TiO2 Nano-layer enhanced fluorescence for simultaneous multiplex

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Biological and Medical Applications of Materials and Interfaces

TiO2 Nano-layer enhanced fluorescence for simultaneous multiplex mycotoxin detection by aptamer microarrays on porous silicon surface Rui Liu, Wei Li, Tingting Cai, Yang Deng, Zhi Ding, Yan Liu, Xuerui Zhu, Xin Wang, Jie Liu, Baowen Liang, Tiesong Zheng, and Jianlin Li ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b01431 • Publication Date (Web): 06 Apr 2018 Downloaded from http://pubs.acs.org on April 7, 2018

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ACS Applied Materials & Interfaces

TiO2 Nano-layer enhanced fluorescence for simultaneous multiplex mycotoxin detection by aptamer microarrays on porous silicon surface Rui Liu

a,c

, Wei Li

b, c

, Tingting Cai

a,c

, Yang Deng a, Zhi Dinga, Yan Liu a , Xuerui Zhua, Xin

Wanga, Jie Liu a, Baowen Liang a,Tiesong Zheng a and Jianlin Li a* a

Department of Food Science and Engineering, Nanjing Normal University, Nanjing 210024, China

b

Department of Electronic and Electrical Engineering, The University of Sheffield, Sheffield, S3 7HQ, United Kingdom

c

These authors contributed to this work equally and should be regarded as co-first authors *Corresponding authors. Tel.: +86 25 83598286; fax: +86 25 83598901. E-mail addresses: [email protected], [email protected]

ABSTRACT: A new aptamer microarray method on the TiO2-porous silicon (PSi) surface was developed to simultaneously screen multiplex mycotoxins. TiO2 nano-layer on the surface of PSi can enhance 14 times fluorescence intensity than that of the thermally oxidized PSi. The aptamer fluorescence signal recovery principle was performed on the TiO2-PSi surface by hybridization duplex strand DNA from mycotoxin aptamer and anti-aptamer respectively labeled with fluorescence dye and quencher. The aptamer microarray can simultaneously screen for multiplex mycotoxins with a dynamic linear detection range of 0.1 to 10 ng/mL for ochratoxin A (OTA), 0.01 to 10 ng/mL for aflatoxins B1 (AFB1) and 0.001 to 10 ng/mL for fumonisin B1 (FB1) and a limit of detection (LOD) of 15.4 pg/mL,1.48 pg/mL and 0.21 pg/mL for OTA, AFB1 and FB1, respectively. The new developed method shows a good specificity and recovery rates. The method can provide a simple, sensitive and cost-efficient platform for simultaneous screening multiplex mycotoxins and can be easily expanded to the other aptamer-based protocol.

KEYWORDS: multiplex mycotoxins, aptamer, porous silicon, titanium dioxide, fluorescence microarrays, simultaneous detection

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INTRODUCTION Mycotoxins are low-molecular-weight secondary metabolites produced by filamentous fungi species, mainly Aspergillus spp., Penicillium spp., and Fusarium spp. growing on agricultural crop in field and during storage

1, 2

. Cereal samples, such as wheat, corn, rice, peanuts, sorghum

and barley are often contaminated by mycotoxins. Hundreds of mycotoxins with different chemical structures have been identified. They can evoke a broad range of toxic properties including carcinogenicity, neurotoxicity, reproductive and developmental toxicity to human being and animals3. To protect the health of consumers, very strict limits of mycotoxins in cereal and feeds have been set in many countries and regions. For example, maximum levels of 4 µg/kg for the sum of aflatoxins B1, B2, G1 and G2, 2 µg/kg for aflatoxins B1 (AFB1) and 5 µg/kg for ochratoxin A (OTA) in cereals and all products derived from cereals and 400-4000 µg/kg for fumonisin B1 (FB1) are allowed by European Commission4. The co-occurrence of mycotoxins has been always found in the same sample since one sample can be infected by the different fungi species and one fungi can simultaneously produce several kinds of mycotoxins. These co-occurrence multiplex mycotoxins show the additional or synergistic toxic effects5. In addition, some mycotoxins are stable to heat. Thus, they are hard to remove once they enter into the food chains. Therefore, the priority measure to prevent mycotoxins from contaminating food chains is to develop the simple, cost-efficient and sensitive detection techniques for multiplex mycotoxins. Compared with each mycotoxin detection in one run, simultaneous detection method for multiplex mycotoxins is obvious advantages in cost and speed of detection. For the multiplex mycotoxin

detection, liquid chromatography-mass

spectrometry

(HPLC-MS/MS)5, 6, gas chromatography-mass spectrometry (GC-MS/MS)7, lateral flow methods1,

8, 9

techniques14,

, solid-phase microarray10,

15

11

, suspension arrays12,

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and label-free optical

have been developed to simultaneous detection for multiplex mycotoxins.

Although chromatography methods show high sensitivity, accuracy and good reproducibility for multiplex mycotoxins, they are quite expensive and require the complicated pretreatment of samples and skilled personnel. Most of the solid-phase microarray techniques, suspension arrays and label-free optical techniques for multiplex mycotoxin detection are based on the principle of immunoassay and require the specific antibodies and artificial antigens which are the complex 2


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preparation process. In addition, the one dimensional glass plane substrate is often used as probe molecule carrier for the common solid-phase microarray techniques, which limits the detection sensitivity because of its low accommodation capability of probe molecules16, 17. Porous silicon (PSi) is a powerful platform for biosensor due to its cost-efficient, versatile fabrication, easy modification, large of surface area and good biological compatibility18, 19. PSi has been used to assay for small molecule16, 17, 20, 21, heavy metal22, protein23-28, DNA18, 29, virus30 and cell31-34. However, fresh surface of PSi is unstable, which has greatly prevented it from being applied in practice35. Although chemical modifications of Si-O and Si-C bond methods have been used to stabilize the surfaces of PSi, the surfaces are still subject to various degrees of corrosion and dissolution under physiological or higher pH conditions36. Recently, we have prepared the stable surfaces of PSi by spinning sol-gel TiO2 on the surfaces of PSi37. The TiO2-PSi surface has been demonstrated to be stable in the pH 2-12 buffer solutions37. In this work, we reported a stable, sensitive aptamer microarray for simultaneous multiplex mycotoxin assay using TiO2-PSi surface. We found the nano-TiO2 layer on the surface of PSi can enhance 14 times fluorescence intensity than that of thermally oxidized PSi. Aptamers, a type single-stranded oligonucleotides, can bind to target molecules with high affinity and selectivity38. Aptamers are considered promising recognition probes alternative to antibodies because their distinct advantages over antibodies in easier synthesis, modification and immobilization, better stability and higher reproducibility39. Aptamers for mycotoxins (such as AFB1, OTA and FB1) have been selected by the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) process and used to detect mycotoxins40. However, the reports on simultaneous multiplex mycotoxins still are few. This work will provide a new platform for simultaneous multiplex mycotoxin assay. In addition, the developed method is easily expanded to other aptamer assay system. EXPERIMENTAL SECTION Experimental Materials AFB1, OTA, OTB and FB1 standard substance were purchased from Pribolab (Singapore). (3-aminopropyl) triethoxysilane (APTES), (3-Glycidyloxypropyl) trimethoxysilane (GPTMS), and Tris(hydroxymethyl) aminomethane (Tris) were bought from Sigma-Aldrich (Shanghai, China). Absolute ethanol, sodium hydroxide, sulfuric acid and hydrogen peroxide were obtained 3



from Nanjing Chemistry Reagents Co., Ltd. (Nanjing, China). Silicon wafers (0.0008-0.0012 Ωcm resistivity, polished on the (100) face, B-doped) were obtained from Siltronix Co. (Archamps, France). 48% Hydrofluoric acid was bought from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). AFB1, FB1, and OTA ELISA kits were bought from Hongdu Biotech Co., Ltd (Shandong, China). Aptamers and anti-aptamers for AFB1, FB1and OTA were as follows: AFB1-aptamer, 5’-NH2-(CH2)6-AGT TGG GCA CGT GTT GTC TCT CTG TGT CTC GTG CCC TTC GCT AGG CCC ACA-Cy3-3’, AFB1anti-aptamer, 5’-BHQ2-TGT GGG CCT AGC GA-3’; FB1-aptamer,5’-NH2-(CH2)6-TCT AAC GTG AAT GAT AGA TTA ACT TAT TCG ACC ATA CAC GTC TGC ATT ACC TTA TTC GAC CAT ATT CCA TTA CGC TAA TTA ACT TAT TCG ACC ATA-Cy3-3’, FB1anti-aptamer, 5’-BHQ2-TAT GGT CGA ATA AGT TAA-3’; OTA-aptamer, 5’-NH2-(CH2)6-GAT CGG GTG TGG GTG GCG TAA AGG GAG CAT CGG ACA-Cy3-3’, OTA anti-aptamer, 5’-BHQ2-TGT CCG ATG C-3’. These aptamers and anti-aptamers were synthesized and modified by Sangon Biotech Co., Ltd. (Shanghai, China). Cereal samples including rice, corn and wheat samples were bought from supermarket in local. Preparation of PSi, Thermally Oxidized PSi and TiO2-PSi. PSi, thermally oxidized PSi and TiO2-PSi were prepared according to reference37. Briefly, P++-type silicon wafers (boron doped, 0.0008-0.0012 Ωcm resistivity) were etched by a Teflon etch cell and a platinum counter-electrode. 1.3 cm×1.3 cm wafer was immersed in a solution 3:1 (v/v) H2SO4:H2O2 for 30 min and then washed with double distilled water for 3 times and dried with a stream of nitrogen. The wafer was etched with 3:1(v/v) mixture aqueous 48% HF and absolute ethanol. The wafer was first cleaned by etching a sacrificial layer of PSi at current density of 100 mA/cm2 for 20 s in dark. The sacrificial layer was removed with an aqueous solution of 1 M NaOH for 1 min. The wafer was washed with ethanol and dried with a stream of nitrogen. The wafer was etched at a certain current density for 20 s and washed with absolute ethanol and then dried with a stream of nitrogen. The PSi sample was thermally oxidized in tube furnace at 500 °C for 1 h. A TiO2 sol-gel was prepared with a solution of 5:30:1 titanium butoxide: ethanol: triethanolamine (v/v). 100 µL of the TiO2 sol-gel was deposited on the surface 4


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of the thermally oxidized PSi (PSiO2) by spin coating at 10 ×g for 20 s. The sample was calcined in tube furnace at 500 °C for 1 h. The deposition process was repeated for three times on each sample. Characterization of TiO2-PSi surface. The spectra of white light reflected from the wafer film were obtained with Y type fiber and spectrograph in 0.1 M, pH7.4 phosphate buffer solution (PBS) according to previous description36, 37.The scanning electron microscopy (SEM) images of the samples were taken with FEI XL30 microscope equipped with a field emission gun and trough-the-lens detector at an accelerating voltage of 5 kV. The photographs of the samples were taken with common digital camera. The characterizations of TiO2-PSi surface were seen in Figure S1. Modification of TiO2-PSi surface. TiO2-PSi is immersed in 5% GPTMS toluene solution at room temperature for 12 h and then respectively washed with toluene, absolute ethanol and double stilled water. Immobilization of oligonucleotides on the TiO2-PSi surface and detection of fluorescence intensity. The certain concentration aptamers and anti-aptamers of OTA, AFB1 and FB1 in binding buffer solution (0.01mol/L, pH8.0 Tris-HCl, 120 mmol/L NaCl, 20 mmol/L CaCl2, 5 mmol/L KCl, 20 mmol/L MgCl2) were respectively heated in water bath at 88 °C for 5 min. The equal of volume aptamers and anti-aptamers were mixed and incubated at 37 °C for 1 h. 0.2 µL of the mixture was dropped on the surface of TiO2-PSi surface modified with GPTMS. The sample was put in hybridization chamber at 37 °C for 12 h. Then, the sample was respectively washed with PBS, PBST and Tris-HCl solution for 3 times. Finally, the sample was dried with nitrogen. The fluorescence intensity (I1) of the sample was obtained by LuxScan 10k (Captial Bio Crop., China) at an excitation wavelength of 550 nm and an emission wavelength of 570 nm. To investigate the stability of fluorescent signals on the TiO2-PSi surface, the fluorescent intensities of samples were recorded each 20 min for successive 100 min. The stability of fluorescent signals was calculated in according to the percentage between the fluorescence value of different time and the initial fluorescence value. The three-dimensional image of fluorescence sample was taken by confocal laser scanning microscopy (Nikon, Ti-E-A1R) at an excitation wavelength of 550 nm. Multiplex detection for OTA, AFB1 and FB1. 5



100 µL, a series of concentrations of OTA, AFB1 and FB1 standard substances or real sample extraction were dropped on the surface of TiO2-PSi immobilized with the hybridized aptamers of OTA, AFB1 and FB1. The samples were incubated at 37 °C for 1 h, then washed with double distilled water and dried with nitrogen. The fluorescence intensity (I2) of the wafer was obtained by LuxScan 10k at an excitation wavelength of 550 nm and an emission wavelength of 570 nm. The calibration curves for the three mycotoxins were established through fluorescent values of ∆ I=I2-I1 as vertical axis and the different concentrations of mycotoxins as horizontal axis. The specificity for the multiplex mycotoxin assay The specificity for the multiplex mycotoxin assay was performed among OTA, AFB1 FB1 and OTB. 100 µL,1 ng/mL OTA, OTB, AFB1 and FB1 standard substances were respectively dropped on the surfaces of the wafers immobilized with the hybridized aptamers of OTA, AFB1 and FB1. The incubations between mycotoxins and aptamers were the same as the above detection process. The fluorescence intensities of the samples were taken according to the above method. The application of the developed method in the real samples. The cereal samples including rice, wheat and corn were pretreated according to our previous method12. In brief, these samples were ground by a high-speed disintegrator. The samples which the particle size was less than 1 mm were used as the spiked samples. 5 g cereal sample was put in a 100 mL conical flask with cover. The different concentrations of mycotoxin standard substances in the 100% methanol were respectively added into the sample and then completely mixed with the samples. These spiked samples were put in a fume hood overnight to evaporate the solvent. 1g NaCl and 25 mL 80% methanol- water (v/v) were added in the sample and completely mixed. The sample was homogenized by a homogenizer (FJ-200 Shanghai Specimen Model Factory, Shanghai, China) at 820×g for 1 min. The sample was shaken at 5×g at 25 °C for 30 min and filtrated with Whatman filter paper. The filtrate was passed through 0.45µm filter member. The filtrate was collected for mycotoxin detection. The control samples were extracted under the same conditions. The samples were measured according to the above method. The traditional ELISA for the samples were performed according to ELISA kit instructions. Data analysis These data were analyzed with Origin 9.3 software. The average value and standard deviation 6


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for each sample were calculated from no less than 4 samples. Safety precaution. The experimenters should avoid direct contact with mycotoxins and laboratory contamination. The sample pretreatment, mycotoxin extraction and detection should be carried out in a fume hood. Experimenters should equip with laboratory coat, safety glasses, gloves and mouth-muffle. RESULTS AND DISCUSSION The enhance fluorescence characteristics of TiO2-PSi surface. 0.2 µL of 16 µmol/L aptamer (for OTA) labeled with Cy3 was respectively immobilized on the surfaces of PSiO2 and TiO2-PSi modified with GPTMS. The fluorescence intensities of samples and control samples were detected by the fluorescence scanner. Figure 1a and b show that the fluorescence intensity of the sample on the TiO2-PSi surface is remarkable higher than that on thermally oxidized PSi surface. Figure 1c displays that the fluorescence intensity of the sample on the TiO2-PSi surface almost is 14 times than that on thermally oxidized PSi surface in the same conditions. There is low fluorescence background signal on the surfaces of both wafer samples. The result can be attributed to the following reasons: one is that the surface of TiO2-PSi is more stable than that of PSiO237; another is that the surface of TiO2-PSi has more active-sites for DNA immobilization than that of PSiO2; The emission intensity of dye has been increased because the polar TiO2 surface enhances the delocalization of the π electrons and lowers the highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) energy levels of dye41. Figure 1d shows that the stability of fluorescence signal on the TiO2-PSi surface. As time from 0 to 100 min, the fluorescence value decreased by 11.6%. The fluorescence decrease may be attributed to the self-quench of fluorescence dye and many times excitation during detection. Figure 1e shows that the three-dimensional confocal laser scanning image of the fluorescence aptamer molecules immobilized on the surface of TiO2-PSi. The result indicates the aptamer is bound to both on the inner pore wall and the outer surface of TiO2-PSi. Compared with general microarray plane carrier, the porous structure carrier can accommodate more probe molecules both on its internal and outer surface, which is beneficial to the detection sensitivity.

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PSiO2

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Figure 1. The fluorescence characteristics of PSiO2 and TiO2-PSi surfaces. The images of fluorescence scanning of aptamer labeled with cy3 immobilized on the surface of TiO2-PSi (a) and the surface of thermally oxidized PSi (b); The fluorescence intensities and background fluorescence intensities for aptamer labeled with cy3 immobilized on the surfaces of TiO2-PSi and thermally oxidized PSi (c); The fluorescence stability of aptamer labeled with cy3 immobilized on the surface of TiO2-PSi as time (d); The three-dimensional image of fluorescence sample (e). The principle of simultaneous multiplex mycotoxin assay on the TiO2-PSi surface.

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Scheme 1. The principle of simultaneous multiplex mycotoxin assay on the TiO2-PSi surface. The principle of simultaneous multiplex mycotoxin assay on the TiO2 surface is shown in Scheme 1. PSi was prepared by electrochemistry etching method. After PSi has been thermally oxidized in tube furnace in air at 500 °C for 1 h, PSiO2 has been stabilized with TiO2 sol-gel method37. Aptamers of OTA, AFB1 and FB1 and their anti-aptamers (partial complementary strands) have been respectively hybridized in test tube. The different positions of TiO2-PSi surface are used to encode the different mycotoxin aptamer probes. These hybridization duplex strands have been immobilized on the surfaces of TiO2-PSi surface through chemical bond linkage between GPTMS and amino-groups of oligonucleotides. The hybridization duplex strands on the surface of TiO2-PSi show the fluorescence quenching because cy3 at the 3’end of aptamers and the quencher (BHQ2) at the 5’ end of their anti-aptamers is very close. When OTA, AFB1 and FB1 are appear on surface of the wafer, the fluorescence signals are activated because the mycotoxins displace their anti-aptamers and bind their aptamers. The different positions of aptamer probes will report which kind of mycotoxins and the fluorescence signal intensity will give the quantity of the different mycotoxins in sample. For multiplex mycotoxin assay based on microarray methods, most of them have been established by the immunoassay principle on one dimensional plane substrate4, 9


10, 11, 42, 43

.


Generally, they require artificial antigens or mimotopes, antibodies and secondary antibody labeled with dye. The preparation for these reagents are complex process, especial for monoclonal antibodies for mycotoxins. In addition, the detection procedure is time-consuming and complicated. Aptamer techniques can overcome the disadvantages of immunoassay methods. Compared with the common microarrays on one dimensional plane substrate, porous silicon surfaces show many advantages in the factors: it has large internal surface area (hundreds of m2/cm3) which can be highly efficient for biomolecule immobilization44; the surface of PSi is easy to chemically modify; PSi has a good compatible with optics, microelectronics and micro-electromechanical systems (MEMS)18, 37, 45, 46. The TiO2-PSi has more stable surface than PSiO2 and can further improve signal/noise and detection sensitivity. The condition optimization for multiplex mycotoxin assay. The influence factors of fluorescence signal including the concentration of aptamer, anti-aptamer, hybridization time, temperature, incubation time between mycotoxins and hybridization duplex strands have been optimized. The results are shown in Figure S2. The optimal conditions are as follows: 15 µmol/L,16 µmol/Land 12 µmol/L the concentration of aptamer for OTA, AFB1 and FB1, respectively; 75 µmol/L, 48 µmol/L and 48 µmol/L the concentration of anti-aptamer for OTA, AFB1 and FB1, respectively; hybridization time for 120 min in vitro, hybridization temperature 25 °C; incubation time for 120 min at room temperature. Calibration curve for multiplex mycotoxin assay.

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Figure 2. The calibration curves for multiplex mycotoxin assay. The relationships between the fluorescence intensities and the concentrations of mycotoxins (a, b and c for OTA, AFB1 and FB1, respectively.); The detection linear ranges for mycotoxins (d, e and f for OTA, AFB1 and FB1, respectively.). Under the optimal conditions, the different concentrations of OTA, AFB1 and FB1 standard substrates were detected by the developed method. The relationship between fluorescence intensities and the different concentration of mycotoxins is shown in Figure 2. As the increase of the concentration of the three kinds of mycotoxins, the fluorescence intensities increase and reach to a plateau (Figure 2 a, b and c). The increase of fluorescence intensities is in agreement with the principle of the designed method. The detection linear ranges are shown in Figure 2d, 2e and 2f, respectively. For OTA, AFB1 and FB1, the detection linear ranges vary from 0.1 to 10 ng/mL, 0.01 to 10 ng/mL and 0.001 to 10 ng/mL, respectively. The limit of detection (LOD) are respectively estimated to be 15.4 pg/mL,1.48 pg/mL and 0.21 pg/mL for OTA, AFB1 and FB1 according to the 3 times of signal/noise. The LODs are low than the previous reports on the 11



surface of agarose-modified glass slides11, diamino-poly(ethyleneglycol) functionalized glass slides4, 96 well microtitre plate47 and epoxy slides42. The characteristic comparison of them for multiplex mycotoxin detection was summarized in the Table S1. The high sensitivity result can be attributed to the factors:1) aptamer-based principle;2) porous structure for probe immobilization;3) TiO2 nanolayer enhanced signal/noise. The specificity for multiplex mycotoxin assay. FB1

2800 OTA


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Figure 3. The specificity for multiplex mycotoxin assay. The specificity for multiplex mycotoxin assay was evaluated among OTA, OTB, AFB1 and FB1. 100 µL,1 ng/mL OTA, OTB, AFB1 and FB1 were respectively dropped on the samples which immobilized with the three kinds of the hybridized duplex stands of aptamers. The fluorescence intensities of the samples were obtained and the result was shown in Figure 3. Among the multiplex mycotoxin samples, the fluorescence intensities of the samples can increase dramatically only when the mycotoxin molecules met their corresponding aptamers. The increased fluorescence intensities for positive samples are 5-12 times than that of negative samples. Between the structural analogue of OTA and OTB, the developed method also can recognize its target molecule. The results show the developed method has a good specificity for OTA, AFB1 and FB1. The specificity mainly depends on the specificity of aptamers of mycotoxins. Evaluation in the real sample detection. To evaluate the developed method for multiplex mycotoxin assay, 0.1, 1 and 10 ng/g multiplex 12


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mycotoxins were respectively spiked in the wheat, corn and rice samples and they have been detected with the new method. The recovery rates of the samples are shown in Figure 4a and Table S2. Among the recovery rates of samples, six of them are lower than 80%, one sample (0.1 ng/g corn) at 55.33%, most of them are more than 80%. The recovery rates can be accepted for the screening multiplex mycotoxins. We also detected the recovery rates of the same samples with traditional ELISA method. They are shown in Figure 4b and Table S2. From Figure 4a,4b and Table S2, the recovery rates for the different cereal samples both the developed method the traditional ELISA are mostly consistent.

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1 1 0.1 0.1 10 10 10 0.1 1 The concentration of mycotoxins spiked in the samples(ng/g) Figure 4. The recovery rates of multiplex mycotoxins in the spiked wheat, corn and rice samples at the 0.1, 1 and 10 ng/g concentration of mycotoxins, respectively (n=3). a, The recovery rates of the developed method; b, The recovery rates of the traditional ELISA. These results indicate that the developed method has a great potential in simultaneously screening for the multiplex mycotoxins by aptamer technique, although a large of application in real samples still require to be further valuated by the standard method (such as HPLC-MS/MS). Compared with the classic solid-phase immunomicroarrays, the new established method for multiplex mycotoxin screening is simple, sensitive and cost-efficient. CONCLUSION In this work, we designed a new aptamer microarray for multiplex mycotoxin screening on the TiO2-PSi surface. TiO2 nano-layer on porous silicon surface can enhance fluorescence signal intensity and signal/noise. Three kinds of mycotoxins including OTA, AFB1 and FB1 have been used as a model for simultaneous screening multiplex mycotoxins on the TiO2-PSi surface. The detection conditions have been optimized. The new developed method showed high sensitivity, wide detection linear ranges and good recovery rates. The aptamer microarray on the stable TiO2-PSi surface can provide a simple, sensitive and cost-efficient platform for simultaneous screening multiplex mycotoxins. The new method can be easily expanded to the other 14


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aptamer-based multiplex screening protocol. ASSOCIATED CONTENT Supporting Information Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author Tel.: +86 25 83598286. Fax: +86 25 83598901. E-mail: [email protected]. Notes The authors declare no competing ﬁnancial interest. Acknowledgments The work was supported by National Natural Science Foundation of China no.31471642.

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TiO2 Nano-layer enhanced fluorescence for simultaneous multiplex

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