Rapid Detection Of Dimethoate In Soybean Samples By Microfluidic Paper Chips Based On OilSoluble CdSe Quantum Dots

2. Materials and Methods
2.1. Materials
The used substrate papers, glass fiber film (SB08, CB06, BT53, RB45), polyester fiber film (VL78), and special absorbent paper (SX18) were purchased from Shanghai Jinbiao Biological Technology Co., Ltd. (Shanghai, China), cadmium oxide, selenium powder, liquid paraffin, oleic acid, oleyl-amine, tetra-methoxy-silane(TMOS), tetra-ethoxy-silane (TEOS), tetra-propoxy-silane (TPOS), 3-aminopropyltriethoxylsilane (APTES), trimethoxy-silyl-propane-thiol (MPTMS), and vinyl-trimethoxy-silane(VMS) were supplied by Aladdin (Shanghai, China); urea, sodium dodecyl sulfonate (SDS), sodium chloride, trichlorfon, acephate, fenthion and dimethoate were supplied by Sigma-Aldrich (Shanghai, China). Soybean samples were purchased from a local market in Xi’an (Shaanxi, China). All the water used in the experiment was ultrapure. All reagents used were of at least analytical grade.

2.2. Synthesis of CdSe QDs
According to the previously published method [20], CdSe QDs were synthesized in a cost-effective and environmentally friendly manner by appropriately modifying the reaction time and temperature. The reaction was carried out under the protection of nitrogen, and when the reaction stopped, the mixture was cooled to room temperature, then washed with methanol to precipitate CdSe QDs, and centrifuged at 9000× g for 10 min. After several repeated extraction, CdSe QDs were dispersed in dichloromethane and stored in the dark at 4 °C for later use. The whole formation process of CdSe QDs can be summarized in the following equations, where Equation (1) was the general reaction equation [20]. CdO + acid + Se oxidant → △ CdSe + oxided products

CdO + Oleic acid → △ Cd − complex

n 4 Se + R − CH 2 CH 2 nCH 3 → Dehydrogenation R – CH 2 ( CH 2 CH = CHCH 2 ) n 4 CH 3 + n 4 H 2 Se

2.3. Characterization of CdSe QDs
The fluorescence intensity of CdSe QDs was measured by fluorescence spectrophotometer (F-7000, Hitachi, Tokyo, Japan). The ultraviolet and visible (UV-vis) absorption spectra were recorded with a UV-Visible Spectroscopy (U-3900, Hitachi, Tokyo, Japan). The morphology of CdSe QDs was characterized by transmission electron microscopy (TEM), and high-resolution TEM (HRTEM) was performed on field emission transmission electron microscope (JEM-2800, JEOL, Tokyo, Japan) operating at 200 kV. X-ray powder diffraction (D8 Advance, Brook, Karlsruhe, Germany) was employed to obtain the XRD pattern of CdSe QDs. The test conditions were as follows: Cu target Kα radiation (λ = 0.154 nm), scanning step size 0.02, scanning rate 0.02 sec/step, test interval 10°–60° (2θ).

2.4. Construction of a Rapid Detection Device for Paper Chip
2.4.1. Preparation of paper@QDs
After washing with deionized water and drying, the square fiberglass paper (4–6 sheets of 1.0 cm × 1.0 cm) were put into petri dish. 15 mL CdSe QDs solution was added, then oscillated for 12 h at room temperature in darkness. Next, 60 μL TEOS was added as protective material. In order to keep the fluorescent stability in the process of synthesis, it is necessary to control the reaction surface of the paper chip always facing upward.

2.4.2. Preparation of paper@QDs@MIPs
Twelve mg dimethoate and 194 μL MPTMS (the molar ratio was about 1:20) were added to 10 mL ethanol solution and left overnight at 4 °C in darkness (for more than 20 h). After the reaction, 100 μL NH3·H2O and 50 μL TEOS were added and mixed well. Then the prepared paper@QDs was immersed in the solution subsequently and reacted for 4 h. Finally, the prepared paper chips were washed three times with methanol/HAc mixture (8:2, v/v) to remove the template dimethoate, then washed with methanol alone to obtain the paper@QDs@MIPs (hereinafter referred to as MIPs). The non-imprinted polymer paper@QDs@NIPs (hereinafter referred to as NIPs) were synthesized under the same conditions without the addition of template dimethoate.

2.4.3. Selection of Paper Substrates
To investigate the combination ability of different paper substrates with CdSe QDs, firstly the background fluorescence intensity of six different paper substrates, including glass fiber film (SB08, CB06, BT53, RB45), polyester fiber film (VL78) and special absorbent paper (SX18), was determined. Next, 15 mL of CdSe QDs, oscillating, were incubated with aforesaid different papers in petri dish overnight in darkness. The paper substrates were then eluted with 8 mL of methanol and 2 mL of 0.01 mol/L acetic acid for 1 h and the fluorescence intensity of paper substrates before and after elution was measured.

2.4.4. Investigation of Binding Forces
According to the previously reference [21], paper@QDs (CB06) were treated with 10 mL 0.1 mol/L urea, 0.1% SDS, 0.2 mol/L NaCl solution and ultrapure water for 30 min, respectively. Each group contained three parallel sheets of paper. After the reaction, the fluorescence intensity of paper substrates before and after the treatment was recorded. 2.4.5. Selection of Functional Monomers
Using ethanol as solvent, the concentration of dimethoate was fixed at 60 μM. The mixed solution of dimethoate and TMOS, TPOS, MPTMS, VMS and APTES with a molar ratio of 1:100 was prepared and placed in 10 mL centrifugal tubes. The liquid was fully mixed by shaking for 30 min, and then stood at 4 °C for 12 h to make the dimethoate and silanization reagents interact fully. With the corresponding concentrations of silanization reagent-ethanol solutions as the reference solutions, the effects of different silanization reagents on UV-vis absorption spectra of dimethoate were measured and after the addition of silanization reagents, the ratio of the absorption peak to the initial peak was recorded.

Afterwards, the mixed solutions with different molar ratio of dimethoate and APTES were prepared, and the corresponding concentrations of APTES–ethanol solutions were used as the reference solutions. The absorption spectra of dimethoate solutions with different concentrations of APTES were recorded. Simultaneously, APTES was replaced with MPTMS, and the above steps were repeated to observe the effects of different concentrations of MPTMS on the absorption spectra of dimethoate.

2.5. Characterization of Imprinted Paper Chip
The surface morphology of paper chip in different periods were performed under 20 kV by environmental scanning electron microscope (SEM, Quanta 200, FEI company, Hillsboro, OR, USA). The element scanning and mapping analysis (Zeiss Smart EDX, Carl Zeiss AG, Oberkochen, Germany) was used to verify the existence of the added elements to prove successful modification. Infrared spectra were obtained using an infrared spectrometer (FT-IR, Tensor27, Brook, Karlsruhe, Germany) to determine changes in chemical groups during synthesis. The thermal stability of synthesized MIPs was characterized by a thermal analysis system (10 °C/min, N2) (TG, Q1000DSC + LNCS + FACS Q600SDT, TA Instrument, New Castle, DE, USA). In order to further evaluate the fluorescence stability of MIPs, we recorded the fluorescence intensity after multiple excitations within 2 h. All of the fluorescence spectra were detected by fluorescence spectrophotometer (F-7000, Hitachi).

Determination of response time: 30 μL of 60 μM template solution was added to MIPs. Then the fluorescence intensity (λex = 370 nm) was measured every 5 or 10 min, and the response time was observed for 1 h.

Titration experiment: dimethoate was added to MIPs and NIPs according to the concentration gradient of 0, 5, 10, 20, 40, 60, 80, 100, 120, 150 μM, respectively, and the fluorescence intensity were determined after reaction for 10 min. In addition, a laser confocal microscope (FV1200, OLYMPUS, Tokyo, Japan) was used to observe the changes in fluorescence intensity of MIPs when adding different concentrations of dimethoate.

2.6. Sensitivity and Validation of Method
Determination of selective ability: trichlofon, acephate and fenthion, structural analogues of dimethoate, were selected to form a 100 μM solution. Next, 30 μL were added to MIPs and NIPs, respectively, and the fluorescence intensity of MIPs and NIPs was measured after 10 min reaction. The selectivity of MIPs was investigated by comparing the fluorescence intensity changes before and after adding pesticide.

Detection of dimethoate in practical samples: soybean samples were soaked in deionized water for 30 min before treatment. 10 g soaked soybeans were squeezed into juice and filtered with gauze, then the filtrate was placed in centrifuge tubes and centrifuged at 5000× g for 15 min. 20 mL ethyl acetate and 5 g anhydrous sodium sulfate were added to the supernatant. After eddy mixing, extraction was carried out. The extraction solutions were then blown to nearly dry with nitrogen at 40 °C and the certain concentrations of standard solutions were added. The concentration of dimethoate in the samples were 0, 5, 10 and 20 μmol/L, respectively. Finally, the solutions were filtered with 0.45 μm filter membrane. 30 μL of aforesaid solutions was transferred into the detection area of the microfluidic paper chip and then the fluorescence intensity was measured. The synthesis process of microfluidic paper chip was shown in Figure 1.Meanwhile, the labeled samples were detected by gas chromatography-tandem mass spectrometry (GC-MS) to evaluate the accuracy of the proposed method. The detection conditions were as follows: Agilent HP-5 MS column (30 m 0.25 mm, 0.25 μm, inner diameter), and used helium as the carrier gas at 1.69 mL/min. Injection was made without a split ratio at an injection volume of 1 μL and a temperature of 250 °C. The column temperature was initially 50 °C and held for 1 min, then increased to 125 °C at 25 °C/min, finally to 300 °C at 10 °C/min, and this temperature was maintained for 5 min. The electron energy was 70 eV, and the temperature of the ion source was set at 200 °C.

2.7. Investigation on Possible Quenching Mechanism
In order to study the possible quenching mechanism of CdSe QDs, the transient fluorescence spectrometer (FLS1000, Edinburgh Instruments, Edinburgh, England) was used to determine the fluorescence lifetimes of CdSe QDs before and after adding 60 μM dimethoate solution. The UV-vis absorption spectra of CdSe QDs were recorded at the same time.

Photolysis experiment: CdSe QDs solution treated with and without Cu2+ were prepared and irradiated by ZWF three-use ultraviolet analyzer for 0, 30, 60 and 90 min, respectively, and the UV-vis absorption spectra were recorded.

2.8. Statistical Analysis
The analysis of variance (ANOVA) of the data was used to evaluate the significance in the difference between two methods by the SPSS21 (SPSS Inc., Chicago, IL, USA), and p