用于检测转基因作物的电化学生物传感器: Comparison
Please note this is a comparison between Version 1 by Jie Jayne Wu and Version 4 by Beatrix Zheng.

Food safety issues are directly related to people's quality of life, so there is a need to develop efficient and reliable food contaminants’ detection devices to ensure the safety and quality of food. Electrochemical biosensors have the significant advantages of miniaturization, low cost, high sensitivity, high selectivity, rapid detection, and low detection limits using small amounts of samples, which are expected to enable on-site analysis of food products.食品安全问题直接关系到人们的生活质量,因此需要开发高效可靠的食品污染物检测装置,以确保食品安全和质量。电化学生物传感器具有小型化、低成本、高灵敏度、高选择性、快速检测、使用少量样品的低检测限等显著优势,有望实现食品的现场分析。

  • electrochemistry
  • biosensor
  • food safety
  • high sensitivity
  • high selectivity

1. Introduction简介

Safe food is a fundamental need for human health. Food safety can be affected by harmful substances such as allergens, pathogens (e.g., parasites, bacteria, viruses, prions, etc.), toxic agents or radioactive substances [1]. To safeguard human health, regulatory agencies such as the 安全食品是人类健康的基本需求。食品安全可能受到有害物质的影响,例如过敏原,病原体(例如寄生虫,细菌,病毒,朊病毒等),有毒物质或放射性物质[1]。为了保障人体健康,美国食品药品监督管理局(United States Food and Drug Administration (USFDA), the European Food Safety Authority (EFSA), and the Chinese Food and Drug Administration (CFDA) have imposed limits on the maximum levels of various contaminants in food. Nevertheless, in 2015, the World Health Organization (WHO) estimated that more than 600 million cases of foodborne diseases and 420,000 deaths are likely to occur each year, due to foodborne diseases caused by 31 foodborne pathogens at the global and subregional levels [2]. In agriculture, pesticides control pests and diseases in crops and ensure crop yield and quality. However, the overuse of pesticides can leave residues on crops that threaten human health through the food chain [3]. In addition, many food additive safety incidents had occurred around the world, such as aquatic products containing malachite green, red-hearted duck eggs dyed with Sudan red, melamine milk powder, industrial gelatin yogurt, etc., causing distrust and fear among people [4]. In order to screen and monitor the safety of food and prevent harm from food contaminants, a sensitive and reliable on-site analysis technology for food contaminants is highly desired. At present, there are many mature technologies for food safety detection, such as gas chromatography (FDA)、欧洲食品安全局(EFSA)和中国食品药品监督管理局(CFDA)等监管机构对食品中各种污染物的最高含量进行了限制。然而,2015年世界卫生组织(WHO)估计,在全球和次区域层面,由于31种食源性病原体引起的食源性疾病,每年可能发生超过6亿例食源性疾病病例和42万例死亡[2]。在农业中,农药控制作物的病虫害,确保作物产量和质量。然而,过度使用杀虫剂会在作物上留下残留物,通过食物链威胁人类健康[3]。此外,世界各地还发生了许多食品添加剂安全事件,如含有孔雀石绿的水产品,用苏丹红染色的红心鸭蛋,三聚氰胺奶粉,工业明胶酸奶等,引起了人们的不信任和恐惧[4]。为了筛查和监测食品安全并防止食品污染物的危害,非常需要一种灵敏可靠的食品污染物现场分析技术。
目前,食品安全检测有许多成熟的技术,如气相色谱(GC), high-performance liquid chromatography ()、高效液相色谱(HPLC), gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LCMS), and enzyme-linked immunosorbent assay (ELISA) [5]. However, most of these methods have disadvantages, such as complicated operation, high detection costs, long detection time, and high requirements for the samples tested, which are prone to false positives. To improve this situation, simple, rapid, economical, and portable electrochemical biosensors have attracted much attention. They could not only achieve high specificity and sensitivity, but also enable real-time monitoring in the field. The basic components and principles of an electrochemical biosensor for food contaminants detection are shown in Figure )、气相色谱-质谱(GC-MS)、液相色谱-质谱(LCMS)、酶联免疫吸附法(ELISA)[5]。然而,这些方法大多存在操作复杂、检测成本高、检测时间长、对被测样品要求高等缺点,容易出现假阳性。为了改善这种状况,简单、快速、经济、便携的电化学生物传感器备受关注。它们不仅可以实现高特异性和灵敏度,还可以实现现场实时监测。用于食品污染物检测的电化学生物传感器的基本组件和原理如图1. It can convert the biological signal generated by the specific combination of the target analytes and the sensitive elements into an electrical signal, which is detected by the electrochemical methods. Finally, signal processing is performed by a computer to achieve quantitative or qualitative detection of food contaminants. 所示。它可以将目标分析物和敏感元素的特定组合产生的生物信号转换为电信号,通过电化学方法进行检测。最后,信号处理由计算机执行,以实现食品污染物的定量或定性检测。
Figure 1. Schematic diagram of an electrochemical biosensor for food contaminants detection.
Common常见的电化学方法包括电位法、循环伏安法 electrochemical methods include potentiometry, cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), square wave voltammetry (SWV), and differential pulse voltammetry (DPV). Potentiometry is one of the simplest electrochemical techniques, characterized by a short response time, high selectivity, and extremely low detection limit [6]. Cyclic voltammetry is performed with the applied electrical potential oscillating over a range, while electrochemical impedance spectroscopy is usually performed at a fixed potential over a frequency range [7]. Square wave voltammetry is one of the most advanced and versatile members of the pulse voltammetry technology family, which has high analytical sensitivity and measurement speed [8]. Differential pulse voltammetry is more sensitive than conventional pulse, derivative conventional pulse, and cyclic voltammetry, and is suitable for studying the electrochemical process at the interface of metal(CV)、电化学阻抗谱法 (EIS)、方波伏安法 (SWV) 和差分脉冲伏安法 (DPV)。电位法是最简单的电化学技术之一,其特点是响应时间短,选择性高,检测限极低[6]。循环伏安法是在施加的电势在一定范围内振荡的情况下进行的,而电化学阻抗谱通常在一定频率范围内的固定电位下进行[7]。方波伏安法是脉冲伏安法技术家族中最先进、用途最广泛的成员之一,具有很高的分析灵敏度和测量速度[8]。差分脉冲伏安法比常规脉冲法、导数常规脉冲法和循环伏安法更灵敏,适用于研究金属-electrolyte solution [9]. Although electrochemical biosensors have not been widely used in food safety detection, their significant advantages deserve further investigation.电解质溶液界面处的电化学过程[9]。虽然电化学生物传感器尚未广泛应用于食品安全检测,但其显著优势值得进一步研究。

2. Genetically Modified Crops转基因作物

转基因作物(Genetically Modified Crops (MC)是通过基因工程技术修饰具有目标性状的基因,然后引入受体植物的基因组中的植物[70]。这些外源基因不仅在后代中稳定遗传,而且可以在作物中产生抗虫性、抗除草剂性、抗病性状等有益性状。然而,GMC) are plants in which genes with target traits are modified by genetic engineering techniques and then introduced into the genome of the recipient plant [10]. These exogenous genes are not only stably inherited in the offspring, but can also lead to beneficial traits such as insect resistance, herbicide resistance, and disease resistance in the crop. However, the biosafety of 的生物安全性一直存在争议。目前,GMC has been controversial. Currently, the detection of GMC components mainly includes gene nucleic acid detection, protein detection, and metabolite detection [10]. although traditional detection methods such as 成分的检测主要包括基因核酸检测、蛋白质检测和代谢物检测[70]。虽然PCR, ELISA, and HPLC are mature and reliable, they cannot meet the practical needs of high speed and low cost. Therefore, a fast, accurate, and low-cost field detection platform for transgenic crops is needed. The following is a discussion of electrochemical biosensors for GM soybean and maize detection (Table 1).和HPLC等传统检测方法成熟可靠,但无法满足高速、低成本的实际需求。因此,需要一种快速、准确、低成本的转基因作物田间检测平台。以下是用于转基因大豆和玉米检测的电化学生物传感器的讨论(表8)。
Marcos et al. [11] developed an electrochemical immunosensor for the detection of the transgenic soy protein 等人[71]开发了一种电化学免疫传感器,用于检测大豆种子中的转基因大豆蛋白CP4 EPSPS in soybean seeds, which does not require a labeling and signal amplification system. The sensor schematic is shown in Figure 2. The ,这不需要标记和信号放大系统。传感器原理图如图16所示。首先将CP4 EPSPS antibodies were first modified on the gold electrodes, and the modified gold electrode was incubated in soy protein solutions of different concentrations at 抗体修饰在金电极上,并将修饰后的金电极在不同浓度的大豆蛋白溶液中于37 °C for 孵育30 min. After washing with ultrapure water, the electrodes were immersed in an electrochemical cell containing 。用超纯水洗涤后,将电极浸入含有K4[Fe(CN)铁(中)6] (1 mM) and LiCl (0.1 M) solutions. Electrochemical measurements were performed using SWV, and the peak current was linearly related to the concentration of soy protein in the range of )和氯化锂(0.1 M)溶液。使用SWV进行电化学测量,峰值电流与大豆蛋白浓度线性相关,范围为0.005–0.3 mg/mL, with a detection limit of ,检测限为38 ng/mL CP4 EPSPS (below (低于0.00038% CP4 EPSPS). Since many countries recommend labeling foods containing higher than 0.9% )。由于许多国家建议标记CP4 EPSPS, the detection limit of this sensor meets the detection needs含量高于0.9%的食品,因此该传感器的检测限满足了检测需求。
Figure 216. Schematic diagram of the electrochemical immunosensor for the detection of 用于检测CP4 EPSPS (Adapted with的电化学免疫传感器示意图(经参考文献[71]许可改编)。 permission from Ref. [11]. 2022, Marcos et al.).,马科斯等人)。
Gao et al. [12] also established a label-free electrochemical sensing platform for the detection of transgenic soybean 等[72]还建立了用于检测转基因大豆SHZD32-1. 的无标记电化学传感平台。将大豆Soybean SHZD32-1 seeds were ground into powder and then genomic DNA samples were extracted by a CTAB-based method. GCDs modified on the SPCE surface can be attached to single-stranded DNA probes via Au-S bonds while improving the conductivity of the DNA sensor. After binding of the DNA probes to transgenic soybean DNA, the electron transfer resistance (种子磨成粉末,然后采用CTAB法提取基因组DNA样品。在SPCE表面上修饰的GCD可以通过Au-S键连接到单链DNA探针上,同时提高DNA传感器的电导率。DNA探针与转基因大豆DNA结合后,电子转移电阻(Ret) on the sensor surface was quantified by the )在传感器表面通过Ret response increased with logarithmic increase in target 进行量化响应随着靶DNA concentration over a linear range of 浓度在1.0 × ×10线性范围内的对数增加而增加−7–1.0 × 10−13 M, with a detection limit of ,检测限为 3.1 × 10−14 M. This label-free sensor is made by inserting the SPCE into a handheld EI analyzer is conveniently fabricated, demonstrating simplicity of construction and operation, requiring no additional indicators or cumbersome procedures, and can be used in a friendly manner by non-specialists.这种无标记传感器通过将SPCE插入手持式EI分析仪制成,制造方便,结构和操作简单,不需要额外的指示器或繁琐的程序,并且可以由非专业人员以友好的方式使用。
Cui et al. [13] developed a label-free electrochemical impedance gene sensor using gold carbon dots (等人[73]开发了一种使用金碳点(GCDs) and an easy-to-use portable device. It consists of a handheld electrochemical impedance (EI) analyzer equipped with a coin-sized )和易于使用的便携式设备的无标记电化学阻抗基因传感器。它由配备硬币大小的SPCE. Figure 3 shows the preparation process of this sensor, 的手持式电化学阻抗(EI)分析仪组成。图17显示了该传感器的制备过程,使用GCDs were used to modify a screen-printed carbon electrode and capture probes were immobilized by 修饰丝网印刷的碳电极,并通过Au-S bonding. Transgenic maize sample DNA was extracted using a one-step extraction method with direct plant lysis buffer and amplified by recombinase polymerase amplification (RPA). The capture probes immobilized on the sensor were identical to the forward RPA primer. After the amplification products bound to the capture probes, the EI signal increased due to the formation of a biocomplex that hindered the interfacial electron transfer. The proposed genetic sensor combined with RPA can detect maize Ruifeng12-5 in a linear range of 键合固定捕获探针。采用一步提取法直接提取植物裂解缓冲液,并通过重组酶聚合酶扩增(RPA)扩增转基因玉米样品DNA。固定在传感器上的捕获探针与正向RPA底漆相同。放大产物与捕获探针结合后,由于生物复合物的形成阻碍了界面电子转移,EI信号增加。所提出的遗传传感器结合RPA可以在0.10–5.0% with a detection limit of 0.10%, roughly calculated as 36 copies/µL based on the size of the maize haploid genome. The sensor device is simple to prepare and does not require expensive instruments or specialized personnel and has wide application prospects.的线性范围内检测玉米瑞丰12-5,检测限为0.10%,根据玉米单倍体基因组的大小,大致计算为36拷贝/μL。该传感器装置制备简单,不需要昂贵的仪器或专业人员,具有广泛的应用前景。
Figure 317. Fabrication process of EI gene sensor based on GCD (Reprinted with permission from Ref. [13]. 2022, Cui et al.).
基于GCD的EI基因传感器的制备工艺(经文献[73]许可转载。 2022,崔等人)。
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