桔青霉素(citrinin，CIT)是一种由青霉属(Penicillium)、曲霉属(Aspergillus)和红曲霉属(Monascus)中的部分菌株产生的聚酮类真菌毒素，其主要存在于储存的谷物中[1]。特别在气候较炎热的国家生产的谷物，CIT的污染是一个严重问题[2]。谷物产品和其他具有药用价值的植物在生长、收获、运输和储存过程中可能会被产生CIT的真菌污染[3-4]。当处理和储存条件没有得到严格控制时，水果也易被CIT污染而造成重大损失。根据调查，工业化国家的作物收获后损失约为25%，其他国家超过50%[5]。而且CIT被证明对动物具有较强的肾毒性以及潜在的遗传毒性、胚胎毒性[6]和致畸性[7]。鉴于其对人类健康及经济发展的危害，开发一种灵敏、快速的CIT检测方法显得非常重要。日本厚生省在2000年版的“日本食品添加剂标准”中率先制定出红曲色素中CIT限量标准为0.2 mg/kg，这一标准是当时能够检出的CIT的最低剂量[8]，欧盟规定CIT在红曲霉发酵的大米补充剂中的限量标准为2 mg/kg，中国台湾省制定的《食品中真菌毒素限量标准》规定了食品中CIT的限量，其中红曲色素中CIT的限量为0.2 mg/kg、原料用红曲米的限量为5 mg/kg以下，使用红曲原料制成的食品为2 mg/kg以下[8]。目前在中国大陆地区，还没有建立不同农产品中CIT含量的控制指标。

目前，CIT检测可用的分析方法有薄层色谱法[9]、高效液相色谱法(HPLC)[10]、液相色谱串联质谱法(liquid chromatography-tandem mass spectrometry，LC-MS/MS)[11]、超高效液相色谱和荧光检测法[12]、气相色谱质谱法[13]和酶联免疫分析法(enzyme-linked immunosorbent assay，ELISA)[14]。薄层色谱法虽然操作简单，但是准确性和特异性较差；HPLC等仪器检测方法虽然灵敏度和稳定性较高，但样品前处理操作较为复杂，且需要专业的大型仪器和检测场所，不能满足食品安全快速检测要求。ELISA作为一种灵敏度高、高通量、设备简单、成本低的样品筛查和定量方法，近年来得到了迅速发展和广泛应用。基于辣根过氧化物酶(horse radish peroxidase，HRP)催化底物的比色ELISA法存在灵敏度低、抗基质干扰能力差的局限性。荧光酶联免疫分析法(fluorescein-linked immunosorbent assay，FELISA)与现有的分析平台兼容性良好，被认为是目前有害食源性物质筛选最敏感的方法之一[15-16]。在过去的几十年里，已开发出多种具有高灵敏度的FELISA用于检测目标分子，包括基于碱性磷酸酶的化学发光免疫检测[17]、基于HRP的化学发光免疫检测[18]、基于过氧化氢酶的荧光免疫检测[19]、基于人α凝血酶的荧光免疫检测[20]等，具有更广泛的应用前景。因此，建立一种具有高灵敏度的FELISA方法用于谷物中CIT的定量检测具有较大的应用前景。

胰蛋白酶(trypsin)是一种高特异性的丝氨酸蛋白水解酶，对于含精氨酸(Arg)位点的肽段表现出非常高的水解催化性能[21]。基于trypsin的这个特性，研究人员设计并合成了含氨基保护基的三肽Cbz-Ile-Pro-Arg-OH，再通过酰化反应将Arg的羧基与罗丹明110分子中的2个氨基缩合形成双酰胺底物(Cbz-Ile-Pro-Arg)2-R110，用于trypsin的催化能力测定[22]。在trypsin的催化下，连接在罗丹明110分子两端的酰胺键迅速发生水解断裂，具有微弱荧光的罗丹明110双酰胺底物转变为具有强荧光的罗丹明110分子，荧光信号强度可以得到极强的提高[23]。本研究通过将trypsin和罗丹明110双酰胺底物引入到FELISA中，克服实际样本的基质干扰，以实现检测灵敏度提高的目的。

本研究通过甲醛加成法[24]合成了trypsin-CIT，并将其引入到FELISA信号生成系统实现CIT的快速检测。其原理如图1所示，trypsin-CIT可通过抗原-抗体相互作用被固定在酶标孔中，选择trypsin替代常用的HRP作为标记酶，并且选用仅存在极低荧光的罗丹明110双酰胺衍生物作为trypsin的催化底物。通过CIT与trypsin-CIT对其抗体的竞争关系，被固定下来的trypsin催化罗丹明110双酰胺衍生物裂解而触发荧光信号的产生，实现对CIT的快速检测。本研究与传统ELISA的HRP-TMB显色体系进行了对比，显示出较高的分析灵敏度，并将其应用于检测玉米样本，验证了本方法的检测性能和稳定性。

1 材料与方法

1.1 材料与仪器

胰蛋白酶、链球菌蛋白G, 美国Sigma-Aldrich公司；(Cbz-Ile-Pro-Arg)2-R110赛默飞世尔科技公司； CIT， Fermentek公司；抗CIT腹水， 南昌大学中德联合研究院；脱脂乳， 广东赛国生物科技有限公司；甲醇(色谱纯、分析纯)及常见试剂(分析纯)，西陇科学分有限公司；96孔酶标板， Costar公司。

Waters H CLASS/XEVD TQD， 美国沃特世公司；桔青霉素免疫亲和柱， 北京华安麦克生物技术有限公司；Varioskan LUX多功能酶标仪， 赛默飞世尔公司；超纯水仪， 美国密里博公司。

1.2 trypsin-CIT与HRP-CIT的制备

将5.7 mg trypsin溶解于2 mL浓度为1 mmol/L的HCl(pH 3)中，随后向其中逐滴加入10 mg/mL的CIT甲醇溶液100 μL，混合均匀后加入200 μL体积分数为37%甲醛溶液，37 ℃搅拌反应24 h，即得到trypsin-CIT，同样CIT与HRP反应得到HRP-CIT，反应结束后，置0.01 mol/L PBS中4 ℃透析72 h。

1.3 trypsin催化的荧光底物浓度

罗丹明110双酰胺荧光底物的浓度对于trypsin催化的FELISA信号强度有着较大影响，因此本研究测定了在相同trypsin质量浓度(400 ng/mL)条件下，荧光底物浓度与trypsin浓度的变化规律，获得最佳目标底物浓度。

1.4 trypsin催化的FELISA检测流程

将链球菌蛋白G(proteinG)用PBS(pH 8.6)稀释至25 μg/mL，每孔100 mL，4 ℃过夜包被，去除孔内液体，用含0.05%吐温-20的磷酸缓冲液进行洗板，重复3次，用PBS(pH 7.4)将抗CIT腹水稀释至20 μg/mL，每孔100 μL，37 ℃孵育1 h后洗板，加入300 μL 1%脱脂乳溶液，37 ℃孵育1 h，加入50 μL质量浓度为5.5 μg/mL的trypsin-CIT和50 μL的待测液，37 ℃孵育30 min后洗板，加入浓度为0.312 5 μmol/L的罗丹明双酰胺底物，37 ℃孵育30 min后测定激发波长为498 nm，发射波长为521 nm时的荧光强度。

1.5 trypsin催化的FELISA的精密度与准确性评价

分别通过LC-MS/MS和FELISA的方法对加标的阴性玉米样本进行分析，随后根据GB 5009.222—2016《桔青霉素测定》中的样本提取方法：称取10.0 g加有CIT的玉米样本于锥形瓶中，加入50 mL体积分数70%的甲醇水溶液，在涡旋振荡2 min，5 000 r/min离心，取上清液用快速定性滤纸过滤后待检测。取上清液用0.01 mol/L PBS稀释7倍后，进行FELISA检测；另取1 mL上清液加入49 mL 0.01 mol/L磷酸溶液(pH 7.5)混匀，用微纤维滤纸过滤后取20 mL上述溶液过免疫亲和柱净化，将其以1～2滴/s的流速全部通过亲和柱，再将5 mL 0.01 mol/L磷酸溶液(pH 7.5)也以1～2滴/s的流速通过亲和柱，待液体排干后，用2 mL甲醇以1滴/s的流速进行洗脱，洗脱液收集至样品瓶中用于LC-MS/MS分析。

2 结果与讨论

2.1 trypsin催化的荧光底物浓度

根据图2-a可得，在trypsin质量浓度固定于400 ng/mL条件下，随着底物浓度升高，trypsin催化底物产生的荧光信号也逐渐升高。结合荧光信号强度，底物浓度为0.156 μmol/L时具有明显的荧光信号，故选择了0.156 μmol/L为本实验的最终底物浓度。

2.2 trypsin-CIT与trypsin催化性能对比

在0.156 μmol/L底物浓度下，绘制trypsin、trypsin-CIT浓度与荧光强度关系曲线，如图2-b所示，trypsin和trypsin-CIT质量浓度为25 ng/mL时，trypsin-CIT催化产生的荧光强度为35.42，trypsin催化产生的荧光强度为16.68，可推得酶活损失52.91%。

2.3 trypsin催化的荧光ELISA的检测条件优化

实验优化了包被腹水浓度、trypsin-CIT浓度、pH、甲醇浓度、NaCl浓度、免疫孵育时间和酶催化时间等影响FELISA的几个关键参数，以获得高灵敏度和强荧光信号。

包被抗体和trypsin-CIT的浓度被认为是影响直接竞争ELISA检测灵敏度的最重要因素。为了提高CIT抗体的生物活性，本实验预先将proteinG包被在酶标板上，定向捕获CIT抗体的Fc片段，从而提高CIT抗体的生物活性。因此本实验设置腹水质量浓度为80、40、20、10 μg/mL和trypsin-CIT质量浓度为22、11、5.5、2.75 μg/mL进行正交实验，结果如表1，通过(F为阴性条件下的荧光值，F0为50 ng/mL加标条件下的荧光值)反映本方法对CIT的检测灵敏度，并基于F与F0的最大差值与B0的最高值原则选择最佳腹水浓度与trypsin-CIT浓度，根据最佳信号显示，包被腹水质量浓度为20 μg/mL、trypsin-CIT质量浓度为5.5 μg/mL是最佳条件。

pH、甲醇浓度、NaCl浓度能够通过改变抗原抗体结合位点的活性来影响抗原抗体反应，本实验通过B0=(1-F/F0)×100(F为阴性条件下的荧光值，F0为50 ng/mL加标条件下的荧光值)反映加入标准品后的抑制结果，当B0值越大时，表示本方法对于CIT的检测灵敏度越高。如图3-a所示，在pH 6.5时，B0达到最大值，为最佳pH条件。由于CIT分子具有很强的疏水性，为实现对样本中CIT的高回收率提取，需要用含有一定体积分数甲醇的样本提取液，但是待检测溶液中的甲醇体积分数过高时，抗原抗体的反应会受到极大干扰，因此，研究了含不同体积分数甲醇的待检测溶液进行反应的检测结果，如图3-b所示，当甲醇体积分数升高时，B0会逐渐下降，且在甲醇体积分数>10%时B0从34.09%降至15.55%，基于这一结果，本实验选择甲醇体积分数为10%为免疫检测最佳条件。在免疫反应过程中，一定浓度的NaCl有利于抗原抗体的相互作用，如图3-c所示，随着NaCl浓度的升高，B0也逐渐升高，且当NaCl浓度达到80 mmol/L时，B0达到最高值。为了评价免疫孵育时间和酶催化时间对B0的影响，设置了不同免疫孵育时间(15～75 min)和不同酶催化时间(10～60 min)作为反应条件，30 min是免疫孵育时间最佳条件(图3-d)，酶催化时间达到30 min后，B0进入平台期(图3-e)，因此最佳酶催化时间为30 min。

2.4 trypsin催化的FELISA检测性能评价

以CIT浓度为横坐标，B0为横坐标绘制定量曲线(图4-a)，随着溶液中的CIT质量浓度从1.56～6 400 ng/mL，B0逐渐增加，且在CIT质量浓度为12.5～400 ng/mL时，B0随CIT浓度增加呈线性增加关系，其定量可用方程y=18.606 lnx-18.713(R2=0.992)，检测灵敏度IC20为8.01 ng/mL，IC50为40.16 ng/mL，为了与基于HRP-CIT的传统比色ELISA检测性能对比，设置腹水质量浓度为80、40、20、10 μg/mL，HRP-CIT质量浓度为80、40、20、10 μg/mL进行正交实验同样优化了二者浓度，结果如表2，根据竞争抑制率B0=(1-OD阴性/OD阳性)×100(OD阴性为阴性条件下的吸光度，OD阳性为加标200 ng/mL条件下的吸光度)，由最高竞争抑制率所对应的条件得出最佳腹水质量浓度为40 μg/mL，HRP-CIT质量浓度为40 μg/mL，同时基于此条件构建了基于HRP-CIT的传统比色ELISA，并绘制了其定量曲线(图4-b)，其定量可用方程y=13.476 lnx-11.975(R2=0.971 2)，检测灵敏度IC20为10.73 ng/mL，IC50为110.08 ng/mL，对比可得，本实验构建的FELISA检测灵敏度是传统比色ELISA的1.34倍，IC50是传统比色ELISA的2.741倍。

用本方法进一步检测质量浓度为1 μg/mL的常见真菌毒素CIT、脱氧雪腐镰刀菌烯酮(deoxynivalenone，DON)、玉米赤霉烯酮(zearalenone，ZEN)、黄曲霉毒素B1(aflatoxin B1，AFB1)、黄曲霉毒素B2(aflatoxin B2，AFB2)、黄曲霉毒素G1(aflatoxin AFG1，AFG1)和伏马毒素B1(fumonisin B1，FB1)，结果如图5所示，与常见真菌毒素无明显的交叉反应，表明本方法检测CIT具有较好的特异性。

为了评价该方法检测实际样本的可行性，将不同质量浓度的CIT添加至玉米样本中，加标量分别为3.5、1.75、0.88、0.22 mg/kg，结果如表3，FELISA批内回收率分别为97.05%、98.97%、110.58%、90.94%，批间回收率分别为90.10%、109.11%、101.63%、93.46%，且批内、批间测定的标准偏差值均小于15%。检测结果与液相色谱串联质谱法作为参考方法进行对比，表4结果显示trypsin催化的FELISA检测结果与LC-MS/MS结果具有良好的一致性。以上结果表明，trypsin催化的FELISA具有良好的回收率和准确性，可对玉米样本中的CIT进行定量检测。

3 结论

本研究基于trypsin催化罗丹明110双酰胺荧光底物的特性，结合CIT和CIT标记的trypsin与CIT抗体的相互竞争作用关系，建立了一种FELISA方法用于检测玉米中的CIT。在最佳条件下：抗CIT腹水质量浓度20 μg/mL、trypsin-CIT质量浓度为5.5 μg/mL、pH 6.5、甲醇体积分数10%、NaCl浓度80 mmol/L、免疫反应时间30 min、酶催化时间30 min，该方法检测CIT的最低灵敏度可达8.01 ng/mL，IC50可达40 ng/mL，是HRP催化的比色ELISA检测灵敏度的1.34倍，IC50的2.741倍，此外，该方法具有良好的特异性、重现性和精密度。如果使用CIT的单克隆抗体，检测灵敏度还可以更高，为更快速、灵敏定量检测CIT提供了新的选择。

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Fluorescence ELISA based on trypsin-catalyzed rhodamine 110 bis-amide derivative for the detection of citrinin

ZOU Hui1,2, NIE Lijuan1,2, XIONG Yonghua1,2, LI Xiangmin1,2*

1(State key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047, China) 2(Jiangxi-OAI Joint Research Institute, Nanchang University, Nanchang 330047, China)

ABSTRACT Citrinin (CIT) is a fungal toxin that is widely found in crops such as maize and rice and is toxic to animals in terms of nephrotoxicity, genotoxicity, embryo toxicity and teratogenicity. In addition, CIT contamination in crops such as maize and fruit has caused large losses to the production economies of industrialized countries. In view of its harmful effects on human health and economic development, it is important to develop a sensitive and rapid method for the detection of CIT. Currently, Japan and the European Union have issued limits for CIT, while mainland China has not yet issued limits for CIT and has only issued methods for its detection. In the national standard detection method, it is necessary to clean up with an immunoaffinity column or a solid phase extraction column, and then use HPLC with fluorescence detector to determine it. Based on the limitations of large instrumental methods and the current situation of CIT contamination, it is necessary to develop a simpler and more sensitive method for the determination of CIT in cereals. As a sensitive, high-throughput, simple and low-cost method for sample screening and quantification, enzyme-linked immunosorbent assay (ELISA) has been rapidly developed and widely used in recent years. Traditional ELISA methods are based on horseradish peroxidase-catalyzed chromogenic substrates and thus quantitative detection of the analytes by colorimetric signals, which are susceptible to large interferences during the detection of complex substrates, thus affecting the accuracy of the results. The ELISA method, which uses fluorescence as the signal output, is considered to be one of the most sensitive methods for the screening of hazardous foodborne substances due to its ability to resist substrate interference and its high sensitivity. In order to achieve high sensitivity, a fluorescent ELISA(FELISA) method was constructed for the detection of CIT in maize by introducing trypsin, which has high specificity for the arginine site in the peptide, and a rhodamine 110 bis-amide derivative, which is attached to both ends of the fluorescent molecule through the arginine-containing peptide, as the enzyme and substrate in the ELISA method as follows: non-fluorescent rhodamine 110, the bis-amide fluorescent derivative was hydrolyzed by trypsin, the arginine present between the rhodamine 110 molecule and the two terminal peptide chains, and the resulting rhodamine 110 molecule was highly fluorescent. Trypsin-CIT was synthesized by formaldehyde addition, retaining 47.09% of the enzymatic activity, and after immobilization of the CIT antibody on the enzyme plate by protein G, using the ELISA was established using the competitive binding relationship between trypsin-CIT and CIT and CIT antibodies. When both were present in the assay system and the concentration of trypsin-CIT was certain, the number of trypsin-CIT bound to CIT antibodies was stronger because of the stronger binding ability of CIT and CIT antibodies, when the concentration of CIT gradually increased, the number of trypsin-CIT bound to CIT antibodies Therefore, the concentration of rhodamine bis-amide derivatives was firstly optimized in this experiment, and the concentration of the derivative that was catalytically hydrolyzed to produce the strongest fluorescence signal at a certain enzyme concentration was selected as the optimal substrate concentration, and in order to obtain the best performance of the assay, the concentration of the derivative that was catalytically hydrolyzed to produce the strongest fluorescence signal was determined by B0=(1-F/F0)×100. In order to obtain the best performance of the assay, B0=(1-F/F0)×100 (F is the fluorescence value under negative conditions, F0 is the fluorescence value under 50 ng/mL spiking conditions) was used to reflect the performance of the established method. The optimal conditions were: 20 μg/mL of encapsulated ascites, 5.5 μg/mL of trypsin-CIT, pH 6.5, 10% of methanol, 80 mmol/L of NaCl, 30 min of immunoincubation time and 30 min of enzyme catalytic time. The standard curve can be expressed by the equation y=18.606 ln x-18.713(R2=0.992). The quantitative range of the method is 12.5-400 ng/mL, with a minimum sensitivity of 8.01 ng/mL and an IC50 of 40 ng/mL. The IC50 was up to 40 ng/mL, which is 1.34 times the sensitivity and 2.741 times the IC50 of the horseradish peroxidase-catalyzed colorimetric ELISA. Subsequently, six common mycotoxins were assayed at high concentrations and the results showed that the method had no significant cross-reactivity with the common mycotoxins and had good specificity. The results showed that the spiked recoveries of the method ranged from 90.10%-110.58% and the intra- and inter-batch coefficients of variation ranged from 3.65%-14.08%, indicating that the method has good reproducibility and precision. The results showed that the method is suitable for the rapid and sensitive quantification of different concentrations of CIT in maize. In conclusion, this experiment has established a FELISA method for the determination of CIT in maize by introducing fluorescence signals, which has good performance and application prospects and provides a reference for subsequent use in the determination of other targets.

Key words FELISA; trypsin; rhodamine 110; citrinin

DOI:10.13995/j.cnki.11-1802/ts.030039

引用格式:邹回，聂丽娟，熊勇华，等.胰蛋白酶催化罗丹明110双酰胺衍生物荧光酶联免疫吸附方法检测桔青霉素[J].食品与发酵工业,2022,48(11):246-252.ZOU Hui, NIE Lijuan, XIONG Yonghua, et al.Fluorescence ELISA based on trypsin-catalyzed rhodamine 110 bis-amide derivative for the detection of citrinin[J].Food and Fermentation Industries,2022,48(11):246-252.

第一作者：硕士研究生(李响敏讲师为通信作者，E-mail：lixiangmin73@163.com)

基金项目：“十三五”国家重点研发计划项目(2018YFC1602203)

收稿日期：2021-11-11，改回日期：2021-12-30