5443bp size, lacking IL-2 encoding DNA. Different factors such as electrode activation conditions and washing strategy were tested in order to eliminate the nonspecific adsorption of pET21a(+). We have found that the PGE activation for 300s produces a condition in which desorption of nonspecifically adsorbed plasmids from the electrode surface can be achieved by 300s washing of the electrode in 20mM Tris-HCl buffer solution (pH 7.0) containing 20mM NaCl. Diagnostic performance of the biosensor is described and the detection limit is found to be 10.31pg/microL.