Electronic SupportingMaterialsonthe Microchimica Acta publication entitled

A conventional chemical reaction for use in an unconventional assay: A colorimetric immunoassay for aflatoxin B1 by using enzyme-responsive just-in-time generation of a MnO2 based nanocatalyst

WenqiangLai1,*·QiaoZeng2· JuanTang 3· MaoshengZhang 1Dianping Tang4

1Key Laboratory of Modern Analytical Science and Separation Technology, College of Chemistry and Environment, Minnan Normal University, Zhangzhou 363000, People’s Republic of China

2Ji’an Vocational Polytechnic College, Ji’an 343000, People’s Republic of China

3Ministry of Education Key Laboratory of Functional Small Organic Molecule, Department of Chemistry and chemical engineering, Jiangxi Normal University, Nanchang 330022, People’s Republic of China

3Key Laboratory of Analysis and Detection for Food Safety (Ministry of Education & Fujian Province), Institute of Nanomedicine and Nanobiosensing, Department of Chemistry, Fuzhou University, Fuzhou 350108, People’s Republic of China

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Optimization of assay conditions

Furthermore, we also investigated the dynamic response curve (absorbance vs reaction time) at 652 nm between TMB,KMnO4 and Mn(II) (Fig. S1). As shown inFig. S1, the absorbance rapidly increases with the increasing reaction time, and reached to the steady-state equilibrium within one minute.To ensure adequate reaction between KMnO4-Mn(II) and TMB, 2 min was used for color development in this wok.

To achieve a good colorimetric immunoassay with high sensitivity, one important precondition was that the KMnO4-Mn(II)-TMB system should be highly efficient toward the KMnO4-responsive ascorbic acid-based colorimetric platform in the presence of low-concentration AOx. To adequately complement the catalytic efficiency of AOx, the concentration of Mn(II) should be optimized. Firstly, we investigated the effect of Mn(II) concentration on the TMB-KMnO4-Mn(II) system.In this case, 1.0 mM TMB and KMnO4 was used for color development. As shown in Fig. S2, the absorbance initially rapidly increased with the increasing Mn(II) concentration, and then tended to become slow after 2.0 mM. To improve sensitivity,consider absorbance and avoid the excessive Mn(II), 2.0 mM Mn(II) was used for the development of colorimetric assay.

Toward bioactive AOx enzyme, the catalyticefficiency toward AA relied on the catalytic time. At room temperature (RT), we monitored the effect of different incubationtimes between AOx and AA on the absorbance. Asindicated from Fig. S3, the absorbance increased within theinitial 15 min and then tended to slightly decrease. Therefore, 15 min was selected forenzymatic reaction in this work.

Monitoring of enzymatic activity with theTMB-KMnO4-Mn(II) system

Furthermore, we utilized the TMB-KMnO4-Mn(II) system for the detection ofAOx activity by using AA as the enzymatic substrate. The assayprinciple was schematically illustrated in Scheme 1. As seen fromFig. S4, the absorbance increases with the increasing AOx activity,indicating that the system could be employed for quantitativemonitoring of AOx activity. Favorably, a low detection limit couldbe estimated to 15 mU mL-1 AOx at the 3sblank criterion. Theresults also suggested that the absorbance of using the TMB-KMnO4-Mn(II)system could be significantly changed only if the level of AOx inthe sample was lower than 20 mU mL-1, which also provide a vitalprecondition for the development of high-efficient TMB-KMnO4-Mn(II)-based colorimetric immunoassay.

Fig. S1Effect of different reaction times between TMB, KMnO4 and Mn(II) [1.0 mM KMnO4 and 2.0 mM Mn(II) used in this case] on the absorbance.

Fig. S2Absorbance intensity of the TMB-KMnO4-Mn(II) system toward Mn(II) standards with variousconcentrations [1.0 mM KMnO4 used in this case].

Fig. S3Effect of reaction time of AOx with the colorimetric immunoassay[2.0 U mL-1AOx used in this case].

Fig. S4Catalytic reactivity of the AOx with different concentrations in the TMB-KMnO4-Mn(II)system.

Fig. S5Specificity of our developed strategy toward target AFB1 (20 ng mL−1), AFB2 (200 ng mL−1), AFG1 (200 ng mL−1), AFG2 (200 ng mL−1), OTA (200 ng mL−1) and OA (200 ng mL−1).

Table S1 comparison of the just-in-time generation of MnO2-based visual colorimetric immunoassay on analytical performance with other AFB1 detection schemes

Method / Indicator / Linear range / LOD / Ref.
Potentiometric immunosensor / AuNP-mAb / 0.1 – 5.0 ng mL-1 / 87 pg mL-1 / [1]
Fluorescence aptasensor / CdTe quantum dots / 1.0–1.0 × 105 ng mL-1 / 0.3 ng mL-1 / [2]
Lateral flow immunodipstick / Silver core and gold shell / - / 0.1 ng mL-1 / [3]
Photoelectrochemical immunoassay / CdTe quantum dots / 0.01 – 15 ng mL-1 / 3.0 pg mL-1 / [4]
Voltammetric immunoassay / Methylene blue / 0.01 – 30 ng mL-1 / 4.8 pg mL-1 / [5]
Fluorescence aptasensor / Ag nanoparticles / 0.005– 1.0 ng mL-1 / 0.3 pg mL-1 / [6]
Electrochemical aptasensor / Label-free / 0.125 – 16 ng mL-1 / 0.12 ng mL-1 / [7]
Chemiluminescence immunoassay / 2',6'-dimethylcarbonyphenyl / - / 0.01 ng mL-1 / [8]
Amperometric immunosensor / Label free / 0.2 – 30 ng mL-1 / 0.12 ng mL-1 / [9]
Lateral flow assay / NaYF4:Yb3+, Er3+ / - / 2.5 ng mL-1 / [10]
Colorimetric immunoassay / Au(III)-ABTS / 0.01– 100 ng mL-1 / 7.8 pg mL-1 / [11]
Colorimetric assay / Gold nanoparticles / - / 10 pM / [12]
Colorimetric immunoassay / DNAzyme / 0.1 – 10000 ng mL-1 / 0.1 ng mL-1 / [13]
Colorimetric immunoassay / KMnO4-Mn(II)-TMB / 0.1– 100 ng mL-1 / 0.1 ng mL-1 / This work

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