Ultrathin Self-Assembled Diphenylalanine Nanosheets through A Gold

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Ultrathin Self-Assembled Diphenylalanine Nanosheets through A GoldStabilized Strategy for High-Efficiency Adsorption/Desorption/Ionization Siming Huang, Guosheng Chen, Ruoheng Ou, Su Qin, Fuxin Wang, Fang Zhu, and Gangfeng Ouyang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b01855 • Publication Date (Web): 18 Jun 2018 Downloaded from http://pubs.acs.org on June 20, 2018

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Analytical Chemistry

Ultrathin Self-Assembled Diphenylalanine Nanosheets through A Gold-Stabilized Strategy for High-Efficiency Adsorption/Desorption/Ionization Siming Huang, Guosheng Chen, Ruoheng Ou, Su Qin, Fuxin Wang, Fang Zhu, Gangfeng Ouyang* MOE Key Laboratory of Bioinorganic and Synthetic Chemistry/KLGHEI of Environment and Energy Chemistry, School of Chemistry, Sun Yat-sen University, Guangzhou 510275, China. * Corresponding author: +86-20-84110845; [email protected] (G. Ouyang). ABSTRACT: Herein, the ultrathin and robust diphenylalanine (FF) self-assembled nanosheets were fabricated by a gold-stabilized strategy for the first time, using a facile electrospray method followed by a thermal treatment process. The key for the goldstabilized mechanism was explored, demonstrating that the synergy of the stable binding and steric effect between gold nanoparticles (AuNPs) and the exposed amino groups of FF nanosheets, led to strong thermal stability and solvent resistance of the composites. Contributing to the features of remarkable accessible surfaces and strong laser light absorption ability of this FF/Au nanosheets, two robust functional devices, i.e. solid-phase microextraction (SPME) fiber and surface-assisted laser desorption/ionization (SALDI) platforms, were in-situ prepared for in vitro and in vivo biological analysis. The findings indicated that the fabricated platforms possessed two advantages: 1) rapid absorption/desorption speed (within 5 min); 2) remarkable enhancement of ionization efficiency with two orders of magnitudes. As a result, the extraction efficiency of the SPME fiber and the quantitation ability of SALDI platform were significantly improved. This study not only demonstrated that FF/Au composites could be prepared through an electrospray method followed with thermal-treatment to serve as promising adsorption/desorption/ionization materials for specific applications, but also provided useful strategy to advance the ideas for future combination of SPME with LDI technique.

INTRODUCTION In general, sample preparation and quantitative determination are two essential steps within an analytical methodology. Solid-phase microextraction (SPME), as a solvent-free sample preparation method integrating sampling, isolation, enrichment and introduction into one step, has gained wide acceptance in the environmental, biological, food and clinic areas, etc.1-5 Arguably, fiber coating is the core throughout the SPME technique.6 Thus, the fabrication of novel coating materials, possessing robust, economical features and providing fast mass transfer rate, is of significant importance. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS), as a detection tool, has been widely used for the identification of macromolecules. However, its adoption for the analysis of small molecules has been heavily restricted, because the usage of organic matrices leads to severe background interferences in low m/z region (m/z < 1000 Da) and spot-to-spot signal heterogeneity. Consequently, surface-assisted laser desorption/ionization (SALDI) technique as a matrix-free LDI method has been developed to overcome the limitations in MALDI in low m/z region.7 Recently, a series of functional surfaces such as metal-based, carbon-based, polymers-based, and semi-conductor based nanomaterial have been developed to enhance analyte signal and minimize background interferences.8,9 In SALDI analysis, the quantitation still remained a continuous challenge mainly because of the inhomogeneity of nanomaterials deposited onto

the substrate, and low accessible surface areas of the fabricated SALDI surface for capturing target analytes. It is no doubt that the fabrication of functional materials with even and high accessible surface areas would be helpful to improve not only the assistance effect of the surface for the desorption and ionization of target analytes, but also the quantitation ability of SALDI method. Obviously, developing novel functional materials as both the SPME fiber coating and the SALDI platform for the highefficiency adsorption/desorption/ionization of target analytes, may open up effective ways for high-efficiency analytes determination in real-world applications. Besides, it could advance the ideas for future combination of SPME with LDI technique. The self-assembly of small organic molecules such as saccharide, amino acid and nucleic acid is an intriguing phenomenon, encouraging scientists to utilize these bio-inspired building blocks to create advanced nanomaterials.10,11 These versatile materials have opened new horizons in the development of drug delivery, therapeutic and antibiotics, catalysis, optical devices, and micro/nanomotors etc.12-14 Among them, diphenylalanine (FF), is the first reported dipeptide building block.15 Since the emergence of FF, extensive efforts have been devoted to organize the FF-based building blocks into diverse micro/nanostructures such as spherical vesicles, micro/nanotubes, nanowires, nanosheets and nanofibrils for various applications.16,17 Generally, the self-assembly behavior of FF could be controlled by modifying the assembly conditions.

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It has been proven that changing the solution conditions, such as solvents, pH and temperature, light, interface, vapor and addition of enzymes or co-assembled components, is an efficient way to obtain different morphologies.18-20 In addition, FF could be manipulated into thin films on different substrates (SiO2, Au, Pt, PDMS, etc.) using vapor deposition methods or solvent vaporization.21,22 However, the self-assembled FF structures obtained from the commonly used solventcontrolled methods are sensitive to the surroundings,23 which seriously restricted their potential applications in sensor, biological, optical and electronic fields. Additionally, most researches mainly focused on the structural design and modulation of the spherical, tubular and wire-like structures due to their corresponding useful properties for a definite application.16,17,24-27 Up to now, rare researches would pay attention to the nanosheet-like FF structures, which might feature high accessible surface area and thus could offer highly competent performance in adsorption, energy transfer, and sensing.28-30

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Herein, we proposed a new strategy called gold-stabilized approach for the stable ultrathin FF/Au nanosheet design, and explored the mechanism of gold-stabilization effect for FF nanosheets (Scheme 1). The results indicated the proposed nanosheets dominated surprising stability compared to the FF structures obtained from previously solvent-controlled methods. Given inspiration from the stable ultrathin nanosheet structures of FF/Au and laser light adsorption ability of AuNPs, we for the first time fabricated two kinds of functional platforms, namely, stainless steel wire (SSW) based SPME fiber and ground steel plate (GSP) based SALDI device, respectively. The platforms showed excellent adsorption/desorption (within 5 min) and ionization performance (two orders of magnitudes higher than that without the addition of AuNPs) owing to the formed ultrathin nanosheet structure and strong laser adsorption, which realized rapid and high-efficiency in vitro and in vivo analysis of target analytes in complex samples with a small volume.

Scheme 1. (A) Schematic illustration of formation of the FF/Au self-assemblies; (B) the molecular arrangement of FF/Au selfassemblies after electrospray preparation, of which the AuNPs could not interact with the amino group because of the steric effect; (C) the molecular arrangement of FF/Au self-assemblies after electrospray preparation followed with thermal treatment, of which the AuNPs could interact with the amino groups that exposed outside the layer.

EXPERIMENTAL SECTION Preparation of FF/Au composite-based functional platforms. The FF/Au-based coatings were self-assembled on the surfaces of the stainless steel wires (SSWs) and ground steel plate (GSP) using an electrospray method based on a previous report,31 to fabricate robust functional platforms for microextraction adsorption and SALDI application (Figure 1). The electrospray solution was obtained by dissolving 13% (w/w) FF monomer, 15 mg HAuCl4 and 30 mg Vc in 1.5 mL HFIP

solvent. After sonication treatment for 40 min at 60 ºC, the color of mixture changed from clear to brick-red, indicating that the Au3+ was reduced to Au nanoparticles. It demonstrated the solution was ready for electrospraying. The pumping speed for electrospraying was set at 0.2 mL/h according to a series of optimization experiments. And the distance and voltage between the spinneret and SSW or GSP were 17 cm and 20 kV, respectively. The spraying duration time was 30 min for SALDI plate, 1 hour for SPME fiber, respectively. Afterwards, the SSWs and GSP with FF/Au coatings were treated at 120

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ºC for 30 min, and then washed with water. Subsequently, the adsorption efficiency of FF/Au composite based SPME fiber was investigated for the analysis of estrogens, and the enhancement effect of the prepared SALDI plate was studied for the analysis of steroids. In addition, to enhance the attachment

of the coatings on the surface of SSWs when inserting the SPME fiber into the fish muscle, a thin layer of PDMS (neutral silicone sealant dissolved in o-xylene) was formed onto the SSWs before proceeding the electrospray.

Figure 1. Scheme of preparation procedures of FF/Au nanosheets based functional platforms using an electrospray method (A); Use of the FF/Au based SPME fiber for the rapid in vitro and in vivo sampling of five estrogens in bovine serum, human serum, human urine and fish samples (B); Use of the FF/Au based SALDI platform for the rapid identification of four steroids without sample pretreatment (C).

In-vitro SPME procedure. Bovine serum was purchased from Sigma Aldrich (Shanghai, China). Human urine and serum were obtained from Centre for Disease Prevention and Control of Guangdong Province (Guangzhou, China). The samples were stored at -20 ºC in the refrigerator prior to the real sample analysis. A volume of 2 mL biosample was filtrated using a microporous membrane filter with 220 µm pore diameter; then the as-prepared FF/Au SPME fiber was directly immersed to samples for extraction for 2 min (Figure 1B). Subsequently, the fiber was exposed to 200 µL acetonitrile for desorption for 10 min. After the desorption process was finished, the solutions were ready for LC-MS/MS analysis. The standard curves obtained by using PBS buffer solution as matrix were employed to the quantification of target estrogen analytes. In vivo fish sampling. Immature tilapias (Oreochromis mossambicus) were obtained from a local fishery. The tilapias were reared in an aquarium containing 40 L of dechlorinated tap water for a week before experiment. The in vivo fish sampling procedures were conducted as follows. Firstly, the fish was anaesthetized in the municipal water containing 0.1% (v/v) eugenol. Secondly, the stainless steel needle of a 1-mL syringe was pierced into the dorsal-epaxial muscle of the anaesthetized fish to a 2 cm-depth, then taken out of the muscle, of which the hole acted as the fiber guide for the subsequent sampling. Thirdly, the as-obtained FF/Au based fiber was inserted into the muscle along the hole, until the fiber coating was entirely exposed in the muscle (Figure 1B). This process was simultaneously conducted on both sides of the dorsal-epaxial muscle of the fish with two SPME fibers for mutual references. After the fibers were successfully placed into the muscle, the fish was put into the municipal water for 5 min sampling. After the sampling finished, the fibers were withdrawn from the muscle carefully in case of the coating

damage. The fibers were rinsed with pure water and dried with a dust-free paper. Finally, the fibers were desorbed in 200 µL of acetonitrile for 10 min with a vibration speed of 500 rpm. The sampling-rate calibration method was used for quantification,32 which was described in the text in Supporting Information. SALDI. Four steroids including androstenedione, testosterone, androsterone, and hydroxyprogesterone were chosen as the target analytes. Since the ionization mode of the five estrogens used in SPME procedures was negative ion mode, we chose four steroids including androstenedione, testosterone, androsterone, and hydroxyprogesterone as the target analytes in SALDI analysis. The structures of these four steroids were similar to the estrogens, but they were ionized in positive ion mode. In order to improve the signal intensity, an in-situ derivatization method was employed. The detailed SALDI procedures were described as follows. 2 µL of PBS buffer solution containing target analytes or real serum and urine samples was directly deposited onto the FF/Au-based SALDI plate, after the solution was dried under RT condition, the spot was washed with DI water to remove the impurities. Then 1 µL of 0.1% hydroxylamine hydrochloride solution was deposited on the same spot for derivatization reaction. Finally, the SALDItime of fight mass spectrometry (TOF MS) measurements were carried out for detection when the samples were absolutely dried (Figure 1C). Reagents material, characterization and instruments, parameters of LC-MS/MS analysis, sampling-rate calibration for in vivo sampling, and liquid extraction. The details of the other experimental procedures were described in the Supporting Information.

RESULTS AND DISCUSSION



Characterization of the self-assembled FF/Au nanosheets. The self-assembled FF/Au composites were prepared by an electrospray method, followed by thermal treatment (Figure 1). The preparation of FF precursor solution and AuNPs were integrated into one step based on a previous report.31 Firstly, to confirm the structure, component and thermal stability of the FF/Au self-assemblies, the techniques of thermogravimetry (TG) analyzer, Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), ultraviolet-visible (UV-Vis) spectroscopy and transmission electron microscope (TEM) were employed. From the FTIR spectra of the thermal treated FF and FF/Au self-assemblies (Figure 2A, blue and red lines), they shared the same vibration band at 1668 cm-1 that belonged to the stretching vibration of the C=O bond. The bending N-H vibration, aromatic amine C-N vibration, and NH vibration appeared around 1457, 1336, and 3211 cm-1, respectively. However, the peaks at 3260, and 1560 cm-1 assigned to N-H bond of -NH2, and the peaks around 3100-2800, 1385 and 1253 cm-1 assigned to the O-H and C-O bonds of COOH (Figure 2A, green line), which belonged to the FF dipeptide (Figure S2), were not observed compared to the peaks of thermal-treated FF or FF/Au self-assemblies (Figure 2A, blue and red lines). The results suggested that the FF dipeptide molecules might rearrange in the period of thermal treatment. Then the TG analyses were conducted to support the assumption. In Figure 2B, we noted that an irreversible phase transition of the FF/Au composites occurred at 120 ºC, and the same transition of FF self-assemblies at 150 ºC as well. However, there was no phase transition for the FF/Au self-assemblies

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which has been threated at 120 ºC before the TG analysis. During the thermal treatment, the cyclization reaction between the -NH2 and -COOH groups within FF dipeptide molecule occurred, resulting in the loss of H2O molecule (Figure S2), in which the actual amount of water loss was almost the same to the calculated amount of water loss (error between experiment and calculation < 10%).33 The result indicated that the FF dipeptide molecules rearranged during thermal treatment. The XRD pattern of the FF/Au nanosheets (Figure 2C, dark cyan line) was similar to the previously reported flake-like FF structure, in which an orthorhombic crystal structure was observed.20 Additionally, the bands of 2 θ = 38.18 º, 44.39 º, corresponded to the Bragg reflections from the planes of Au (1 1 1), Au (2 0 0), respectively,34 affirmed the existence of AuNPs. Meanwhile, the TEM images (Figure 2E) clearly showed the AuNPs were merged in the backbone into the FF nanosheets, and confirmed the gold crystalline structure attributed to (2 2 0) plane (Figure 2E (c)). Furthermore, as shown in Figure 2D (blue line) of the solid UV-Vis spectroscopy, the peaks associated to FF were in the range of 200 nm to 300 nm due to the strong absorption of aromatic rings and amide groups in the UV region, and the peak around 500 nm to 600 nm assigned to AuNPs also proved the coexistence of AuNPs and FF nanosheets.35 Additionally, the self-assembled FF/Au composites exhibited good thermal stability that could endure high temperature at over 300 ºC (Figure 2B). The above results demonstrated the successful preparation of FF/Au self-assemblies with high thermal stability.

Figure 2. Characterization of FF and FF/Au self-assemblies. (A) FTIR spectra; (B)TG analysis ; (C) XRD patterns; (D) Solid UV-Vis spectra. Noted that herein the notes of “without heat”, “heat” “heat+water” in the figures were represented to the different treatment for the as-obtained material after the electrospray preparation. (E) TEM images: (a) the image of the as-obtained FF nanosheets with AuNPs; (b) the image of AuNPs with higher resolution corresponded to (a); (c) the image of AuNPs showing the crystal structure; (d, e, f): the mapping images of C and Au elements

As a matter of fact, spherical and wire-like mixed structures of FF/Au composites were obtained after the electrospray without further thermal treatment (Figure 3), which was re-

sulted from π-π stacking interaction between interlayers, then followed by the bending and closure of layers.36,37 However, they were unstable and easily dissolved in different solutions


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such as deionized (DI) water, methanol and acetone (Figure S3A), because of the interference of solvents to disturb the inter/intra-molecular noncovalent interactions.38 However, the morphologies of the spherical/wire-like FF and FF/Au changed to fibril and nanosheet structures, respectively after heating (Figure 3A and 3B). It is worth noting that the morphology of FF/Au self-assemblies differed from that of FF self-assembled structures, where the FF/Au self-assemblies tended to grow into ultrathin nanosheet structures. Importantly, the chemical stability of thermal treated FF/Au composites increased dramatically. They were insoluble and exhibited the same morphology after being washed by different solvents (Figure 3B and Figure S3B-3C). Nevertheless, the structure of thermal treated FF, without the addition of AuNPs, was destroyed when being washed by hydrogen-bonding capable solvents (Figure 3A and Figure S4).The results demonstrated that the thermal-treated FF/Au nanosheets possessed remarkable chemical stability, and the AuNPs played a vital role on the structure stability of FF/Au self-assemblies. Noted that the scanning electron microscope (SEM) images were obtained by using the materials prepared on Al substrates for the ease of operation. However, the property of substrate would not influence the synthesis of FF/Au nanosheets, since the morphologies of FF/Au deposited on the SSW and GSP were the same as those on the Al substrate (Figure 4B and Figure S5).

Figure 3. (A) SEM images of FF nanomaterials obtained from electrospray method (a), after heat treatment (b), and aqueous treatment (c), respectively; (B) SEM images of FF/Au selfassemblies obtained from electrospray method (a), after heat treatment (b), and aqueous treatment (c), respectively. Especially, the images inserted in pictures a, c and e were corresponding contact angle.

Au-stabilized mechanism. As mentioned above, the introduction of AuNPs was demonstrated to be an important role on the stability of FF/Au self-assemblies. As far as we know, AuNPs have been extensively employed for the preparation of organic-inorganic hybrid materials with enhanced properties for various applications.39 Additionally, it has been reported that the AuNPs could be strongly absorbed by amino groups due to electrostatic interaction.40 Herein, we proposed a possible regulation mechanism to explain the formation and stabilization of FF/Au nanosheets. Firstly, from the XRD patterns (Figure 2C, blue and green lines), we found that the watertreated FF structures exhibited different diffraction peaks compared with those of water-untreated FF self-assemblies, changing from unstable nanowire-amorphousness mixture to stable brick-like structure,41 which was in good agreement with the morphologies displayed in SEM images (Figure

3A(b-c)). The phenomenon occurred because the water molecules could interact with the FF molecules through hydrogen bonding, further changed structure and morphology of FF assemblies.42 On the contrary, in the presence of AuNPs, water-untreated/treated materials shared exactly same diffraction peaks (Figure 2C, red and dark cyan lines). And these FF/Au self-assemblies remained the same morphology before and after water treatment (Figure 3A(b-c)). In addition, to gain a more direct insight into the Au-stabilized FF nanosheets, the TEM imaging and mapping experiments of C and Au elements were carried out. We found that the AuNPs about 30-60 nm in diameter were distributed through the FF nanosheets (Figure 2E (a)), and the element mapping images of C and Au (Figure 2E (e,f)) proved the coexistence of FF self-assemblies and AuNPs. It is reasonable to suppose that the interaction between FF nanosheets and AuNPs occur through electrostatic attraction, enabling the improved solvent resistance of the FF nanosheets. Subsequently, to prove the interaction between FF and AuNPs, the solid UV-Vis spectroscopy analyses were employed. As shown in the solid UV-Vis spectra (Figure 2D), the peak at 226 nm assigned to FF blue-shifted to 210 nm when the AuNPs were within the FF nanostructures. These results suggested that the AuNPs could interact with the FF nanosheet, and thus stabilized the architecture of the FF assembly. Given the aforementioned phenomenon, it is reasonable to assume that, in the first stage, AuNPs could not affect the selfassembled behavior of FF under high voltage electrospray, since the morphologies of the FF and FF/Au were similar. However, during the thermal process, the interaction between amino groups of FF and AuNPs occurred, because the molecular rearrangement of FF molecule from linear to cyclic structure resulted in the amino groups exposed outside the layers (Scheme 1C). The stable binding between gold and amino groups hindered the structure rearrangement through inter/intra hydrogen bonds. Meanwhile, the steric effect of the AuNPs prevented the participation of varied solvents from molecular reorganization. Such synergy of the stable binding and steric effect between AuNPs and the exposed amino groups of FF nanosheets guaranteed the strong resistance against environmental factors. The schematic illustration for the formation of self-assembled FF/Au was also exhibited in Scheme 1. Wettability of the FF/Au nanosheets-based film. Given the robust ultrathin nanosheets, it is reasonable to suppose that the platform based on FF/Au composites might be potentially promising for high-efficiency adsorption. Concerning the importance of hydrophobicity for adsorption in complicated matrix,43 the wettabilities of the three obtained FF/Au composites were explored by measuring the contact angles. As depicted in the insert figures of Figure 3B, significant increase of contact angle was observed, revealing the transformation of FF/Au surface from hydrophilic (the insert in Figure 3B(a)) to hydrophobic (the insert in Figure 3B(b), contact angle = 78.5 °) and superhydrophobic (the insert in Figure 3B(c), contact angle = 138.5 °) features. The hydroxyl group in the surface of the FF/Au composite would be reduced after heat treatment (Figure S3) and the polarity of the FF/Au composite would be lowered. Subsequently, water treatment would remove the residual salts produced during the in situ reduction process of Au3+, resulting in superhydrophobic surface of the FF/Au composite. This property combined with the ultrathin nanosheet structure made FF/Au composites excellent candi-



date for hydrophobic analytes adsorption and separation from complex biosamples, meanwhile avoiding the attachment of fouling organisms. FF/Au nanosheet-based SPME fiber for the analysis of estrogens. As a proof of concept, the SPME fiber based on the ultrathin FF/Au nanosheets was in-situ prepared on the surface of a 127 µm-thick SSW. A homogeneous coating was successfully obtained, which the thickness of the fiber coating was about 10 µm (Figure 4A). Five estrogens, including diethylstilbestrol (DES), estrone (E1), α-estradiol (E2), estriol (E3) and 17α -ethinylestradiol (EE2) were chosen as target analytes, because of their adverse effect against humans and animals at low-ng/L concentrations. The extraction kinetics of the fiber coating for five estrogens in PBS solution and semi-solid agar were demonstrated in Figure 4B and 4C, respectively, which all showed remarkable fast mass transfer procedures within 5 min, as well as high analyte loading capability. It indicated the ultrathin nanosheets architecture was highly favorable for high-efficiency adsorption. Additionally, the home-made fiber coating exhibited much higher extraction efficiency than that of commercial polydimethylsiloxane (PDMS) fiber (Figure S6A). Especially for DES and E1, the extracted amounts of FF/Au coating were 15 to 21 times higher than those in PDMS fibers. The possible reason for the fast kinetics of the FF/Au SPME fiber was ascribed to the easy accessibility of the ultrathin layer of the coating for the analytes via π-π stacking interaction. In addition, the almost vertical arrangement of the nanosheets on the SSW increased the available surfaces for adsorption. Moreover, the structure of the FF/Au coating was robust enough from the evidence that the morphology of the coating remained almost the same before and after being inserted into the fish muscle for in vivo sampling (Figure 4A(b-c)). The desorption profile, biofouling resistance, method performance including linear range, limit of detection (LODs, S/N=3), limit of quantification (LOQs, S/N=10), intra-fiber reproducibility and inter-fiber repeatability of the fabricated FF/Au coatings for the analysis of five estrogens were provided in the Supporting Information (Figure S6, S7 and Table S2), of which extraordinary low-level LODs (0.38-0.58 ng L1 ), good repeatabilities (RSD, 2.9%-17 %) were achieved. These findings demonstrated that the ultrathin FF/Au nanosheets promised to be evolved as an appealing platform for bioanalysis.

Figure 4. (A) SEM images of FF/Au nanosheets-based SPME coating on SSW: (a) the image of the SPME fiber with the magnification times of ×400; (b) the image of the coating with the magnification times of ×3k before the SPME fiber was used for in vivo fish sampling; (c) the image of the coating with a magnifica-

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tion of ×5k after the SPME fiber was inserted to the fish muscles for in vivo fish sampling. (B) and (C) Extraction time profiles of five estrogens, of which the concentration of each analyte was 10 µg L-1 in PBS solution (B) and semi-solid agar (C), respectively.

From the above results, the robust ultrathin FF/Au nanosheets evolved as excellent micro-platform for the rapid, high efficient and sensitive analysis of estrogens, was presented. In practical applications, the prepared SPME fibers were deployed in the application of complex biosamples including bovine serum, human serum and human urine with a small volume (2-5 mL), and in vivo fish sampling as well. The detected concentrations (Table S2) of target analytes in bovine serum, human serum and human urine ranged from 2.40 to 142 ng L-1, 2.00-111 ng L-1, 5.80 ng L-1 to 232 ng L-1, respectively, with good recoveries from 65.4% to 99.1% (Tables S3, S4 and S5). Among all the samples, E1 showed the highest concentration, in accordance with previously reported values.44,45 On the other hand, as for in vivo fish sampling, the determined concentrations (11.0 ng kg-1 to 310 ng kg-1, details of the quantitative method shown in section 1.4 and Table S6 in the Supporting Information) of five estrogens were compared with those obtained by traditional liquid extraction method (Table 1). The RSDs values were obtained between 7.07% and 35.4%, which would be acceptable because of the complicated matrices of fish muscles and the difference between individual fish.2 The results suggested the reliability of the proposed SPME fiber for rapid in vivo analysis. Table 1. The concentrations (ng kg-1) of five estrogens in fish muscle determined by in vivo SPME sampling and solvent extraction. DES

E1

E2

E3

EE2

Solvent extraction

310 (±99.2)

11.0 (±1.87)

29.0 (±6.67)

180 (±18.0)

210 (±31.5)

In vivo sampling

460 (±101.2)

14.0 (±2.24)

44.0 (±14.5)

300 (±36.0)

190 (±34.2)

Mean

385

12.5

36.5

240

200

RSD/%

27.5

17.0

29.0

35.4

7.07

FF/Au nanosheet-based SALDI platform for the analysis of anabolic steroids. Given the remarkable fast adsorption performance of the ultrathin FF/Au nanosheets and the laser absorption/transfer capability of AuNPs, we anticipated the proposed FF/Au coating was potential to evolve as a highefficiency platform for laser desorption/ionization (LDI). As a proof of concept, a novel SALDI platform based on FF/Au nanosheets was fabricated through the same electrospray method, and four anabolic steroids were selected as the model analytes (Figure S8). In the current method, we believed that the features of FF/Au coating, including homogenous surface, fast mass transfer of analytes from sample to FF/Au surface, and high laser light absorption of AuNPs, promised it as reliable SALDI platform for the quantification of small molecules. In our study, in order to enhance the ionization efficiencies of the four steroids, an in situ derivatization method was utilized (Figure S9). Firstly, the shot-to-shot and point-to-point repeatabilities were investigated. Excellent repeatabilities varying from 6.20% to 18.3% were achieved (Table S7), which indicated that the obtained surface was well homogenous. Then the individual linearity of the steroids was studied with the concentration level from 5 µg L-1 to 500 µg L-1. From Figure 5A and 5B, we could find that good linearity of each ana-


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lyte was observed. And the limit of detection of each analyte was calculated to be 1.5 µg L-1 (S/N = 3). Combing with the good repeatabilities, the results suggested that the quantitative analysis of small molecules could be achieved using the proposed SALDI platform. Additionally, we compared the desorption/ionization efficiencies of the FF/Au based platform with the individual FF and Au thin film based platforms, which were all prepared using the same electrospray method. The desorption/ionization efficiencies of target analytes using FF/Au based platform were 2-3 times higher than those of AuNPS thin film based platform, and two orders of magnitudes higher than those of FF based platform without the addition of AuNPs (Figure 5C). The results indicated that the AuNPs played the dominant role on the laser absorption and transfer for further analyte desorption and ionization. Furthermore, the FF ultrathin nanosheet structure offered more binding sites to capture the target analytes from samples, leading to the higher efficiencies of FF/Au based coating compared with individual AuNPs coating.

The reasonable mechanism of the laser desorption/ionization process could be resulted from: 1) the homogenous surface, fast mass transfer merits of the FF nanosheet contributed to the fast adsorption and desorption; 2) the surface plasmon resonance (SPR) phenomenon of AuNPs, which exhibited a broad SPR band from visible light to ultraviolet.46 And this feature could result in the rapid heat accumulation and dissipation of AuNPs under laser irradiation. Subsequently, the heat accumulation could cause the proton transfer from capping agents to the target analytes.47 Herein, since FF nanomaterials were assembled from amino acids, it could be acted as Au capping agent, which were effective additives for protonation of organic compounds. Therefore, in general, the synergy effect of AuNPs and FF contributed to the desorption and ionization of target analytes. Furthermore, the findings indicated that the present FF/Au nanosheets based SALDI platform was more suitable for the small analytes that ionized in positive ion mode.

Figure 5. (A) TOF-MS spectra of four anabolic steroids obtained when investigating the linear ranges; (B) The linear ranges of the four anabolic steroids corresponding to the previous mass spectra, which the concentrations of target analytes were varied from 5 µg L-1 to 500 µg L-1; (C) Comparison of ionization efficiencies of FF, AuNPs and FF/Au based SALDI platform; (D) Applications of the FF/Au nanosheets based SALDI platform to real human urine and serum samples. Noted that numbers 1 to 4 here were expressed as the ions of androstenedione ([M+2NH2+2H]+, 320), testosterone ([M+NH3OH+H]+, 323), androsterone ([M+NH3OH+H]+, 325), and hydroxyprogesterone ([M+NH2+H]+, 347), respectively.

Finally, to demonstrate the applicability of the current method in real complex biosamples, human serum and urine were used as model samples. As depicted in Figure 5D, the character peaks of the analytes could not be found, indicating that the concentrations of the analytes were below the linear range. However, obvious peaks were observed when the real samples were spiked with 15 µg L-1 of the target analytes.

Although only 2 µL of each samples were used, satisfactory recoveries ranging from 65.1% to 81.4% (Table S8) were achieved. All of these results implied that the proposed SALDI platform provided an efficient candidate for rapid determination of small molecules in complex matrices with an extremely low volume (µL level), but without tedious sample pretreatment.



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CONCLUSION In summary, ultrathin gold-stabilized FF nanosheets were prepared for the first time via an electrospray method followed by thermal treatment. The synergy of the stable binding and steric effect between AuNPs and the exposed amino groups of self-assembled FF nanosheets contributed to the high stability of the nanosheets. To make full use of this ultrathin nanosheet structure and strong laser light adsorption of AuNPs in potential applications for adsorption/desorption/ionization, two robust functional platforms including SPME fiber and SALDI plate were fabricated. The prepared SPME fiber was successfully applied to real complex biological samples, exhibiting rapid and high-efficiency adsorption capability. In addition, in SALDI applications, the homogenous FF/Au film achieved the quantitative determination of small molecules in complex matrices without tedious sample preparation. This study not only demonstrated that FF/Au composites could be prepared to serve as promising adsorption/desorption/ionization materials for specific applications, but also provided useful strategy to advance the ideas for future combination of SPME with LDI technique.

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ASSOCIATED CONTENT

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Supporting Information

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The Supporting Information is available free of charge on the ACS Publications website. Experimental section including reagents and material, characterization and instruments, parameters of LC-MS/MS analysis, sampling-rate calibration for in vivo sampling, and liquid extraction. Characterization of FF/Au based coating. FF/Au based SPME fiber for the analysis of five estrogens. SALDI TOF-MS analysis of four anabolic steroids.

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AUTHOR INFORMATION Corresponding Author

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[email protected] (G. Ouyang).

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Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

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ACKNOWLEDGMENT This research was supported by projects of National Natural Science Foundation of China (21527813, 21477166, 21677182 and 21737006), and the Natural Science Foundation of Guangdong Province (S2013030013474).

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Table of Contents


Ultrathin Self-Assembled Diphenylalanine Nanosheets through A Gold

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