Visual electrochemiluminescence biosensing of aflatoxin M1 based on luminol-functionalized, silver nanoparticle-decorated graphene oxide

Seyyed Mehdi Khoshfetrat a, Hasan Bagheri b,*, Masoud A. Mehrgardi a a Department of chemistry, University of Isfahan, Isfahan 81746-73441, Iran.b Chemical Injuries Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran.

1. Materials and Reagents.

Tris(2-carboxyethyl)phosphine (TCEP), tetraethyl orthosilicate (TEOS), aminopropyl triethoxysilane (APTES), sodium chloride (NaCl), potassium chloride (KCl), potassium dihydrogen phosphate (KH2PO4), disodium hydrogen phosphate (Na2HPO4), potassium ferrocyanide (K4Fe(CN)6), potassium ferricyanide (K3Fe(CN)6), sodium borohydride (NaBH4), potassium permanganate (KMnO4), hydrogen peroxide (H2O2), hydrazine and nitric acid (HNO3) were analytical grade and obtained from commercial sources (Sigma-Aldrich or Merck).

2. Instrumentation and Physicochemical Characterization.

Morphological characterization and size distribution analysis of the nanomaterials was performed using a high-resolution transmission electron microscope (Philips/CM20) operating at 300 kV. Field emission scanning electron microscope microscopy (FESEM) images were acquired using a scanning electron microscope (Hitachi S4160, Japan). X-ray diffraction (XRD) patterns of the samples were recorded on a Bruker D8/Advance X-ray diffractometer (Germany) with Cu-Ka radiation at 40 kV and 40 mA. Fourier transform-infrared (FT-IR) spectra were recorded using on a JASCO, FT/IR-6300 (Japan) spectrophotometer. The homemade ECL instrumental setup in our research laboratory was used for all the related experiments. Ultraviolet-visible (UV-vis) spectra were recorded using a Agilent 8453 UV-Vis diode array spectrophotometer (Agilent, USA). A DC power supply (EPS-600Z, Paya Pajohesh,Iran) was used to provide the necessary potentials. Electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) were performed electrode by an Ivium potentiostat/galvanostat (Vertex, Ivium Technologies, Netherlands) at a conventional three-electrode system where a platinum wire, an Ag/AgCl/KCl (3 mol L−l) electrode and small piece of an archival gold recordable compact disc-recordable were employed as an auxiliary electrode, a reference electrode, and working electrode, respectively.

3. Synthesis of Gold-Coated Fe3O4 NPs (GMNPs).

3.1. Synthesis of Fe3O4 NPs.

Fe3O4 NPs were prepared using co-precipitation method based on the procedure of Massart (Bee et al. 1995). FeCl3.6H2O (8.5 g) and FeCl2.4H2O (3 g) were dissolved in deoxygenated HCl (40 mL, 0.4 M) at room temperature. NH3 (400 mL, 0.7 M) solution was quickly added to the mixed solution under argon with vigorous stirring. After 30 min, magnetite precipitates were collected with a permanent magnet under the reaction flask and washed with deionized water and re-dispersed in 150 mL of deionized water.

3.2. Synthesis of the core–shell Fe3O4@SiO2.

The core–shell Fe3O4@SiO2 was prepared via hydrolysis of TEOS in a basic solution via Stöber's method with slightly modification. Briefly, 20 mL suspension of Fe3O4 NPs were added into 200 mL 2-propanol and sonicated for 30 min, followed by the addition of 2.24 mL ethylene glycol, 10 mL ammonia (28%), 20 mL water and 1.3 mL TEOS under vigorous stirring for 24 h. The black products were sequentially washed with water and ethanol, and dried at room temperature for 6 h.

3.3. Aminopropyl terminated Fe3O4 (Fe3O4@SiO2/NH2).

3 gr Fe3O4@SiO2 along with 8 mL APTES were added into 30 mL anhydrous toluene with stirring overnight under argon condition (Stöber et al. 1968). The final product was magnetically separated, completely washed with acetone and dichloromethane, and finally dried under vacuum. 20 mg Fe3O4@SiO2/NH2 was dispersed in 30 mL water at pH 4 in an ultrasound cleaning bath for 30 min. Subsequently, 10 mL HAuCl4 aqueous solution (1.71 mM) was added to the MNPs. The gold NPs has been deposited on the MNPs surface by the reduction of auric acid using 10 mL NaBH4 solution (0.1 M) under ultrasonic exposure. Consequently, the nanoparticles were magnetically separated and washed using PBS 1X (pH 7.4).

3.4. Synthesis of gold coated Fe3O4 NPs (GMNPs).

20 mg Fe3O4@SiO2/NH2 was dispersed in 30 mL water at pH 4 in an ultrasound cleaning bath for 30 min. Subsequently, 10 mL HAuCl4 aqueous solution (1.71 mM) was added to the MNPs. The gold NPs has been deposited on the MNPs surface by the reduction of auric acid using 10 mL NaBH4 solution (0.1 M) under ultrasonic exposure (Khoshfetrat and Mehrgardi 2017). Consequently, the nanoparticles were magnetically separated and washed using PBS 1X (pH 7.4).

4. Investigation of mechanism preparation of GMNPs.

Silica-coated Fe3O4 NPs were synthesized in an aqueous phase. Silica shell not only is used as a protective agent to prevent the agglomeration of the formed Fe3O4 NPs due to negatively charges Si-OH groups, but also provide silanol groups for further modification. Second, amino terminate functional groups on the surface of Fe3O4@SiO2 were carried out using APTES. Finally, Au coated Fe3O4 nanoparticles (Fe3O4@Au) were fabricated by reducing HAuCl4 in acidic media. To this end, positive charges onto the surface of the Fe3O4@a-SiO2

NPs were implemented by adding diluted HNO3 solution. It causes that the -CH2NH3+ groups

interact with AuCl4- via electrostatic attraction. The resulting microspheres were redispersed

in 0.1 M NaBH4 as reducing agent solution for reduction of AuCl4-1. The color of the

suspension changed to wine red, indicating the formation of gold nanoparticles on the MNPs.

5. Preparation of the Gold BPE Array and Fabrication of the Cell.

An array of 7 parallel gold BPEs with dimensions of 25.0×25.0 mm was created by scratching with a pin. Six parallel and equally spaced scratches with lengths of 25.0 mm were created on the polymer-coated surface of the gold CD (25.0×30.0 mm). After removing the protective polymeric layer using concentrated HNO3, the array surface was electrochemically cleaned at a potential from 0 to -1.5 V in 0.01 M NaOH and from 0 to +1.5 V in 0.05 M H2SO4. Finally, the unscratched area was carefully cut down to disconnect the electrodes from each other. For visual imaging, a glass cell with 8.0 cm length, 2.5 cm width, and 2.5 cm height was designed. The cell was divided into two chambers containing the cathodic and anodic channels. The separated channels were connected by the gold BPE array for spatial resolution analysis. Two driving plate electrodes (304 stainless steel sheets) were situated at the ends of the cell, and a potential of 5.0 V was applied to these electrodes using a regulated DC power supply. The ECL signals were recorded using a PMT detector at a set potential of 800 V. In this case, the experiments were performed in a single-channel homemade Teflon cell that was divided into two chambers containing two reservoirs with dimensions of 2 cm × 2 cm × 1 cm and a channel between the two reservoirs (4 cm in length, 0.27 cm in width). In the last step, the cell was covered with a lid. An optical fiber with a 1.2 mm diameter was inserted through a hole in the top of the lid into the anodic compartment to transfer the ECL signal to the PMT detector. In both the visual imaging and PMT detectors, the anodic and cathodic chambers were filled with 1 mM PBS solution containing 2 mM H 2O2 and 10 mM thionine solution, respectively.

6. Optimization of the Factors Influencing the ECL Signal.

The predictive equation is as follows:

R = +0.71 - 0.73B + 0.13C + 0.15D + 0.083AC - 0.19AD - 0.27BD - 0.088 A 2 + 0.21B2 - 0.064D2 where A, B, C, and D are the aptamer concentration (Capt), the GMNP amount (NPs), pHads and pHdet, respectively. The ANOVA results showed that the effects were significant (p value < 0.050, 95% confidence level). The validity of the model was confirmed using F = 38.15 (p-value < 0.0001) and a “lack of fit F-value” of 0.80. As shown in Table S2, the R 2

value, a measure of the amount of variation around the mean explained by the model, was 0.982, which implied that 98.2% of the variation could be explained by the prediction model. Furthermore, the variation around the mean of the number of factors, R2

Adj, and the new data, R2

Pred, explained by the model were 0.9672 and 0.9476, respectively, indicating a high degree of correlation between the observed and predicted values and an adequate fit of the prediction

model. The signal-to-noise ratio (S/N) is characterized by the term “adequate precision.” A ratio greater than 4.0 indicates that the range of the predicted values is larger than the average prediction error and the model is adequate. Low coefficients of variation (C.V. = 5.22%) and prediction error sum of squares (PRESS) values support the precision of the presented model. Leverage (Fig. S2A) and Cook’s distance (Fig. S2B) plots confirmed the reliability of the model.

Table S1. ANOVA table for modeling of AFM1 ECL peak intensities as a function of detection parameters.

p-valueF valueMean SquaredfSum of SquareSource

Significant< 0.000166.580.8197.33Model

< 0.0001245.403.0013.00B-pHads

0.001418.070.2210.22C-NP

0.000424.720.3010.30D-pHdet

0.05624.550.05610.056AC

0.008610.190.1210.12AD

< 0.000149.470.6010.60BD

0.01069.430.1210.12A^2

< 0.000151.660.6310.63B^2

0.04794.950.06110.061D^2

0.012110.13Residual

Not Significant0.81530.488.745E-00370.061Lack of Fit

0.01840.073Pure Error

0.81207.46Cor Total

Table S2. Statistical model parameters from stepwise multiple linear regression (MLR) and analysis of variance (ANOVA) for the detection of AFM1.

Std. Dev. 0.11 R-Squared 0.9820

Mean 0.75 Adj R-Squared 0.9672

C.V. % 14.75 Pred R-Squared 0.9476

PRESS 0.39 Adeq Precision 32.180

7. Synthesis and Characterization of the GO-L-AgNPs Nanocomposite.

The UV-vis spectrum (Figu. S1A) of luminol exhibits distinct absorption peaks at 225, 305 and 360 nm. The yellow-brown GO produces a maximum and a shoulder absorption peak at 230 nm and 300 nm, related to the π-π* transition of C=C bonds and the n-π* transition of C=O bonds, respectively (Guo et al. 2009). The L-AgNPs show two characteristic absorption peaks at 230 nm and 431 nm, which are attributed to the luminol and SPR bands of the AgNPs, respectively (Chai et al. 2010). Coating the L-AgNPs onto the GO sheets blueshifted the GO peak to 217 nm because of the exfoliation of the layered GO sheets by the AgNPs and luminol on the surface (He et al. 2011). The corresponding peak of the AgNPs was redshifted approximately 11 nm compared with the plasmon resonance band of AgNPs at 420 nm because of an electron deficiency, based on Mie theory (Khoshfetrat and Mehrgardi 2014).

Meanwhile, the two luminol peaks underwent modest shifts in the GO-L-AgNP dispersion. These results reflect the decoration of AgNPs and luminol molecules on the GO surface (Zhao et al. 2015). Compared to pristine graphite, the inter-plane distance (d-spacing) of exfoliated GO of 11.4 A (2θ=11.4 Å) was larger than the d-spacing (3.35 Å) of pristine graphite (2θ=26.6 Å) because of the presence of oxygen-containing functional groups in the former (Fig. S1B). In addition to the GO peak, the XRD patterns of the GO-L-AgNPs contain four peaks positioned at 2θ values of 38.2° (111), 44.4° (200), 64.7° (220) and 77.7° (311), corresponding to the face-centered cubic phase of the AgNPs (He and Cui 2012). These results, in agreement with the UV-vis results, indicate that GO was not reduced during the reaction process. Typical SEM and TEM images of the synthesized GO-L-AgNPs are shown in Figures S1C and S1D, respectively. Analysis of the images shows a homogeneous distribution of AgNPs, with an average particle size of 10 nm, on the GO surface.

Fig. S1. A) UV–vis absorption spectra of GO, luminol, L-AgNPs and GO-L-AgNPs in aqueous solution. B) XRD patterns of Graphite, GO and GO-L-AgNPs. SEM (C) and TEM (D) image of GO-L-AgNPs.

Fig. S2. Leverage and (c) cook's distance for modeling of AFM1 ECL peak intensities as a function of detection parameters.

Fig. S3. Nyquist plots (A) and CV (B) recorded on the different modification of bare gold electrode.

Fig. S4. The ECL intensity-Etot plot of anodic modified gold BPE.

Fig. S5. A) Optical graph of the GO-L-AgNPs, B) GMNPs-GO-L-AgNPs, and C) the mixture of Apt-GMNPs-GO-L-AgNPs.

Fig. S6. ECL signals of Apt-GMNP-GO-L-AgNPs on the anodic BPE in the solution of 1mM PBS, pH=9 (trace a) and PBS (1mM, pH=9)+2 mM H2O2 (trace b) for 1h.

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