Fluorescence Probe Based on an Amino-Functionalized Fluorescent

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Fluorescence Probe Based on Amino-Functionalized Fluorescent Magnetic Nanocomposite for Detection of Folic Acid in Serum Xiaowan Li, and Ligang Chen ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b10163 • Publication Date (Web): 31 Oct 2016 Downloaded from http://pubs.acs.org on November 1, 2016

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ACS Applied Materials & Interfaces

Fluorescence Probe Based on Amino-Functionalized Fluorescent Magnetic Nanocomposite for Detection of Folic Acid in Serum Xiaowan Li, Ligang Chen* Department of Chemistry, College of Science, Northeast Forestry University, 26 Hexing Road, Harbin 150040, China ABSTRACT: A new fluorescence probe constructed with a multifunctional nanocomposite, Fe3O4-ZnS:Mn2+/SiO2-NH2, was successfully synthesized and then used to detect folic acid in real serum samples. The nanocomposite was characterized by fluorescence spectroscopy, transmission electron microscopy, Fourier transform infrared spectroscopy, X-ray powder diffraction and physical

property

measurement

system.

With

the

addition

of

analyte,

the

Fe3O4-ZnS:Mn2+/SiO2-NH2 composite and folic acid formed a new complex because cross-linking of the amino and carboxyl groups participated in the condensation reaction. Then, the energy of quantum dots was transferred to the complex and led to quenching of the fluorescence. Moreover, the fluorescence intensity decreased significantly as the concentration of folic acid increased, and the fluorescence quenching ratio F0/F was related to the folic acid concentration in the range from 0.1 to 5 µg mL-1. This method was used for detecting folic acid in real serum samples and gave recoveries in the range of 89.0%-96.0%, with relative standard deviations of 1.2%-3.9%. The detection limit was 9.6 ng mL-1 (S/N=3). These satisfactory and simple results showed the great potential of this fluorescence probe in the field of pharmaceutical analysis. KEYWORDS: Fluorescence probe; Magnetic separation; Quenching mechanism; Folic acid; Serum

1

ACS Paragon Plus Environment


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1. INTRODUCTION Folic acid (FA, Fig. S1) is a water soluble chemical compound in the vitamin B group.1 FA is beneficial to the production and maintenance of new cells, is conductive to the synthesis of DNA and RNA, and prevents changes to DNA, thus preventing cancer.2 FA deficiency can cause megaloblastic anemia, and it disturbs the synthesis of DNA and cell division, which affects hematopoietic cells and neoplasms.3 FA analytical methods need to be highly sensitive and selective. Nevertheless, FA detection is complicated due to its low thermodynamic and kinetic stability and its multiple forms. Common methods for determining FA include capillary electrophoresis (CE),4 microemulsion electrokinetic

chromatography

spectrophotometry,10

(MEEKC),5

photochemical

chemiluminescence

fluorimetry,11

and

liquid

(CL),6

voltammetry,7-9

chromatography

(LC).12-13

Nevertheless, these methods have some limitations, such as time consuming processes, low sensitivity and high cost, and they require complicated sample preprocessing. Therefore, developing a simple and efficient method for determining FA in real samples is necessary. In recent years, fluorescent probes based on certain nanomaterials have been widely established because of their high sensitivity and rapid analysis. In contrast to organic dyes, quantum dots (QDs) have narrow emission bandwidth and high quantum yield.14-17 However, some QDs also have disadvantages, such as chemical instability, complex conjugation chemistry, and inherently toxic elements (Cd, Pb, Hg, Se and As). ZnS QDs have been of great interest due to their non-toxic elements.18-19 Rajabi et al. proposed Mn2+ as a dopant into ZnS nanocrystals to act as luminescence centers.20 ZnS:Mn2+ QDs have received much attention because of their large exciton binding energy, wide direct band gap, and potential applications. Magnetic nanoparticles (MNPs) have been successfully applied for enrichment, purification and separation of samples because of their unique dispersion capability in aqueous solutions and high separation efficiency using a permanent magnet.21 MNPs have been used for some general applications, for instance, lithium-ion batteries,22 biosensors,23 drug delivery,24 magnetic resonance 2


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imaging (MRI),25 separation and catalysis.26-28 Due to these attractive features, magnetics and fluorescence have received much attention because of their individual advantages.17 On occasion, a suitable combination of two functionalities can complement each other. Therefore, work has been done to prepare a fluorescent magnetic nanocomposite (Fe3O4-QDs).29-30 Fe3O4-QDs could be used for the separation and preconcentration analytes from complex sample and detect them via generated fluorescence signal. The purpose of this work was to establish a rapid and sensitive method to detecting FA in serum samples. First, we constructed a fluorescent magnetic nanocomposite by wrapping ZnS:Mn2+ QDs and Fe3O4 nanoparticles in one silica shell via a reverse microemulsion method. Second, we studied its function as a fluorescence sensor based on a hybrid Fe3O4-ZnS:Mn2+/SiO2-NH2 nanocomposite for the detection of FA. Finally, we studied the mechanism based on fluorescence quenching.

2. EXPERIMENTAL SECTION 2.1. Materials The folic acid (FA) (99.99%) standard, poly (dimethyl-diallyl ammonium chloride) (PDDA), hexyl alcohol, L-cysteine (L-cys), tetramethylammonium hydroxide pentahydrate (TMA), (3-aminopropyl) triethoxy-silane (APTES) (98.0%) and iron chloride hexahydrate (FeCl3·6H2O) were obtained from Aladdin (Shanghai, China). Ferrous chloride tetrahydrate (FeCl2·4H2O)

and zinc

sulfate

heptahydrate

(ZnSO4·7H2O)

were

obtained from

Shuangchuan (Tianjin, China). Sodium sulphide nonahydrate (Na2S·9H2O) (≥ 98.0%) was obtained from Kaitong (Tianjin, China). Manganese chloride (MnCl2·4H2O) was obtained from Bodi (Tianjin, China). Cyclohexane and ammonia hydroxide were obtained from Guangfu (Tianjin, China). Tetraethoxysilane (TEOS), Triton X-100, ethanol and isopropanol were obtained from Kermel (Tianjin, China). Every reagent was analytical grade. Ultra-pure water was made by the system of Milli-Q water (Millipore, Billerica, MA, USA). Equine serum was obtained from HyClone (Logan, UT, USA). The serum was stored at -18 oC in a refrigerator and should not be placed for more than 2 h at room temperature. 3



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A stock solution of FA (100 µg mL-1) was obtained via dissolving FA (10.0 mg) in NaOH (1 mL, 0.1 mol L-1) and diluting to 100 mL in water, after that stored at 4 oC and protected in the dark. 2.2. Apparatus X-ray powder diffraction (XRD) spectrum was measured by Shimadzu XRD-600 diffractometer

(Kyoto,

Japan).

Functional

groups

of

Fe3O4-ZnS:Mn2+/SiO2-NH2

nanocomposite were detected by Fourier transform infrared spectroscopy (FT-IR) (Nicolet, Madison, WI, USA). The morphology characteristics were measured on transmission electron microscope (TEM) (H-7650, Matsudo, Japan). Magnetism was tested with physical property measurement system (PPMS) (Quantum Design Instrument, San Diego, CA, USA). The ultraviolet spectrum was measured with TU-1901 spectrometer (PERSEE, Beijing, China). Hitachi F-4600 fluorescent spectrometer (Tokyo, Japan) was used to fluorescence measurement. 2.3. Synthesis of Fe3O4-ZnS:Mn2+/SiO2-NH2 nanocomposite The synthesis of Fe3O4 nanoparticles was based on a previous report.31 As follows: FeCl3·6H2O (5.4 g) and FeCl2·4H2O (2.2 g) were dissolved in water (100 mL) with rapid stirring. The oxygen in this solution was removed with nitrogen. When the temperature rose to 90 oC, 10 mL NH3·H2O was added to reaction solution. The reaction was carried out for 1.0 h. The Fe3O4 was obtained, separated via external magnetic field and washed using deionized water. The TMA (2.6 g) was added into Fe3O4 nanoparticles (30 mL), and the mixture was ultrasound for 10 min in order to surface functionalization. Then the product was separated via a permanent magnet, washed by deionized water and diluted to 2 mg mL-1. The preparation of Mn-doped ZnS QDs was according to a reported method with some modifications.32 First, ZnSO4·7H2O (25 mmol), MnCl2·4H2O (2 mmol) and water (80 mL) were stirred for 20 min under nitrogen. Afterwards, Na2S·9H2O (25 mmol, 10 mL) was added dropwise to the reaction solution. After 30 min of stirring, L-cysteine (0.1545 g) was dissolved in deionized water (5 mL) and was added slowly to the reaction solution. The 4


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mixture was stirred for 1 h in order to modify the QDs. Ultimately, the modified ZnS:Mn2+ QDs were washed with ethanol and water and diluted to 2 mg mL-1. The Fe3O4-ZnS:Mn2+/SiO2-NH2 nanocomposite was synthesized at room temperature via reverse microemulsion. First, 7.5 mL cyclohexane, 1.8 mL Triton X-100, 1.8 mL n-hexanol, 150 µL TMA-modified Fe3O4 (2 mg mL-1), 600 µL L-cys modified ZnS:Mn2+ QDs (2 mg mL-1) and 60 µL PDDA were added to a flask. After stirring for 30 min, 60 µL NH3·H2O and 100 µL TEOS were added to the microemulsion system for hydrolysis reaction under alkaline condition. The microemulsion was stirred for 24 h while avoiding light. Afterwards, 50 µL APTES was injected into the system. The reaction was continuously stirred for another 24 h. Then, 20 mL acetone

was

added

to

the

mixture

to

break

up

the

microemulsion.

Ultimately,

Fe3O4-ZnS:Mn2+/SiO2-NH2 nanocomposite was obtained and washed with ethanol, isopropanol and water. The fluorescent magnetic nanocomposites were stored in a refrigerator at 4 °C in the dark. 2.4. Fluorescence response to FA PBS buffer solution (1.0 mL, pH 7.4), serum (1.0 mL) and the Fe3O4-ZnS:Mn2+/SiO2-NH2 nanocomposite (1.0 mL, 1 mg mL-1) were added with constant volume of 10 mL centrifuge tube. The mixture was shaken thoroughly and placed at room temperature for 10 min. The Fe3O4-ZnS:Mn2+/SiO2-NH2 nanocomposite associated with FA was separated by a permanent magnet and then re-dispersed in 5 mL PBS buffer (pH 7.4). The fluorescence intensity of the Fe3O4-ZnS:Mn2+/SiO2-NH2

nanocomposite

was

measured

with

a

fluorescence

spectrophotometer. 2.5 Fluorescence measurement Fluorescent detection was tested upon an F-4600 fluorescence spectrophotometer. The excitation wavelength was set at 300 nm. The parameters keeping constant are set as follows: slit widths of 10 nm, excitation voltage of 650 V, scan speed of 240 nm min-1 and quartz cell of 1 cm path length. Moreover, each sample was measured three times in parallel. The quantum yield (QY) was calculated according to the following equation: 5



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QYx = QYr ( Fx / Fr )( Ax / Ar )(nx 2 / nr 2 )

(1)

where F is the measured integrated emission intensity, A is the absorbance at the excitation wavelength and n is the refractive-index of solvent (for experiments using the same solvent, the ratio is 1). The subscript x and r designate sample and Rhodamine 6G (QY=95%),33 respectively.

3. RESULTS AND DISCUSSION 3.1. Synthesis of amino-functionalized fluorescent magnetic nanocomposite Water-in-oil microemulsion, also called reverse microemulsion, has been discovered to be especially efficacious as a nanoreactor for the confined preparation of nanocomposites of different sizes, forms, and functionalities.34-35 In this work, the silica layer was synchronously coated onto water-soluble

Mn-doped

ZnS

QDs

and

Fe3O4

to

construct

a

multifunctional

Fe3O4-ZnS:Mn2+/SiO2-NH2 nanocomposite, which can be used to detect FA in serum samples. As schematically illustrated in Fig. 1, the synthesis reaction and drug analysis consisted of three parts. First, Mn-doped ZnS QDs were obtained by an aqueous synthesis method with L-cys as the stabilizing agent, and Fe3O4 nanoparticles were obtained by a coprecipitation method with TMA as the modifying agent. Second, an Fe3O4-ZnS:Mn2+/SiO2 nanocomposite was obtained via reverse microemulsion using essential nanoparticles to contribute magnetism and fluorescence. Finally, the surface of the Fe3O4-ZnS:Mn2+/SiO2 nanocomposite was modified with APTES, and the NH2 modified nanocomposite was associated with FA, which verified the potential application of the sensor for detection and separation of analytes. In the absence of FA, recombination of the hole and the electron generates a fluorescent signal; the introduced FA acts as an electron acceptor, effectively receiving the conduction band electron from the Fe3O4-ZnS:Mn2+/SiO2 nanocomposite36, and stopping the recombination of the electron and the hole at the interfaces of the Fe3O4-ZnS:Mn2+/SiO2 nanocomposite (Fig. 2), which leads to fluorescence quenching.37 The silica-encapsulated core precursor is non-toxic, relatively stable under biological conditions, and benefits further applications in reverse microemulsion systems.38 Moreover, the well-known 6


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bioconjugation surface chemistry of SiO2 is more likely to be available.39 Silica is regarded as an optimal material for protecting Fe3O4 nanoparticles and ZnS:Mn2+ QDs. We added PDDA to effectively balance between ZnS:Mn2+ QDs and SiO2 electrostatic repulsion, in order to embedding more ZnS:Mn2+ QDs. 3.2. Fluorescence properties of Fe3O4-ZnS:Mn2+/SiO2-NH2 nanocomposites We studied the fluorescence properties of ZnS:Mn2+ QDs and Fe3O4-ZnS:Mn2+/SiO2-NH2 nanocomposites by obtaining the fluorescence spectra in Fig. 3A(a, b). Rhodamine 6 G was used as a reference. The fluorescence QY of ZnS:Mn2+ QDs was 13.2%. The QDs prepared in this study were comparable to previous studies. The QY of ZnS:Mn2+ QDs reported by Azizi et al40 and Kolmykov et al41 was 13% and 11%, respectively. When the SiO2 encapsulated the ZnS:Mn2+ QDs and Fe3O4 nanoparticles, the fluorescence intensity of the QDs was decreased. The QY of the Fe3O4-ZnS:Mn2+/SiO2-NH2 nanocomposite is 9.6%. Some similar studied have been also reported that when QDs were mixed with Fe3O4 or encapsulated with SiO2, the QY would decrease.42-43 However, the fluorescence nanocomposite still has sufficient sensitivity for detection of FA. The Fe3O4-ZnS:Mn2+/SiO2-NH2 nanocomposite has the advantages of fast separation and detection of analytes. As shown in Fig. 3A(c), the fluorescence of the Fe3O4-ZnS:Mn2+/SiO2-NH2 nanocomposite was quenched when it interacted with FA. The inset in Fig. 3A displays the dispersion of Fe3O4-ZnS:Mn2+/SiO2-NH2 nanocomposites in the absence and presence of a permanent magnet under UV illumination. The Fe3O4-ZnS: Mn2+/SiO2-NH2 nanocomposite can be activated with UV light (300 nm), and the emitted fluorescence (595 nm) is quenched with FA. 3.3. Characterization of the Fe3O4-ZnS:Mn2+/SiO2-NH2 nanocomposite TEM images of QDs and the nanocomposite are shown in Fig. 3B and Fig. 3C, respectively. TEM imaging was used to obtain information about the size and structure of ZnS:Mn2+ QDs. The TEM images show that the QDs have dimensions of approximately 6 nm and are nearly spherical particles, and the Fe3O4-ZnS:Mn2+/SiO2-NH2 nanocomposite is approximately 90 nm. XRD spectrum was obtained for the Fe3O4 nanoparticles, Mn-doped ZnS QDs and 7



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Fe3O4-ZnS:Mn2+/SiO2-NH2 nanocomposites (Fig. 3D) for the investigation of their crystalline structure. The XRD pattern of the Fe3O4 nanoparticles in Fig. 3D(a) showed five characteristic diffraction peaks at 2θ = 30.3°, 35.4°, 43.1°, 57.4° and 62.4°, which can be attributed to (220), (331), (400), (511) and (440) positions, which is consistent with the Fe3O4 standard in the database of the Joint Committee on Powder Diffraction Standards (JCPDS card 19-0629). As we can see from Fig. 3D(b), the diffractions at 28.6°, 48.5° and 56.8° are characteristic of (111), (220), and (311) planes, respectively, in cubic zinc blended nanoparticles. In Fig. 3D(c), the X-ray data were consistent with the composite structure of Mn-doped ZnS QDs and Fe3O4. The FT-IR spectrum (Fig. 3E) of the Fe3O4-ZnS:Mn2+/SiO2-NH2 nanocomposite was measured to study the various functional groups. The peaks at 460 and 619 cm-1 were stretching vibrations of Fe-O, and the peaks at 1614 and 3431 cm-1 indicated carboxyl and hydroxyl groups. The peaks at 1066 and 790 cm-1 were characteristic bands of asymmetric and symmetric stretching vibrations, respectively, of a framework of Si-O-Si groups. The C-H stretching at approximately 2926 cm-1 verified the propyl group in APTES. The peak at approximately 3431 cm-1 was the N-H stretching vibration partially overlapped with the O-H stretching vibration. To examine the magnetic properties, the Fe3O4-ZnS:Mn2+/SiO2-NH2 nanocomposite was studied via PPMS. Its saturation magnetization decreased to 13.69 emu g-1 from the 58.18 emu g-1 of Fe3O4 (Fig. 3F). The adsorbent was dispersed in water and collected with an external magnetic field in a short time. Afterwards, it could be readily re-dispersed with a slightly shake, which indicated that the Fe3O4-ZnS:Mn2+/SiO2-NH2 nanocomposite was successfully synthesized with a high magnetic responsivity. The effect of pH on the Fe3O4-ZnS:Mn2+/SiO2-NH2 nanocomposite in a range between 5.5 and 8.5 was investigated. As shown in Fig. 3G, the fluorescence QY of the nanocomposite was low when the pH was less than 7. Similar phenomenon has also been reported in the literature44 which because of decomposition of the QDs to form H2S in the acidic condition.45 When the pH value increased from 6.8 to 7.6, the QY was increased and reached the maximum value. 8


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3.4. Effect of pH The pH not only influenced the fluorescence QY of Fe3O4-ZnS:Mn2+/SiO2-NH2 but also influenced the subsequent fluorescence quenching with FA. As shown in Fig. S2A, under acidic conditions, the fluorescence quenching of the composite is relatively weak. Moreover, the silica layer could be ionized at pH > 7.6, which decreased the association between the silica layers and FA. Furthermore, FA has the potential to slightly denature at lower or higher pH values. Hence, we chose pH 7.4 as the optimal value after comparing the fluorescent intensity in various experiments with Fe3O4-ZnS:Mn2+/SiO2-NH2 in the presence or absence of FA. 3.5. Effect of incubation time The

Fe3O4-ZnS:Mn2+/SiO2-NH2 nanocomposite

sample

was

interacted

with

different

concentrations of FA (0.1, 0.7, 2.0 µg mL-1), which were dispersed in PBS (pH 7.4). The effect of incubation on the fluorescence intensity measured at varying time points (0-60 min) is exhibited in Fig. S2B. The fluorescence intensity of the Fe3O4-ZnS:Mn2+/SiO2-NH2 nanocomposite achieved stability at 10 min when 0.1, 0.7 and 2.0 µg mL-1 of FA were added. The fluorescence intensity maintained equilibrium for up to 60 min. Therefore, we recorded the fluorescence intensity after the system was incubated for 10 min. The incubation time used in this study is comparable to that reported in the literature for the determination of FA using CdTe QDs.46 3.6. Fluorescence-mediated detection of FA based on Fe3O4-ZnS:Mn2+/SiO2-NH2 Fig. 4A shows the fluorescence quenching of the Fe3O4-ZnS:Mn2+/SiO2-NH2 nanocomposite upon association with varying concentrations of FA under the above-mentioned optimal conditions. The effect of FA concentration on Fe3O4-ZnS:Mn2+/SiO2-NH2 nanocomposite quenching was investigated. The fluorescence quenching with different FA concentrations was explained by the Stern-Volmer equation (2): F0 / F = 1 + K SV [Q ]

(2)

In the equation, F0 and F are the fluorescence intensity of the Fe3O4-ZnS:Mn2+/SiO2-NH2 nanocomposite in the absence and presence of FA, respectively. [Q] and KSV are the quenching 9



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agent (FA) concentration and the Stern-Volmer quenching constant, respectively. The quenching intensity of the Fe3O4-ZnS:Mn2+/SiO2-NH2 nanocomposite was increased when the FA concentration increased. The results, shown in Fig. 4B, confirmed a good linear correlation between F0/F and FA concentration, which ranged from 0.1 to 5 µg mL-1. The linear equation is y = 0.7071x + 0.9662 and the determination coefficient (R2) obtained is 0.9989. The limit of detection (LOD) is 9.6 ng mL-1, which is defined as three times the signal to noise ratio. 3.7. Mechanism of Fe3O4-ZnS:Mn2+/SiO2-NH2 nanocomposite detection of FA The fluorescence intensity of the Fe3O4-ZnS:Mn2+/SiO2-NH2 nanocomposite was studied through changing excitation wavelength 300-350 nm. The ZnS QDs have defect feeble blue peak at 460 nm, and a strong orange peak at 595 nm, which is due to the transition of Mn2+ from 4T1 to 6A1.47 When FA was added, fluorescence quenching of the Fe3O4-ZnS:Mn2+/SiO2-NH2 nanocomposite occurred, which may be due to the interaction between the nanocomposite and FA. We think that electron transfer between the QDs and FA caused the fluorescence quenching. Tu et al48 and Wang et al.49 also have reported this electron transfer mechanism. From the absorption spectra of the Fe3O4-ZnS:Mn2+/SiO2-NH2 nanocomposite in Fig. 4C, we can see that the UV absorption band of FA is near the band gap. In addition, the absorption spectrum of FA and the emission spectrum of Fe3O4-ZnS:Mn2+/SiO2 have no overlap. The specific FA detection and recognition processes of the fluorescent magnetic nanocomposite are demonstrated in Fig. 5A. Clearly, it can be seen that the fluorescence quenching phenomenon is a result of the electron-transfer process between carboxyl group on FA and amino group on Fe3O4-ZnS:Mn2+/SiO2, with a complex forming between Fe3O4-ZnS:Mn2+/SiO2 and FA. The energy of the QDs could be subsequently transferred to the complex, thus leading to fluorescence quenching. The number of amino groups grafted onto the nanocomposite surfaces was adjusted by changing the amount of APTES used in the preparation of the Fe3O4-ZnS:Mn2+/SiO2-NH2 nanocomposite, which allowed us to investigate the effect of the amino groups on the quenching of the fluorescence 10


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composite. As shown in Fig. S2C, when a small amount of APTES was added, the fluorescence quenching was relatively weak. This further demonstrated that the fluorescence quenching of the nanocomposite is the result of the interaction between the amino group on the surface of Fe3O4-ZnS:Mn2+/SiO2 and the carboxyl group of FA. Moreover, the molecular orbital theory can be used to explain the fluorescence quenching mechanism. The electrons of QDs are excited from the valence band to the conduction band when they receive a UV photon (Fig. 5B). Then, the excited electrons return to the valence band, and the QDs produce a fluorescent signal. In addition, there is an electrostatic interaction between FA and the amino groups on the QDs in the presence of the FA. The excited electrons are able to turn to the LUMO of the complex directly. The excited electrons on QDs would then go back to the ground state without a fluorescent signal because the energy level of the complex is higher than that of the QDs, which will lead to fluorescence quenching. The fluorescence intensity of the Fe3O4-ZnS:Mn2+/SiO2-NH2 nanocomposite decreased with additional FA. Dynamic and static quenching are common fluorescence quenching mechanisms.50 The Ksv will increase with rising temperature in dynamic quenching, and in static quenching, the reverse occurs. The Stern-Volmer equation was used to analyze the fluorescence quenching mechanism at different temperatures.51 Based on equation (2), we can calculate Ksv of the Fe3O4-ZnS:Mn2+/SiO2 nanocomposite system at three temperatures, as shown in Table 1. It is observed that Ksv decreased with increasing temperature. This indicated that the possible quenching mechanism of Fe3O4-ZnS:Mn2+/SiO2-FA was static quenching, which implied the formation of a non-luminescent ground state complex. This mechanism proposed in this work is consistent with that reported in the literature, which also used Mn-doped ZnS QDs for detection of FA.52 The thermodynamic equations (3, 4) were used to calculate entropy changes (∆So), free enthalpy changes (∆Go) and enthalpy changes (∆Ho).

∆Go = −RT ln KSV

(3)

∆Go = ∆H o − ∆S oT

(4)

11



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By rearranging equations (3) and (4), KSV can also be calculated with equation (5).

ln K SV =

∆S o ∆H o − R RT

(5)

In this equation, R and T are the universal gas constant (8.314 J mol-1 K-1) and quenching temperature, respectively. KSV is the equilibrium constant that represents the quenching constant. The calculation results are shown in Table 1. UV absorption spectroscopy was also used to investigate the interaction between FA and the composite materials. Two absorption peaks at 280 and 348 nm were found for FA (Fig. 4C). The UV absorption peak of Fe3O4-ZnS:Mn2+/SiO2-NH2 was weak. When the two were mixed into a single system, the absorption peak of FA at 348 nm became strong and was red shifted to 370 nm. These experimental results further show that the Fe3O4-ZnS:Mn2+/SiO2-NH2 nanocomposite has a specific interaction with FA. 3.8. Effect of coexistent substances To explore the selectivity of the method using the Fe3O4-ZnS:Mn2+/SiO2-NH2 nanocomposite as a probe to detect FA. The effect of some common metal cations, biomolecules and inorganic ions was studied at the optimal conditions to investigate the potential for practical application. This method showed good selectivity because most of coexistent substances had no effect on the detection of FA (Table S1). 3.9. Serum sample analysis To study the applicability of the method, it was used to detect FA in serum samples. The spiked serum samples were prepared by adding varying amounts of FA. Afterwards, these samples were detected with this method (Table S2). The range of quantitative recovery was 89.0%-96.0%, and the range of the relative standard deviation (RSD) was 1.2%-3.9%. The analytical results of this method were compared with other methods reported4-5, 12, 46, 52-53 for the determination of FA. The results are summarized briefly and shown in Table 2. The recovery, accuracy and LOD of the method were comparable or exceeded those of other methods. Fluorescence analysis has unique advantages, such as simplicity, speed, and low-consumption 12


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compared with HPLC. Fluorescence probes based on QDs for detecting FA have been developed by previous researchers. However, in the research of Wang et al46, toxic CdTe QDs were used. In the research of Geszke-Moritz et al52, Mn-doped ZnS QDs was also used, but the LOD obtained was much higher than our study. It is important that the composites prepared in our work possess magnetism so that it can be directly applied to separation and detection of FA in a complex biological serum sample. When the composite material is associated with FA, it is easy to be separated from the serum matrix under the action of the magnetic field, without extra filtration and centrifugation procedure. In previous work, when QDs were used to detect target analytes in serum, the samples had to undergo complicated pretreatment processes, such as deproteinization with organic solvent, and a high degree of dilution, as much as 100 or 1000 times.54-55 These procedures not only make the process more complicated but also reduce the detection sensitivity.

4. CONCLUSIONS In summary, a fluorescent magnetic multifunctional nanocomposite was successfully synthesized by improved reverse microemulsion and used for detecting folic acid in serum sample. ZnS:Mn2+ quantum dots and Fe3O4 were encapsulated by SiO2 layer, which led to increased stability, reduced toxicity and enhanced biocompatibility. The mechanism of the synthetic nanocomposite for detection of folic acid was studied in detail. The reason for the fluorescence quenching is electron transfer between the nanocomposite and folic acid. Moreover, quenching constants were reduced following temperature increases, which indicated that the likely quenching mechanism of the Fe3O4-ZnS:Mn2+/SiO2-NH2 nanocomposite is static quenching. Under optimum conditions, the linear concentration range of the calibration plot was 0.1-5 µg mL-1 and the limit of detection was 9.6 ng mL-1. The nanocomposite prepared in this work has advantages derived from the use of magnetic materials and quantum dots. Quantum dots are able to act as fluorescent markers, and magnetic materials are easy to handle using a magnetic field. Hence, the nanocomposites possess wide potential applications in drug targeting, biomarker detection and quick bioseparation. 13



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ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: (1) Chemical structure of folic acid (Fig. S1); (2) pH influence, time-dependent and APTES volume response of Fe3O4-ZnS:Mn2+/SiO2-NH2 nanocomposite (Fig. S2); (3) effects of coexistent substances of Fe3O4-ZnS:Mn2+/SiO2 nanocomposite- folic acid (Table S1); (4) analytical results and recovery tests of folic acid in serum samples (Table S2).

AUTHOR INFORMATION Corresponding author * E-mail: [email protected]. Tel.: +86-451-82190679. Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was supported by the Fundamental Research Funds for the Central Universities (No. 2572014EB06), Heilongjiang Province Science Foundation for Youths (No. QC2014C005) and Harbin Science and Technology Innovation Talent Research Special Funds (2016RAQXJ151).

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Figure captions: Fig. 1 Schematic of synthesis of Fe3O4-ZnS:Mn2+/SiO2-NH2 nanocomposite by the reverse microemulsion system. Fig. 2 Mode of interaction of Fe3O4-ZnS:Mn2+/SiO2-NH2 nanocomposite with folic acid. Fig. 3 (A) Fluorescence spectra of Mn-doped ZnS QDs (a) and Fe3O4-ZnS:Mn2+/SiO2-NH2 in the absence (b) and presence (c) of folic acid. (B) TEM image of ZnS:Mn2+ QDs. (C) TEM image of Fe3O4-ZnS:Mn2+/SiO2-NH2 nanocomposite. (D) XRD patterns of Fe3O4 (a), Mn-doped ZnS QDs (b) and

Fe3O4-ZnS:Mn2+/SiO2-NH2

nanocomposite

(c).

(E)

FT-IR

spectrum

of

Fe3O4-ZnS:Mn2+/SiO2-NH2 nanocomposite. (F) Magnetization curves of the Fe3O4 (a) and Fe3O4-ZnS:Mn2+/SiO2-NH2 nanocomposite (b). (G) The effect of pH on the fluorescence QY of Fe3O4-ZnS:Mn2+/SiO2-NH2 nanocomposite. Fig. 4 (A) Fluorescence spectra of Fe3O4-ZnS:Mn2+/SiO2-NH2 nanocomposite with folic acid various concentration. (B) The linear plots of the quenched fluorescent intensity of Fe3O4-ZnS:Mn2+/SiO2-NH2 against the concentration of folic acid. (C) UV spectra of folic acid (curve 1), Fe3O4-ZnS:Mn2+/SiO2-NH2 nanocomposite (curve 2) and Fe3O4-ZnS:Mn2+/SiO2-NH2 nanocomposite associated with folic acid (curve 3), fluorescent emission spectra of Fe3O4-ZnS:Mn2+/SiO2-NH2 nanocomposite (curve 4). Fig. 5 Schematic of QDs fluorescent quenching theory (A) and molecular orbital theory (B).

22


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Fig. 1

23



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Fig. 2

24


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Page 25 of 30

Fluorescence intensity (a.u.)

A 10000

B

a

8000

b 6000

c

4000 2000 0 400

500

600

700

Wavelength (nm)

D

C

a

b c 0

20

40

60

80

100

2θ (degree)

2926.49

50

1065.70

3431.32

40 30

1613.75 1471.15 1384.32

60

789.91 619.44 459.74

70

20 10 0

60 40

a

20

b

0 -20 -40 -60

3500

3000

2500

2000

1500

1000

-20000

500

Wavenumber (cm )

G

-10000

0

10000

Applied magnetic field (Oe)

-1

QY of nanocomposite (%)

-10 4000

F Magnetization (emu g -1)

80

E Transmittace (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


12

9

6

3

0 5.0

5.5

6.0

6.5

7.0

7.5

8.0

8.5

pH

Fig. 3 25


9.0

20000

A

8000 7000

B

Page 26 of 30

5.0 4.5 4.0

6000

3.5

4000

3.0

F0/F

5000 3000

2.5

2000

2.0

1000

1.5

0

1.0 540 560 580 600 620 640 660 680 700

0

Wavelength (nm)

C

1

2

3

1.0

8000 7000

0.8

6000

Absorbance (a.u.)

1

5000

0.6

4

4000

0.4

3000 2000

0.2

3 1000

2 0.0

200

300

400

500

4

FA concentration (µg mL-1)

600

0 700

Wavelength (nm)

Fig. 4

26


Fluorescence intensity (a.u.)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Fluorescence intensity(a.u.)


5

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Fig. 5

27



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Page 28 of 30

Table 1 Thermodynamic parameters of Fe3O4-ZnS:Mn2+/SiO2-folic acid system

T

Ksv

∆Go

(K)

(L mol-1)

(kJ mol-1)

288

5.80×105

-31.8

303

3.72×105

-32.3

323

2.59×105

-33.5

∆Ho

∆So

(kJ mol-1)

(J mol-1 K-1)

-21.5

35.6

28


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Table 2 Comparison of the proposed method with other methods used in the literatures Pretreatment method

Analytical method

Sample

Linear range (µg mL-1)

LOD (ng mL-1)

RSD (%)

Recovery (%)

Ref.

Ultrasonic extraction

Capillary electrophoresis with chemiluminescence

Tablets, apple juices, human urine

0.02-4.4

8.83

1.1-4.9

94.3-110

4

Microemulsion electrokinetic chromatography High performance liquid chromatography High performance liquid chromatography

Tablets

160-240

2.98×103

< 1.2

98-101.6

5

Flour, egg yolk, orange juice

0.001-0.2

2-4.1

< 7.5

98-102

53

Fruit juices

50-2500

1.3

-

98-103.6

12

Fluorescence probe based Mn- doped ZnS QDs

Fluorescence spectroscopy

Aqueous solution

4.41-44.1

4.86×103

-

-

52

Fluorescence probe based CdTe QDs


Aqueous solution

0.22-44.1

41

< 2.7

-

46

Fluorescence probe based Fe3O4-ZnS:Mn2+/SiO2-NH2 nanocomposite


Serum

0.1-5

9.6

1.2-3.9

89-96

This work

Ultrasonic extraction Ion pair-based dispersive liquid-liquid microextraction Enzymatic digestion

29



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TOC

30


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Fluorescence Probe Based on an Amino-Functionalized Fluorescent

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