Novel Magnetic Microprobe with Benzoboroxole Modified Flexible

Subscriber access provided by STEPHEN F AUSTIN STATE UNIV

Article

Novel Magnetic Microprobe with Benzoboroxole Modified Flexible Multisite-Arm for High-Efficiency Cis-diol biomolecules Detection Guosheng Chen, Siming Huang, Xiaoxue Kou, Jin'ge Zhang, Fuxin Wang, Fang Zhu, and Gangfeng Ouyang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b05033 • Publication Date (Web): 08 Feb 2018 Downloaded from http://pubs.acs.org on February 11, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Analytical Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 9 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

Analytical Chemistry

Novel Magnetic Microprobe with Benzoboroxole Modified Flexible Multisite-Arm for High-Efficiency Cis-diol Biomolecules Detection Guosheng Chen, Siming Huang, Xiaoxue Kou, Jin’ge Zhang, Fuxin Wang, Fang Zhu* and Gangfeng Ouyang* MOE Key Laboratory of Bioinorganic and Synthetic Chemistry, School of Chemistry, Sun Yat-sen University, Guangzhou 510275, China. *Corresponding author: +86-20-84110845; [email protected] (F. Zhu); [email protected] (G. Ouyang).

ABSTRACT: Cis-diol biomolecules, such as saccharides and glycoproteins play vital roles in regulating a variety of biological events, including molecular recognition, signal transduction, cell adhesion and immune response. However, saccharides and glycoproteins in living system usually exist in very low abundance along with abundant interfering components. High-efficiency detection of saccharides and glycoproteins is a challenging yet highly impactful area of research. Herein, we reported a novel magnetic microprobe with benzoboroxole modified flexible multisite-arm (PEG 2000 grafted PAMAM dendrimers, the microprobe was denoted as BFMA-MNP) for high-efficiency saccharides detection. The extraction capacity was significantly improved by approximately 2 orders of magnitude, owing to the integration of the enhanced hydrophilicity and multivalency effects in benzoboroxoles and the enhanced accessibility of the binding sites within the PEG 2000 grafted PAMAM dendrimers. As a result, the proposed approach possessed several advantages compared with previous boronic acid based methods, including ultrahigh sensitivity (limit of detection was less than 1 ng/mL), wide linear range (ranged from 0.5 µM to 2000µM), and applicable in physiological pH condition. Furthermore, we established a general BFMA-MNP/glycoproteins/AuNPs sandwich assay to realize the visual glycoprotein qualitative screening for the first time. The unique sandwich assay possessed the dual nature of the magnetic separation by BFMAMNPs and specific coloration by citrate-coated AuNPs. This visual sandwich assay enabled the fast differentiation the existence of glycoproteins in complicated sample without any advanced instruments. We believe the proposed BFMA-MNP microprobe herein will advance the ideas to detect and identify trace saccharides and glycoproteins in important fields such as glycomics and glycoproteomics.

Cis-diol biomolecules, such as saccharides and glycoproteins play vital roles in regulating a series of physiology process, including molecular recognition, signal transduction, cell adhesion and immune response. Abnormal structural changes and abnormal expression of these compounds are closely related to the development of diverse diseases. Therefore, most of them have been considered as biomarkers and therapeutic targets in clinical diagnostics.1-3 However, saccharides and glycoproteins in living system usually exist in very low abundance along with abundant interfering components. Highefficiency approaches for saccharides and glycoproteins screening are highly desirable. Antibodies4,5 and lectins6-8 are commonly used receptors for distinguishing specific cis-diol biomolecules, however, they are difficult to prepare, have poor storage stability and high costly. Such limitation restrict their development as an efficient methodology for glycoproteins or saccharides detection. To address these drawbacks, advanced materials based on boronic acids/its derivatives were elaborately synthesized as the receptors for recognition of cis-diol biomolecules such as saccharides and glycoproteins, owing to the controllable boronate-affinity chemistry.9,10 Such versatile artificial receptors have enabled rapid advances in the development of powerful tools for detecting and quantifying saccharides or glycoproteins of interest for research, medical diagnostics and clinical applications. For instance, Ouyang et al. established the solid

phase microextaction11,12 approach based on boronic acid functionalized carbon nanotube and boronic acid functionalized metal organic framework coating for detection of saccharides; Liu et al. proposed a new molecular imprinting combined with the boronate-affinity techniques13,14 for specific glycoproteins recognition. However, the high-efficiency receptor design relied on the precise spatial location of boronic acid scaffold, and therefore the synthetic routes were usually complicated and tedious.15 In addition, most of the current available approaches require a basic pH condition for recognition.15 These drawbacks limited their adoption for trace-level biomarker screening, gives rise to the risk of degradation and denaturation of labile molecules, especially for glycoproteins, and hindered them to apply in general physiological conditions. Last but not least, a simple visual assay for fast glycoproteins qualitative screening is appealing for the on-site diagnostics, but has not be reported. Therefore, developing a high sensitive approach for saccharides or glycoproteins detection and a simple visual assay for fast glycoproteins qualitative screening are highly desirable. Magnetic nanoparticle are widely used in target extraction and enrichment because of their outstanding convenience for retrieval from sample matrix .16-18 In addition, the modifiable surface and high specific surface area endow it the superior platform for a variety of diverse applications in the field of analytical chemistry.19-21 We also noticed the recent reported

ACS Paragon Plus Environment

Analytical Chemistry 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

boronic acid derivatives, Ortho-hydroxymethyl-phenylboronic acids (benzoboroxoles) showed excellent water solubility and improved cis-diol units binding capacity, even superior to the Wulff-type boronic acid in neutral water.22-25 Inspired by these, We anticipated that the combination of the advantages of magnetic extraction and benzoboroxoles receptor enable the development of a high-efficiency approach for saccharides or glycoproteins detection. In this study, we introduced a reformative boronic acid receptor-benzoboroxole combined with PEG 2000 grafted PAMAM dendrimers onto the magnetic nanoparticle (denoted as BFMA-MNP) (Figure 1A). The extraction and enrichment mechanism towards cis-diols biomolecules was based on the controllable boronate-affinity chemistry. 9,10 Briefly, under the alkaline condition, boronic acids could rapidly bind with cis1,2 or 1,3-diols to form five- or six-membered cyclic esters through covalent interaction. After extraction, the enriched cis-1,2 or 1,3-diols substance within the microprobes was facile to be released when the pH was adjusted to acid condition, and the eluent was quantified by the gas chromatography-mass spectrometry (GC-MS) (Figure 1B). Excitingly, the extraction capacity was significantly improved by approximately 2 orders of magnitude owing to the enhanced hydrophilicity and multivalency effects of the benzoboroxoles and the enhanced flexibility and accessibility of the binding sites within the PEG 2000 grafted PAMAM dendrimers arm. As a result, the proposed approach possesses high sensitivity toward saccharides, even though under physiological pH condition. Furthermore, contributed to high recognition capacity of the BFMA-MNP microprobe, we established a BFMAMNP/glycoproteins/AuNPs based visual sandwich visual assay to selectively screen the glycoproteins in a simple manner, which provided a means for fast differentiation the existence of glycoproteins in complicated sample without any advanced instruments.

Figure 1. Synthesis routes of BRA-MNP (A-a), BFA-MNP (A-b) and BFMA-MNP (A-c); Representation of the BFMA-MNP microprobe based assay for detection of saccharides (B).

Experimental Section Reagents and Materials All the reagents and materials used were provided in supporting information Characterization.

Page 2 of 9

Transmission electron microscopy (TEM) images were taken on a JEM-2010HR microscope operating at 200 kV. FT-IR spectra was carried out on Bruker EQUINOX 55 spectroscopy using the KBr method. Thermogravimetric analyse (TGA) was performed under nitrogen atmosphere (20 mL min−1) with temperature increased with 10 °C min−1 using a TA-Q50 system. The samples were degassed in vacuo at 120 °C for 12 h before TGA analysis. Powder X-ray diffraction (XRD) patterns were collected (0.02°/step, 0.06 seconds/step) on a Bruker D8 Advance diffractometer (Cu Kα) at room temperature. The UV-Vis absorbance measurement was performed with a 3600 spectrophotometer (Shimadzu, Japan). Preparation of MNP microprobe. The details information for the Preparation of the BFMAMNP BFA-MNP and BRA-MNP microprobes was displayed in Supporting Information text. Preparation of the citrated-coated AuNPs probe. 100 mL bright yellow HAuCl4 solution (0.24 mM) was heated until 80 °C in a round-bottom flask, followed by adding 2 mL of sodium citrate (85 mM) dropwise. The reaction was kept at these conditions during 1 h and the color changed from bright yellow to black, and then became deep red. The obtained citrated-coated AuNPs was storage at 4 °C for further use. Selectivity test toward cis-diols. For demonstrating the selectivity of the MNPs microprobes towards cis-diol compounds, adenosine and deoxyadenosine were used as model compounds. 1 mg MNPs was dispersed in 1 mL solution of 1 mg mL-1 adenosine or deoxyadenosine in a plastic tube. The tubes were shaken on a rotator (400 rpm) for 30 min at room temperature. Then the MNPs was collected by a magnet, followed by rinsing with 1.5 mL of the pure water for 6 times each. Afterwards, the MNPs was resuspended in 1 mL of 0.1 M acetic acid solution and eluted for 30 min on a rotator with 600 rpm speed. Finally, MNPs was separated by a magnet and the eluents were collected by pipetting carefully. The eluents were used for UV analysis. For the binding dynamic and maximum binding capacity test, the experiments were operated in the same manner with slight modification. In the binding dynamic test, the reaction times were set at 5.0, 10.0, 20.0, 30.0 and 60.0 minutes while in the maximum binding capacity test, the reaction time was set at 12h. Other steps were the same as that in the selectivity test. The MNPs based assay for saccharides. The saccharide mixed standard was prepared in 0.1 M phosphate buffer solution (pH=7.4). Typically, 1 mg MNPs was dispersed in 1 mL saccharides standard with different concentration through ultrasonic. The tubes were shaken on a rotator (600 rpm) for 30 min at room temperature. Then the MNPs was collected by a magnet, followed by rinsing with 1.5 mL of the pure water for 4 times each. Afterwards, the MNPs was resuspended in 1 mL of 0.1 M acetic acid solution and eluted for 30 min on a rotator with 600 rpm speed. Finally, MNPs was separated by a magnet and the eluents were collected by pipetting carefully. The eluents were used for GC-MS analysis.


Page 3 of 9 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

Analytical Chemistry For interference test, the amino acids mixed standard (including tryptophan, proline, glycine, valine, methionine, aspartic acid, lysine and lauric acid), aliphatic acids (including lauric acid, myristic acid and paimitic acid), glutathione and uric acid were pre-added into the saccharides standard, respectively. All the interfering biomolecules were as the same concentration as the saccharides. The other steps were identical with that mentioned in the MNPs based assay for saccharides. The sandwich visual assay for glycoproteins. Typically, 500 µL glycoproteins (dissolved in 0.1 M phosphate buffer solution, pH=7.4) with different concentrations were added into 0.5 mg BFMA-MNPs in a 2 mL transparent vial, followed by ultrasonic for 2 min. Then, the well dispersed solution was shaken on a rotator (600 rpm) for glycoproteins extraction. After 5 min extraction, the MNPs was collected by a magnet and rinsed with 1.5 mL of the pure water for 6 times each, ensured that all the free glycoproteins were removed. Immediately, 500 µL prepared AuNPs solution was introduced, followed by 1 min sonication. Finally, the vital was placed on a magnet. GC-MS analysis The procedure of GC-MS for saccharides analysis was according to the method reported by our group.11 The eluent was firstly undergone a derivatization by acetic anhydride prior to GC-MS analysis. The detection of the derived saccharides was performed on an Agilent 6890N gas chromatograph equipped with a MSD 5975 mass spectrometer. A split/splitless-type injector was used for sample introduction. Chromatographic separation was carried out with a HP-5MS capillary column (30 m × 250 µm × 0.25 µm, Agilent Technology, CA, USA). The inlet temperature was 240 °C, and the oven temperature program was as follows: The initial oven temperature was 140 °C (held for 0. 5 min), ramped at 30 °C/min up to 190 °C (held for 5 min), and ramped at 2 °C/min up to 210 °C (held for 2 min). Helium was used as carrier gas at a constant flow rate of 1.2 mL/min. The MSD was operated in the electron impact ion (EI) mode with a source temperature of 230 °C. The electron energy was 70 eV and the filament current was 200 A.

flexible multisite-arm magnetic nanoparticle microprobe (BFMA-MNP), in which the flexible multisite-arms (PEG 2000 grafted PAMAM dendrimers (ethylenediamine core, generation 4.0, 64 surface amino groups)) acted as a bridge connected MNP and benzoboroxole, as shown in Figure 1A. We anticipated the introduction of the PEG 2000 flexible arms and PEG 2000 grafted PAMAM dendrimers arms could exponentially enhance the flexibility and accessible binding sites of the benzoboroxole, thus increasing the binding performance toward cis-diol units of saccharides and glycoproteins at physiological pH condition. The prepared MNPs was characterized using transmission electron microscopy (TEM), Fourier transform infrared (FTIR) and thermogravimetric analysis (TGA), which confirmed that three kinds of MNP were successfully synthesized according to our expectation. As can be seen from Figure 2A-C and Figure S1, TEM images revealed that the MNPs were well shaped with a diameter of about 100 nm. Importantly, the grafted PAMAM dendrimers were observed under TEM image, which coated onto the MNPs with a ≈3 nm thickness (Figure 2D and Figure S2). Meanwhile, FT-IR and TGA indicated that the benzoboroxole and the different kinds of arms were successfully grafted onto the MNPs (Figure 2E and Figure 2F). In FT-IR spectrum, a strong adsorption peak at 580 cm-1 and two peaks at 1,640 and 1,048 cm-1 were observed for the four types of MNPs, which can be ascribed to Fe-O vibrations and the presence of the N-H and C-N, respectively. In addition, the peak at 1547 cm-1 was ascribed to N-H deformation vibration of –CONH-, which indicates the MNPs were modified with PAMAM dendrimer. The peak at 1358 cm-1 was associated with the C-B vibrations, implying that the benzoboroxole was present on the MNPs surface. Furthermore, the XRD test demonstrated the crystal structure of MNPs were remained after grafting (Figure S3) and the contact angle test confirmed the hydrophilicity of BFA-MNP and BFMA-MNP (Figure S4) . Besides, the magnetic separation properties were desirable (Figure 2G and Figure S5).

Results and discussion Preparation and characterization The receptor types and the accessible binding sites are two significant factors influencing the host-guest recognition. Branched PAMAM dendrimers-assisted boronic acids receptor were proposed for improving the accessible binding sites.26,27 However, the rigid structure of PAMAM dendrimers is unfavourable for effective enrichment and quick equilibration. To simultaneously improve the flexibility and accessible binding sites of benzoboroxole, herein we synthesized three different kinds of benzoboroxole modified magnetic nanoparticle microprobe, including 1) benzoboroxole modified rigid arm magnetic nanoparticle microprobe (BRA-MNP), in which benzoboroxole was directly modified onto the MNPs; 2) benzoboroxole modified flexible arm magnetic nanoparticle microprobe (BFA-MNP), in which the flexible arms (NH2-PEGCOOH, 2000 molecular weight) acted as a bridge connected MNP and benzoboroxole, and 3) benzoboroxole modified

Figure 2. The TEM images of BRA-MNP (A), BFA-MNP (B) and BFMA-MNP (C and D); FT-IR spectra of NH2-MNP, BRA-MNP, BFAMNP and BFMA-MNP (E); TGA curves of the prepared MNPs (F), Photographs showing dispersion and magnetic separation of the BFMA-MNP (G).

Specifity and binding capacity of the microprobes As the selectivity of the microprobe is a critical concern of the methodology, the selectivity of the prepared BFA-MNP and BFMA-MNPs toward cis-diol units was firstly examined. Adenosine, which contains a similar cis-diol structure with saccharides/glycoproteins and has UV absorbance at about 260 nm, was used as a test compound. While deoxyadenosine,



containing consistent molecular structure with adenosine except the cis-diol moiety, was selected as an interferent (Figure 3A). As shown in Figure 3B, 3C and Figure S6, both BFMAMNP and BFA-MNPs selectively captured adenosine but excluded deoxyadenosine. Such high selectivity towards cis-diol units also demonstrated that the enrichment mechanism of cisdiol substances was in accordance with the controllable boronate-affinity chemistry,9,10 in which the benzoboroxole groups bond with cis-diols units to form five- or six-membered cyclic esters through covalent interaction. In addition, the binding capacity of BFMA-MNPs and BFA-MNPs towards adenosine were calculated to be 3.24 µmol/g and 1.01 µmol/g respectively, and the binding capacities of BFMA-MNP was higher than that of the only PAMAM-assisted boronic acid MNP.26 Importantly, the binding capacities of BFA-MNP and BFMAMNP toward cis-diol unites at physical pH were comparable with those at basic condition (Figure 3D and 3E), benefiting by 1) the enhanced hydrophilicity of the benzoboroxoles receptor compared with conventional boronic acid receptors;22 and 2) the unusually small C-B-O dihedral angle of benzoboroxoles28 opens up the cone angle in the resulting tetrahedral diol-boronate complex, which made it may appear to complex hexopyranosides mainly using their 4,6-diol compared to the usual boronic acids.22 Thus, All the undermentioned tests were operated in physical pH condition. We further compared the binding strengths of BFA-MNP and BFMA-MNP, and BRA-MNP was used as a contrast. As seen in Figure 3F and Figure S7, the binding dynamics test showed that the binding strengths displayed the order of BFMA-MNP>BFA-MNP>BRA-MNP, suggested that the introduction of the flexible arm and flexible multisite-arm result in a significantly positive influence on the binding properties. As a result, the maximum binding capacity of the BFMA-MNP towards cis-diol units was improved by approximately 50 times, as compared with BRA-MNP (Figure 3G).

Saccharide analysis based on BFMA-MNP microprobe Saccharide is a class of significant important cis-diol biomolecules in living system, and plays a crucial role in most of the biological and pathological processes. However, saccharides are hydromimetic, blending easily into a background of water molecules.29,30 Recognition and quantify of saccharides in water solution is a challenging yet highly impactful area of research. The assays based on the pH controllable boronateaffinity chemistry of BFA-MNP and BFMA-MNP were proposed for three typical saccharides (including glucose, mannose and galactose) recognition, in which the captured saccharides were eluted under acid condition and then quantified by GC-MS.

Figure 4. The linear ranges of the BFA-MNP (A) and BFMA-MNP (B) based assay toward saccharides.

Figure 5. The binding strength of BFMA-MNP (A) and BFA-MNP (B) toward saccharides when other potential interfering biomolecules were coexisted; The minimum detectable concentrations of the BRA-MNP (C), BFA-MNP (D) and BFMA-MNP (E) based assay for saccharides recognition.

Figure 3. The molecule structures (A) of adenosine and deoxyadenosine; UV-vis analysis of the eluents, which was pre-extracted by BFMA-MNP (B) and BFA-MNP (C) from adenosine and deoxyadenosine (1 mg/mL each), respectively; the binding capacities of BFMA-MNP (D) and BFAMNP (E) toward adenosine in different pH conditions; the binding dynamics (F) and maximum binding capacities (G) of BFMA-MNP, BFA-MNP and BRA-MNP, respectively.

The linear ranges of the proposed assays using BFA-MNP ranged from 25 µM to 1000 µM (Figure 4A), while the linear ranges was further improved using the BFMA-MNP microprobe, which ranged from 0.5 µM to 2000µM (Figure 4B). We found that the extraction capacities toward the three saccharide displayed the order of galactose>mannose>glucose, the reason is that the generally observed binding affinity of boronic acids with monosaccharides follows the order of galactose>mannose>glucose.31 Notably, All the test were operated under physical pH condition. In addition, we also studied the anti-interference abilities of the BFA-MNP and BFMAMNP microprobe toward potential interfering substances coexisting in biofluids, including various amino acids, aliphatic acids, glutathione and uric acid. As shown in Figure 5A and 5B, the anti-interference abilities of the MNP microprobes were desirable because of the high selectivty of the benzo-


Page 4 of 9

Page 5 of 9 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

Analytical Chemistry boroxole recopter towards cis-diols units. Moreover, the minimum detectable concentrations using these three kinds of MNP microprobes were also measured, as shown in Figure 5D. In the assay based on BFA-MNP, the minimum detectable concentrations were approximately 0.01 µg/mL for galactose and mannose respectively, while 0.1 µg/mL for glucose. In the assay based on BFMA-MNP, the minimum detectable concentrations were less than 1 ng/mL for both three saccharides (Figure 5E). Noteworthy, these minimum detectable concentration for saccharide was lower than most previous boronic acid based approach (Table S1). As a comparison, the concentration less than 0.1 µg/mL was undetectable using the BRA-MNP based assay (Figure 5C). Clearly, owing to the exponential enhancement of the accessibility of binding sites as well as the flexibility within BFMAMNP, the sensitivity of the BFMA-MNPs based approach towards saccharide was improved by about 2 orders of magnitude, as compared with the BRA-MNPs without additional flexible multisite-arm modification. Visual sandwich assay for glycoproteins screening. Glycoproteins have important roles containing molecular recognition, signal transduction, cell adhesion and immune response in biological processes. Most of the glycoproteins have been considered as biomarkers and therapeutic targets in clinical diagnostics. 32, 33 However, glycoproteins in living system usually exist in very low abundance along with abundant interfering components. Herein, we firstly study the capacity of the BFMA-MNPs for glycoproteins analysis, in which the glycoproteins (transferrin (TRF) and horseradish peroxidase (HRP)) were extracted by the BFMA-MNPs and the eluents were quantified by high performance liquid chromatography (HPLC) coupled with photodiode array detector (PDAD) (Supporting information text). The results showed that the linear ranges ranged from 1 nM to 200 nM for HRP and 5 nM to 400 nM for TRF (Figure S8). Such desirable linear ranges were owing to the abundant flexible binding sites onto the BFMA-MNPs. It is well known that AuNPs show a red-to-blue color change in response to a dispersion-to-aggregation state change, and the AuNPs based colorimetric assay offers new opportunitites for the visualization of the targets due to their high sensitivity, designability, and low technical demands.34 We attempt to construct a visual approach based on AuNPs for glycoproteins qualitative screening without any advanced instruments. The principle of the visual approach was shown in Figure 6A. Briefly, the magnetic microprobe BFMA-MNP firstly captured the cis-diols units of glycoproteins on its surface. Application of a magnetic field draws the BFMA-MMPs to the wall of the reaction tube in a matter of seconds, allowing the separation of all of the glycoproteins from the complicated mixture. Such fast magnetic separation is facile to avoid the interference caused by other biomacromolecules such as nonglycoproteins, DNA and polypeptide. Washing the aggregate structures in pure water and then introducing the citrate-coated AuNPs microprobe, the AuNPs and the BFMA-MMPs sandwich the glycoproteins target owing to protein corona on citrate-coated AuNPs through multisites chemical and physical adsorption,35-37 When applying a magnetic field, the AuNPs precipitate in a matter of seconds. The aggregation and precipitation of AuNPs result in the color change from red to purple,

and then become colorless, which can be monitoring by the naked eye. Herein, approximately 20 nm citrate-coated AuNPs microprobe was firstly synthesized and characterized by transmission electron microscopy (TEM) and UV-Vis spectrum (Figure S9). The prepared citrate-coated AuNPs could well dispersed in the water and was unable to aggregate by itself (Figure S10). We next initially demonstrated the BFMAMNPs/glycoproteins/AuNPs sandwich assay could induce the color change of the AuNPs solution, HRP (a glycoprotein containing nine glycosylation sites occupied by eight or nine identical hybrid glycans) was chosen as the typical glycoproteins. As shown in Figure 6A-1, when AuNPs solution was added to the tube and sonication in matter of seconds, the color began to fade. Over time, the solution became colorless. UV-Vis spectrum showed that the plasmon band at 528 nm moved to 588 nm (Figure 6B), similar to the case where the aggregation of AuNPs was induced by the cross-linking of DNA.38,39 To demonstrate the AuNPs were aggregated onto the BFMAMNPs due to the BFMA-MNPs/ glycoproteins/AuNPs sandwich-structure formed, we firstly confirmed the adsorption of the glycoproteins onto AuNPs through the form of proteins corona. As shown in Figure S11A, the fluorescence of HRP was quenched through Förster resonance energy transfer (FRET) when the AuNPs was added. Such quenching was also observed on the other glycoprotein TRF (Figure S11B), suggested that the glycoproteins could be adsorbed onto the AuNPs through the form of proteins corona. We then collected the precipitate for TEM imaging. As predicted, we clearly observed the aggregated AuNPs was formed onto the MNPs (Figure 6C), wherein the glycoproteins surrounded (Figure 6D). Clearly, the BFMA-MNPs/ glycoproteins/AuNPs sandwich-structure was formed and leaded to the aggregation of AuNPs (Figure 6E). Notably, the whole visual assay could complete within 10 min.

Figure 6. (A) Representation of the BFMA-MNP/glycoproteins/AuNPs sandwich visual assay for visual detection of glycroproteins; photograph (B) of the solutions containing the mixtures of BFMA-MNP and AuNPs after applying a magnet, in which the BFMA-MNP pre-extracted the HRP (1), BSA (2), RNase A (3), glucose (4), mannose (5), adenosine (6) and deoxyadenosine (7), and the corresponding UV-Vis spectrum (C) of the supernatant; The TEM (D) and the amplifying TEM (E) images of the precipitate collected from HRP sandwich visual assay; The schematic diagram (F) of the MNPs/glycoproteins/AuNPs sandwich structure.



Selectivity of the visual sandwich assay for glycoproteins We tested the selectivity of this visual assay for glycoproteins by using other abundant interfering biomolecules in place of glycoproteins, including bovine serum albumin (BSA, nonglycoproteins), Ribonuclease A (RNase A, non-glycoproteins), glucose, mannose, adenosine, and deoxyadenosine at the same concentration (0.1 mg/mL). None of these interfering biomolecules could induce the color fade (Figure 6A-2 to 6A-7), and the UV-Vis spectrum showed that only one plasmon band at 528 nm was observed (Figure 6B). Firstly, the BFMA-MNPs microprobe possessed high selectivity toward cis-diols substance, therefore the non-glycoproteins (BSA and RNase A) could be excluded by the BFMA-MNP microprobe in the magnetic separation step (Figure S12). Even though the concentration of them increased to 10 times, no color change phenomenon was observed (Figure S13). Secondly, glucose, mannose and adenosine are typical saccharide possessed the similar cis-diol units formed in the glycan chain of glycoproteins, but the biomicromolecules didn’t possess the protein corona effect on the AuNPs, therefor they still couldn’t induct the aggregation and precipitation of AuNPs through a magnetic field, even though the concentration was increased to 10 times (Figure S13). Clearly, such “belt-and-braces approach” with the magnetic recognition and protein corona effect microprobes ensured the high selectivity toward glycoproteins. Mechanism and general applicability of the visual sandwich assay for glycoproteins To exploit the mechanism of specific glycoproteins visual assay, we further operated other two control experiments. One was that mixing the BFMA-MNP and citrate-coated AuNPs solution without the glycoproteins, the red color was still remain, even though the exposure time was expand to 24 h at the room temperature (Figure S14). The other one was that mixing the AuNPs solution and glycoproteins HRP without the BFMA-MNP, the red color was still unable to fade. Even though the concentration of HRP was increased to as high as 2 µM (Figure S15A), the UV-Vis spectrum also confirmed the non-aggregation of the AuNPs (Figure S15B). It indicated that only the unique BFMA-MNP/glycoproteins/AuNPs sandwich structure could induce the color change. To further confirm the mechanism of visual assay was proceeded according to our expectation, the visual assay for other glycoprotein, Ribonuclease B (RNase B possesses one glycosylation site, to which five high-mannose glycan isoforms may be attached), was conducted. We chose RNase B as another typical glycoprotein because the structural difference between RNaseA (non-glycoprotein) and RNase B (glycoprotein) is only one glycan chain. In the similar visual assay, the color of the AuNPs changed from red to purple when 0.4 µM RNase B was added (Figure 7C). Along with increasing the concentration of RNase B from 0.4 µM to 8 µM, the color changed from purple to blue, and then became colorless, while further increasing the concentration of RNase B, the color remained colorless (Figure 7C). Such phenomenon of the color change was also observed in the HRP visual assay (Figure 7E). It means that the unique BFMA-MNPs/glycoproteins/AuNPs sandwich-structure induced the AuNPs aggregation and the aggregation strength was depended on the concentration of glycoprotein, which also confirmed by the TEM imaging (Figure S16) and UV-Vis spectrum (Figure 7D and 7F). It is

worth mentioning that the minimum fading concentrations were 8 µM for RNase B while 0.5 µM for HRP, causing by the much more glycosylation sites formed in HRP. As a comparison, the color of the AuNPs solution still remained red even though the applied concentration of RNase A was raised from 0.4 µM to 40 µM (Figure 7A and 7B). These findings confirmed the mechanism of the glycoproteins visual assay was well proceeded according to our expectation. This visual sandwich assay was generally applicable for glycoproteins screening. Transferrin (TRF, a sialylated glycoprotein containing two glycosylation sites for the attachment of two identical sialylated glycans) was chosen as another target for the demonstration, because TRF contains a different type of glycan. More importantly, TRF exists in mammalian systems and its levels in human serum are associated with several diseases, such as iron deficiency anemia, protein malnutrition, and atransferrinemia. Along with increasing the concentration of TRF from 0.063 µM to 0.63 µM, the color changed in sequence from red to purple, and then became colorless, while further increasing the concentration of TRF, the color remained colorless (Figure S17A). In addition, the UVvis spectrum of the supernatant and the TEM image of the precipitate demonstrated the aggregation of the AuNPs onto MNPs (Figure S17B and S17C). These results demonstrated the generality of the new strategy. Moreover, it should be noted that the minimum fading concentration could be well improved through decreasing the dosage of AuNPs solution. As shown in Figure S18A, the minimum fading concentration for HRP was improved to be 12.5 nM when the dosage of AuNPs solution was 2mL. Further decreasing the dosage to 1mL, minimum fading concentration was as low as 2.5 nM (Figure S18B). We believe that the high-efficiency magnetic microprobe was responsible for the high sensitivity of the assay for glycoproteins, and the general applicability and controllable limit of fading suggested that the proposed BFMAMNP/glycoproteins/AuNPs sandwich visual assay maybe perform in the biosample with relatively complex mixtures

Figure 7. The photographs of the proposed visual sandwich assay for RNase A (A), RNase B (C) and HRP (E) in different concentrations, and the corresponding UV-Vis spectrum of the collected supernatant from RNase A (B), RNase B (D) and HRP (F) visual assay


Page 6 of 9

Page 7 of 9 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

Analytical Chemistry Real-sample application Given the ultra-sensitivity of saccharide determination with the proposed BFMA-MNP microprobe. To demonstrate the applicability for real sample analysis, the BFMA-MNP microprobe was firstly adopted for glucose detection in bovine serum, in which the bovine serum was diluted by 100 times. The results shown in Figure 8A was well in good agreement with the values measured by a commercial blood glucose monitor. Furthermore, the potential of BFMAMNP/glycoproteins/AuNPs visual sandwich assay for realsample application was demonstrated by the screening of glycoproteins directly from bovine serum. Bovine serum is a complicated matrix containing several kinds of glycoproteins such as α2-macroglobulin (α2MG), fetuin at relatively low abundance, as well as coexist alongside abundance interferential molecules, such as BSA, amino acid, and sugars. Clearly, the sensitive visual sandwich assay could screen the glycoproteins even though diluting the bovine serum 1000 times (Figure 8B).

Figure 8. (A) Glucose detection using the BFMA-MNP microprobe and a commercial blood glucose monitor approach; (B) The visual sandwich assay for the glycoproteins screening in bovine serum.

Conclusion In conclusion, we have demonstrated the new BFMA-MNP microprobe could highly enhance the recognition capacity toward saccharides owing to the synergistic effect of benzoboroxole and flexible multisite-arm. The proposed BFMAMNP based assay for saccharides detection possessed ultrahigh sensitivity, wide linear range and the applicable in physiological pH conditions. In addition, the established BFMAMNP/glycoproteins/AuNPs sandwich visual assay featured the glycoprotein qualitative screening in a simple manner, but the further quantitative analysis still remained challenging at the present stage. Such convenient and visual assay enables the fast differentiation of the existence of glycoproteins in complicated sample without any advanced instruments. We anticipate this BFMA-MNP microprobe approach to be promising tools for important applications such as glycomics, glycoproteomics and clinical diagnostics.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Reagents and materials, preparation of the MNP probes, HPLCPDAD analysis, TEM images for MNP probes, XRD test, contact angle test, photograph of magnetic separation, selectivity towards cis-diols, extraction kinetics of adenosine, linear ranges for glycoproteins, UV-Vis spectrum and photograph of AuNPs, fluorescence spectrums for glycoproteins, photograph and corresponding TEM images and UV-Vis spectrum for visual assay, the comparison of the methodology (PDF).

AUTHOR INFORMATION Corresponding Author [email protected] (F. Zhu); [email protected] (G. Ouyang).

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

ACKNOWLEDGMENT This research was supported by projects of National Natural Science Foundation of China (21377172, 21225731, 21477166, 21527813, 21677182), and the Natural Science Foundation of Guangdong Province (S2013030013474).

REFERENCES (1) Rudd, P. M.; Elliott, T.; Cresswell, P.; Wilson, I. A.; Dwek, R. A. Science 2011, 291, 2370 – 2376. (2) Timmer, M. S.; Stocker, B. L.; Seeberger, P. H. Curr. Opin. Chem. Biol. 2007, 11, 59–65 (3) Morikawa, Y.; Heallen, T.; Leach, J.; Xiao, Y.; Martin, J. F. Nature 2017, 547, 227–231. (4) McGarry, R. C.; Helfand, S. L.; Quarles, R. H.; Roder, J. C. Nature 1983, 306, 376–378. (5) Mouquet, H. et al. Proc. Natl. Acad. Sci. USA 2012, 109, E3268– E3277. (6) Fuchs, A.; Lin, T. Y.; Beasley, D. W.; Stover, C. M.; Schwaeble, W. J.; Pierson, T. C.; Diamond, M. S. Cell Host Microbe 2010, 8, 186–195. (7) Nakata, E.; Koshi, Y.; Koga, E.; Katayama, Y.; Hamachi, I. J. Am. Chem. Soc. 2005, 127, 13253–13261. (8) Ke, C.; Destecroix, H.; Crump, M. P.; Davis, A. P. Nat. Chem. 2012, 4, 718–723 (9) James, T. D.; Sandanayake, K. R. A. S.; Shinkai, S. Angew. Chem. Int. Ed. 1996, 35, 1910–1922. (10) Li, D. J.; Chen, Y.; Liu, Z. Chem. Soc. Rev. 2015, 44, 8097– 8123. (11) Chen, G.; Jun, Q.; Xu, J.; Fang, X.; Liu, Y.; Liu, S.; Wei, S.; Jiang, R.; Zeng, F.; Luan, T.; Zhu, F.; Ouyang, G. Chem. Sci. 2016, 7, 1487-1495. (12) Chen, G.; Fang, X.; Chen, Q.; Zhang, J.; Zhong, Z.; Xu, J.; Zhu, F.; Ouyang, G. Adv. Funct. Mater. 2017, 27, 1702126. (13) Liu, J.; Yin, D.Y.; Wang, S.S.; Chen, H.Y; Liu, Z. Angew. Chem. 2016, 128, 13409–13412. (14) Xing, R.; Wang, S.; Bie, Z.; He, H.; Liu. Z. Nat. Protoc. 2017, 12, 964-987. (15)Wu, X.; Li, Z.; Chen, X.; Fossey, J. S.; James, T. D.; Jiang, Y. Chem. Soc. Rev. 2013, 42, 8032—8048. (16) Sanchez-Gonza ́ lez, J.; Tabernero, M. J.; Bermejo, A. M.; B ́ ermejo-Barrera, P.; Moreda-Piñeiro, A. Talanta 2016, 147, 641−649. (17) Tang, S.; Chia, G. H.; Chang, Y.; Lee, H. K. Anal. Chem. 2014, 86, 11070−11076. (18) Jia, Y.; Yu, H.; Wu, L.; Hou, X.; Yang, L.; Zheng, C. Anal. Chem. 2015, 87, 5866−5871 (19) Wang, H.; Cocovi-Solberg. D. J.; Hu, B.; Miro, M. Anal. Chem. 2017, 89, 12541-12549. (20) Ashley, J.; Wu, K.; Hansen, M. F.; Schmidt, M. S.; Boisen, A.; Sun, Y. Anal. Chem.2017, 89, 11484-11490. (21) Naous, M.; Garcia-Gomez, D.; Lopez-Jimenez, F. J.; Bouanani, F.; Lunar, M. L.; Rubio, S. Anal. Chem. 2017, 89, 1353-1361. (22) Dowlut, M.; Hall. D. G. J. Am. Chem. Soc. 2006, 128, 4226-4227



(23) Li, H.; Wang, H.; Liu, Y.; Liu, Z. Chem. Commun. 2012, 48, 4115-4117 (24) Lü, C.; Li. H.; Wang. H.; Liu, Z. Anal. Chem. 2013, 85, 23612369

Glaser, C. A.; Honnorat, J.; Hoftberger, R.; Iizuka, T.; Irani, S. R.; Lancaster, E.; Leypoldt, F.; Pruss, H.; Rae-Grant, A.; Reindl, M.; Rosenfeld, M. R.; Rostasy, K.; Saiz, A.; Venkatesan, A.; Vincent, A.; Wandinger, K. P.; Waters, P. Dalmau, J. Lancet Neurol. 2016, 15, 391

(25) Bie, Z.; Chen, Y.; Li, H.; Wu, R.; Liu, Z. Anal. Chim. Acta 2014, 834, 1-8

(34) Jiang, Y.; Zhao, H.; Lin, Y.; Zhu, N.; Ma, Y.; Mao, L. Angew. Chem. Int. Ed. 2010, 49, 4800 – 4804.

(26) Wang. H.; Bie, Z.; Lü, C.; Liu, Z. Chem. Sci. 2013, 4, 4298-4303

(35) Monopoli, M. P.; Walczyk, D.; Campbell, A.; Elia, G.; Lynch, I.; Bombelli, F. B.; Dawson, K. A. J. Am. Chem. Soc. 2011, 133, 25252534.

(27) Li, T.; Yu, Z.; Zhang, L.; Wang, C.; Deng, S.; Huo, X.; Tian, X.; Zhang, B.; Ma, X. Anal. Chim. Acta 2017, 988, 58-65 (28) Zhdankin, V. V.; Persichini, P. J., III; Zhang, L.; Fix, S.; Kiprof, P. Tetrahedron Lett. 1999, 40, 6705-6708. (29) Davis, A. P.; Wareham, R. S. Angew. Chem., Int. Ed. 1999, 38, 2978–2996. (30) Kubik. S. Angew. Chem., Int. Ed. 2009, 48, 1722–1725. (31) Lorand, J. P.; Edwards, J. O. J. Org. Chem. 1959, 24, 769–774.

Page 8 of 9

(36) Treuel, L.; Brandholt, S.; Maffre, P.; Wiegele, S.; Shang, L.; Nienhaus, G. U. ACS Nano 2014, 8, 503-513 (37) Wang, L.; Li, J.; Pan, J.; Jiang, X.; Ji, Y.; Li, Y.; Qu, Y. Zhao, Y.; Wu, X.; Chen, C. J. Am. Chem. Soc. 2013, 135, 17359-17368. (38) Storhoff, J. J.; Lazarides, A. A.; Mirkin, C. A.; Letsinger, R. L.; Mucic, R. C. ; Schatz, G. C. J. Am. Chem. Soc. 2000, 122, 4640 – 4650; (39) Liu, J.; Lu, Y. J. Am. Chem. Soc. 2003, 125, 6642 – 6643.

(32) Narimatsu, H. Expert Rev. Proteomics, 2015, 12, 683 (33) Graus, F.; Titulaer, M. J.; Balu, R.; Benseler, S.; Bien, C. G.; Cellucci, T.; Cortese, I.; Dale, R. C.; Gelfand, J. M.; Geschwind, M.;


Page 9 of 9 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

Analytical Chemistry For TOC only


9

Novel Magnetic Microprobe with Benzoboroxole Modified Flexible

Recommend Documents