Ultrasensitive Electrochemical Detection of Glycoprotein Based on

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Ultrasensitive Electrochemical Detection of Glycoprotein Based on Boronate Affinity Sandwich Assay and Signal Amplification with Functionalized SiO@Au Nanocomposites 2

Min You, Shuai Yang, Wanxin Tang, Fan Zhang, and Pin-Gang He ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b00444 • Publication Date (Web): 10 Apr 2017 Downloaded from http://pubs.acs.org on April 11, 2017

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ACS Applied Materials & Interfaces

Ultrasensitive Electrochemical Detection of Glycoprotein Based on Boronate Affinity Sandwich Assay and Signal Amplification with Functionalized SiO2@Au Nanocomposites Min You, Shuai Yang, Wanxin Tang, Fan Zhang* and Pin-Gang He* College of Chemistry and Molecular Engineering, East China Normal University, 500 Dongchuan Road, Shanghai 200241, P. R. China.

KEYWORDS: glycoproteins, boronate-affinity sandwich assay, oriented surface imprinting, multiple signal amplification, electrochemical sensor.

Abstract: Herein we propose a multiple signal amplification strategy designed for ultrasensitive electrochemical detection of glycoproteins. This approach introduces a new type of boronate-affinity sandwich assay (BASA), which was fabricated by using gold nanoparticles combined with reduced graphene oxide (AuNPs-GO) to modify sensing surface for accelerating electron transfer, the composite of molecularly imprinted polymer (MIP) including 4-Vinylphenylboronic acid (VPBA) for specific capturing glycoproteins, and SiO2 nanoparticles carried gold nanoparticles (SiO2@Au) labeled with 6-ferrocenylhexanethiol (FcHT) and 4-mercaptophenylboronic acid (MPBA) (SiO2@Au/FcHT/MPBA) as tracing tag for binding glycoprotein and generating

electrochemical

signal.

As

a

sandwich-type

sensing,

the

SiO2@Au/FcHT/MPBA was captured by glycoprotein on the surface of imprinting film for further electrochemical detection in 0.1 M PBS (pH 7.4). Using horseradish

ACS Paragon Plus Environment


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peroxidase (HRP) as a model glycoprotein, the proposed approach exhibited a wide linear range from 1 pg/mL to 100 ng/mL, with a low detection limit of 0.57 pg/mL. To the best of our knowledge, this is first report of a multiple signal amplification approach

based

on

boronate-affinity

molecularly

imprinted

polymer

and

SiO2@Au/FcHT/MPBA, exhibiting greatly enhanced sensitivity for glycoprotein detection. Furthermore, the newly constructed BASA based glycoprotein sensor demonstrated HRP detection in real sample, such as human serum, suggesting its promising prospects in clinical diagnostics.

1. Introduction Glycoproteins, widely distributed in the cytosolic proteins and membrane-bound proteins, participate in various biological processes such as molecular recognition, cell signaling, cellular component, immune response, and enzymatic reaction, etc.1-2 Protein glycosylation is the most common post-translational modification in higher organisms including human, and the functionality of glycoproteins is associated with many significant metabolic progresses of life. Moreover, glycoproteins are vital marking composition in various diseases and clinical diagnostics.3 Until now, quantities of glycoproteins have been used as conventional disease biomarkers for early detection of pathological processes, while increasing glycoproteins have been proposed as potential biomarkers.4-5 Those clinical diagnostics approaches using glycoproteins as disease biomarkers are novel, interesting, and promising for the detection of diseases such as diabetes, rheumatoid arthritis, cardiovascular disease,


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hereditary diseases, as well as various types of cancer. The general approaches, used for detecting glycoproteins, utilize the interaction of lectins with glycans and the specificity binding of antibodies.6 However, these biomolecules are related to obvious disadvantages, such as poor stability, difficult purification, and high cost. Therefore, developing efficient and reliable approaches for sensitive and selective glycoproteins detection in clinical diagnostics is of great importance. Considering the importance of selectivity and sensitivity toward the efficiency of detection approach, molecular imprinting, an important technology to create economical and stable synthetic receptors with antibody-like binding properties or enzyme-like catalytic activities, can play significant role.7-9 Due to ease of preparation, stability at harsh condition and selective template recognition, MIP offer numerous applications such as separation,10 molecular sensing,11 and catalysis.12 However, molecular imprinting of biological macromolecules, especially proteins, is challenging because of conformational changes in proteins under harsh imprinting conditions and difficulty in removing target protein from the imprinted cavities. To overcome these issues, researchers have proposed a variety of approaches including surface imprinting,13 hierarchical imprinting,14 microcontact imprinting,15 epitope imprinting,16 nanotechnology-based imprinting,17 Pickering emulsion imprinting and so on.18-20 Boronate affinity-based molecular imprinting is a brand new, facile, and reliable approach for imprinting glycoproteins.6,13,21-23,27,52 Boronate affinity has attracted tremendous attention due to its unique reversible covalent binding with compounds



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containing cis-diol such as sugars, small biological molecules, ribonucleotides, and glycoproteins.21 The boronate-affinity MIP exhibits several excellent features including pH based tunable affinity (e.g., pH ≥ 5 binding; while pH < 3 release), high specificity, high affinity, and excellent anti-interference.6,13,22 Boronate affinity MIP, as a plastic antibody, has recently been found to be promising for constructing antibody-free, enzyme-free immunoassay, an appealing alternative of antibodies for immunoassay.23-25 Liu’s group developed a variety of boronate affinity-based MIP grafted on different substrates via oriented surface imprinting and further employed them to fabricate highly selective and sensitive glycoproteins assay, and they first termed the BASA.5,21-23,26-27 Chen’s group combined surface imprinting with nanomaterials to construct nanostructured imprinted materials that exhibited a high surface-to-volume ratio, convenient template removal, and large binding capacity required for glycoproteins analysis.28-30 Ai’s group prepared boronate affinity-based surface imprinted polymer using electrochemical polymerization combined with nanomaterials for signal amplification and obtained a highly sensitive glycoprotein sensor.31 In this work, we firstly proposed a new ultrasensitive electrochemical assay for glycoprotein detection based on dual functionalized SiO2@AuNPs and boronate affinity-based oriented surface imprinting. Scheme 1 illustrates the principle of the proposed approach. As shown in Scheme 1A, the signal amplification tracing tag SiO2@Au/FcHT/MPBA was prepared by an appropriate in situ synthesis of AuNPs on SiO2 nanoparticle surface and subsequent functionalization of nanoparticles by FcHT


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and MPBA, which were used for generating electrochemical signal and binding glycoprotein, respectively. Scheme 1B illustrates the preparation of boronate affinity-based oriented surface imprinting film. The AuNPs-GO was immobilized on the glassy carbon electrode (GCE) surface using chitosan, and MPBA on AuNPs-GO was used to capture target glycoprotein, and then fabricate the MIP film. Scheme 1C illustrates the fabrication of MIP-target-SiO2@Au sandwich fabrication and the electrochemical detection process of glycoproteins. Using HRP as a representative target, we investigated the effects of imprinting conditions and characterized the properties and performance of the prepared MIP. This approach further combined the AuNPs-GO accelerated electron transfer with the glycoprotein binding (with boronic acid, imprinting cavities, and SiO2@Au/FcHT/MPBA) to generate a multiple signal amplification strategy for ultrasensitive electrochemical sensing approach. This novel BASA-based glycoprotein sensor exhibited excellent selectivity, sensitivity, stability, and reusability, showing potential applications in clinical diagnostics.



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Scheme 1. A step-by-step illustration of the proposed approach. (A) The preparation of SiO2@Au/FcHT/MPBA. (B) The preparation process of the boronate affinity-based glycoprotein-imprinted electrode. (C) Fabrication of boronate affinity sandwich assay and the electrochemical detection of glycoproteins procedure.

2. Experimental section 2.1. Materials and methods Graphite powder (analytical grade), glucose, sodium dodecyl sulfate (SDS), chloroauric acid (HAuCl4·4H2O), trisodium citrate, sodium borohydride (NaBH4), ethanol, cyclohexane, n-hexanol, tetraethoxysilane (TEOS), Triton X-100, NH3·H2O, methacrylic acid (MAA), and azobisisobutyronitrile (AIBN) were purchased from Sinopharm Chemical Reagent Co. Ltd (Shanghai, China). Horseradish peroxidase, thrombin

(TB),

3-aminopropyltrimethoxysilane

(APTMS),

ethylene

glycol

dimethacrylate (EGDMA), 4-mercaptophenylboronic acid, 4-vinylphenylboronic acid, and 6-ferrocenylhexanethiol were purchased from Sigma-Aldrich (USA). Bovine serum albumin (BSA) was obtained from Sangon Biotechnology Inc. (Shanghai, China). Human α-fetoprotein (AFP) and carcinoembryonic antigen (CEA) were purchased from Shuangliu Zhenglong Biochem Laboratory (Chengdu, China). Other chemicals used were of analytical grade and were purchased from Sinopharm Group Chemical Regent Co., Ltd. (Shanghai, China). MAA was distilled under reduced pressure to remove the polymerization inhibitor. Human blood serum samples were collected from a local pathology laboratory and stored at 4 °C. All other solvents were


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of analytical reagent grade and used as received. Phosphate buffer solution (PBS, 0.1 M, pH = 7.4), prepared with ultrapure water from a Millipore Milli-Q system, was employed as the supporting electrolyte. 2.2. Instruments Fourier transform infrared (FT-IR) spectroscopic characterization were performed using NEXUS 670 FT-IR spectrometer (Nicolet, USA). Surface morphological images and energy-dispersive spectroscopy (EDS) spectra were obtained by a HITACHI S-4800 scanning electronic microscope (SEM, Hitachi Co. Ltd., Tokyo, Japan), Transmission electron microscopic (TEM) images were acquired using JEOL JEM-2100. Atomic force microscopy (AFM) images were captured using a Veeco Nanoscope IIIa MultiMode AFM microscope in tapping mode. Electrochemical measurements were carried out using a CHI 820b electrochemical workstation (CH Instruments Co., Shanghai, China). A conventional three-electrode system was used with a modified or unmodified glass carbon electrode (3 mm in diameter) as working electrode, while Ag/AgCl electrode with saturated KCl solution as reference electrode and platinum wire as auxiliary electrode in a 10 mL of glass cell containing electrolytic solution. 2.3. Preparation of tracing tag SiO2@Au/FcHT/MPBA Scheme

1A

schematically

describes

the

preparation

of

tracing

tag

SiO2@Au/FcHT/MPBA. The Au NPs and SiO2 nanoparticles were prepared according to a previous literature with some modifications,32-33 and the details are given in the supporting information (SI). The preshrunk SiO2 nanaoparticles were aminated prior



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to Au-coating. After dispersing 0.05 g of SiO2 nanoparticles in 50 mL of absolute ethanol by ultrasonication, 0.05 mL of APTMS was added, and the mixture was then vigorously stirred under N2(g) overnight at room temperature. The aminated particles were purified by repeated centrifugation and resuspension in ethanol and then twice in water and were finally dispersed in 50 mL of ethanol. In 45 mL of the gold-seed solution, 5 mL of aminated nanoparticles were uniformly immersed with stirring for 10 h. The faintly pink color of the solution disappeared gradually as the Au nanoparticles started adsorbing onto the surface of SiO2 NPs. The purple nanoparticles were then collected via centrifugation, washed by sonication in deionized water at least three times and finally, dispersed in 5.0 mL of water for further use. After 30 min of ultrasonication, 5.0 mL of the first gold-seeded silica cores solution was placed into 100 mL round-bottom flask, and then 50 mL of an Au-plating solution (0.1% w/w HAuCl4 in 0.40 mM NH2OH·HCl) was added into the flask with continuous stirring for 30 min. The colloidal solution color turned a deep purple as Au shells were chemically deposited onto the surface of the silica nanoparticles. The SiO2@Au NPs were collected by centrifugation and resuspension repetitively with deionized water and ethanol at least three times. The modified nanoparticles were finally dispersed in 5.0 mL of ethanol. The tracing tag SiO2@Au/FcHT/MPBA was prepared by mixing 1.5 mL of 1.0 mg/mL SiO2@Au NPs in ethanol with 0.5 mL of 6.0 mM FcHT and 2.0 mM MPBA in ethanol with shaking for 24 h under nitrogen atmosphere. The final product was


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rinsed with copious amounts of hexane, ethanol, and water and finally, suspended in 1.5 mL ultrapure water. 2.4. Fabrication of the boronate affinity-based MIP modified electrodes AuNPs-GO was prepared according to a previously reported method with some modification.34-35 The preparation of GO is provided in the SI. After the GO was dried in vacuum, 10 mg GO were dispersed in 100 mL aqueous solution containing 0.25 mM trisodium citrate by sonicating for 1 h. Next, 1.0 mL 1% HAuCl4·4H2O were added to the suspension with gentle stirring for 10 min. After cooling to 0 °C, 3.0 mL 0.1 M NaBH4 solution was slowly added to the mixture with stirring. After stirring for 2 h, the black solid was collected via centrifugation at 10,000 rpm, washed thrice by sonication in ultrapure water, and then dried in air overnight at 80 °C. Prior to modification, the GCE (3 mm in diameter) was successively polished to a mirror finish using 1.0 µm and 0.05 µm alumina slurry. After the electrode was rinsed thoroughly with ultrapure water and dried under nitrogen flow, 5 µL chitosan solution (1%) was first dropped on the pretreated GCE electrode and dried in air for 1 h. Then 5 µL dispersion of AuNPs-GO composite was cast onto its surface and dried in air. Next, the modified electrode was immersed in an ethanol solution containing 200 µM MPBA at room temperature for 12 h. Next, the modified electrode was washed with ethanol and water to remove residual reagents and was dried in air. To form a thin template layer, the boronic acid-functionalized electrodes were immersed into a solution, containing 0.1 mg/mL HRP and 0.1 M phosphate buffer (pH 8.0) for 30 min, followed by rinsing with 0.1 M phosphate buffer (pH 8.0). The template-anchored



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electrodes were immersed into a pre-polymer solution mixture containing 2.0 mM VPBA, 1.0 mM MAA, 5.0 mM AIBN and 1.25 mM EGDMA at room temperature for 12 h. Finally, the electrodes were rinsed with 0.1 M HCl containing 10% SDS (w/v) to remove the template. For comparison, non-imprinted polymer (NIP) covered electrodes were prepared following the same processing procedure except immobilizing the template onto the boronic acid-functionalized electrodes. 2.5. Electrochemical detection procedure of the boronate affinity sandwich assay HRP solutions (5 µL) of certain concentrations containing 100 mM phosphate buffer (pH 7.4) was added onto each MIP-modified electrode, and each one was then incubated for 45 min in a humidity chamber. Next, the MIP-modified electrodes were washed with acetonitrile-water solution (30:70, v/v) for 5 min. Captured glycoprotein on the MIP-modified electrode was incubated with 5 µL of SiO2@Au/FcHT/MPBA for 10 min. The resulting electrodes were gently washed with 30:70 v/v acetonitrile-10 mM phosphate buffer (pH 9.0) for 5 min, dried and then detected by the differential pulse voltammetry (DPV) in 0.1 M PBS (pH 7.4). The pulse amplitude, pulse period, and pulse width of DPV were set at 50 mV, 0.2 s, and 50 ms, respectively.

3. Results and discussion 3.1. Preparation and characterization of the tracing tag SiO2@Au/FcHT/MPBA The FcHT and MPBA, self-assembled on the surface of SiO2@Au NPs via Au-thiol bonding, formed two kinds of capacities for the fabrication of boronate affinity


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sandwich assay.36-38 To confirm successful synthesis of the SiO2@Au/FcHT/MPBA, SEM and TEM micrographs were taken to characterize SiO2 NPs, SiO2@Au NPs, and SiO2@Au/FcHT/MPBA (Figure 1). As shown in Figure 1A, the pure SiO2 NPs exhibited uniform size distribution and smooth surfaces with an average diameter of about 200 nm. The AuNPs coated SiO2 NPs showed many small-sized AuNPs scattered on the surface of SiO2 NPs (Figure 1B), indicating the successful formation of SiO2@Au NPs. When compared with the functionalized SiO2@Au NPs image (Figure 1C), we observed that the SiO2@Au/FcHT/MPBA exhibited relatively blurred edges

and

partially

melted

AuNPs,

indicating

successful

modification.

Energy-dispersive spectroscopy analysis (Figure 1D) of the SiO2@Au/FcHT/MPBA clearly revealed their elemental composition Si and Au signals confirmed the SiO2@Au NPs, while the Fe and C signals proved the presence of FcHT and MPBA on the Au surface. To further verify successful surface modification with FcHT and MPBA, we recorded FT-IR spectra of the nanoparticles. The FT-IR spectrum of SiO2@Au NPs and SiO2@Au/FcHT/MPBA are provided in the SI (Figure S1). We found asymmetric and symmetric stretching vibrations of the υa (CH2) and υs (CH2) of FcHT at 2927 cm-1 and 2855 cm-1, respectively.39 In comparison with the FT-IR spectrum of SiO2@Au NPs, the intensities of υa (CH2) and υs (CH2) of the SiO2@Au/FcHT/MPBA FT-IR spectrum were significantly strengthened, while these peaks were invisible in the FT-IR spectrum of SiO2@Au NPs. The other two peaks associated with the ferrocene moiety were clearly observed at 1393 cm-1 (υ, C-C) and 819 cm-1 (δ, C-H). The variation of the intensities was directly related to the mole



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fraction of the FcHT, so the changes observed clearly indicated that the surface of SiO2@Au NPs was successfully modified with FcHT. Furthermore, the successful functionalization of MPBA was evidenced by the presence of the B-O stretching band 1347 cm-1 of aryl-boroxines, and the board band at 3217 cm-1 indicated the presence of free hydroxyl functional groups of boronic acid on the surface of SiO2@Au NPs. The FT-IR results confirmed the successful modification of FcHT and MPBA on SiO2@Au NPs, which could bind with glycoproteins and generate electrochemical signal.

Figure 1. SEM and inset TEM images of (A) SiO2 NPs, (B) SiO2@Au NPs, and (C) SiO2@Au/FcHT/MPBA,

and

(D)

a

representative

EDS

spectra

of

SiO2@Au/FcHT/MPBA. The sensitivity of BASA might be affected by the ratio between FcHT and MPBA, which in turn could affect the quantity of the electrochemical tracing tag binding with HRP and the strength of electrochemical signal. Figure 2 shows that the current


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response reaches a maximum when the ratio of FcHT and MPBA is 3:1. However, when the ratio between FcHT and MPBA increases from 3:1 to 5:1, the intensity of current decreases. The reason could be the reduction of boric acid groups on SiO2@Au NPs reduced resulting in the relative decrease in the quantity of the electrochemical tracing tag binding with HRP. The results show that 3:1 is an appropriate

ratio

for

FcHT

and

MPBA

to

fabricate

nano-probe

SiO2@Au/FcHT/MPBA.

Figure 2. Influence of the ratio of FcHT and MPBA in SiO2@Au/FcHT/MPBA on the intensity of current. 3.2. Preparation and characterization of the boronate-affinity molecularly imprinted film 3.2.1. Characterization of MIP film The boronate-affinity molecularly imprinted film was characterized by FT-IR spectroscopy taken in KBr pressed pellets. Figure 3 presents the FT-IR spectra of NIP, MIP after removal of the template, and MIP prior to washing. The peak at



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approximately 1722 cm-1 can be attributed to the stretching of C=O, while the peak at 1160 cm-1 can be ascribed to the characteristic of C-O stretching.40 The existence of these two peaks provides the obvious evidence of carboxyl group in all the three polymers. However, the peak intensity of MIP prior to washing is the strongest, indicating the imprinting of glycoprotein in the polymer. Furthermore, the bands at 3429 cm-1 and 1451 cm-1 in unwashed MIP can be attributed to the N-H stretching of the amino group in HRP, and as evident in Figure 3, these bands were relatively weak in the spectra of NIP and MIP after removal of the template. The FT-IR results confirm the successful imprinting of glycoprotein template in the polymer. Moreover, the MIP film exhibited excellent adsorption performance towards glycoprotein, as evidenced by the electrochemical behavior of MIP film (Section S2 of SI).

Figure 3. FT-IR spectra of NIP, MIP after removal of the template HRP, and MIP prior washing.


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Figure 4 shows AFM images of NIP film (Figure 4A) and MIP film (Figure 4B) in 2 × 2 µm2 scanned area after removing the template, which suggest a successful deposition of the polymer film on the AuNPs-GO surface. Obviously, the surface of MIP film presents a relatively rough morphology with a large number of cavities whose dimensions are in the range of 25 ± 7 nm across and 1.5 ± 0.7 nm deep (Figure 4B). Whereas, the surface of NIP film is relatively smooth with a large number of small cavities whose dimensions are in the range of 12 ± 6 nm across and 0.7 ± 0.3 nm deep (Figure 4A). The morphologies of MIP and NIP sufficiently differ from one to another and MIP surface shows the cavities in the expected size range as Ref 27 reported that the size of HRP is 2.05 nm, while NIP surface completely absence those features. Hence, both FT-IR results and AFM images strongly corroborate successful synthesis of MIP film.

Figure 4. Atomic force microscopy images for the pores’ size and distribution analysis of (A) NIP film and (B) MIP film after removing the template together with a cross-section profile across the indicated line.



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3.2.2. Fabrication of the MIP film We investigated the elements of boronate affinity imprinting film, composed of the 4-mercaptophenylboronic acid modified electrode surface and MIP film prepared from two polymerizable monomers MAA and MPBA,41-43 using HRP as a representative

target

and

demonstrated

adsorption

behavior

in

terms

of

electrochemical detection of the target protein. In boronate affinity-based surface imprinting, the performance of imprinted polymer is significantly influenced by thickness and chemical properties of the imprinting layer. To obtain excellent binding properties of imprinted cavities towards target glycoprotein, imprinting layer should be of appropriate thickness and hydrophilic, thus preventing nonspecific adsorption toward proteins. Unlike the previous approaches of boronate affinity-based surface imprinting where VPBA has been used as the only monomer, our study has used self-copolymerization of VPBA and MAA in water to form the imprinting layer. There are two reasons for adding MAA in this approach. Firstly, the imprinting layer formed by self-polymerization of VPBA alone exhibits relatively poor hydrophilicity, while the presence of MAA as a co-monomer significantly improves hydrophilicity by reducing nonspecific adsorption. Secondly, the carboxyl group on MAA can interact with the amino acid residual of proteins via hydrogen-bonding. Thus, the presence of methacrylic acid can improve the affinity of imprinted cavities towards target glycoprotein. One of the important factors, influencing the adsorption of HRP onto the MIP surface and in turn the current response, is the concentration of MPBA used for the


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modification of the surface of electrode. Figure 5A clearly shows that the modified electrode with MPBA works better than unmodified electrode. The MIP layer exhibits increased HRP adsorption as the MPBA concentration increases from 10 µM to 200 µM and then gradually decreases. However, the excess MPBA might be preventing electron transfer, resulting the decrease in HRP adsorption beyond 200 µM of MPBA.

Figure 5. (A) Current intensity of the boronate affinity sandwich assay for HRP (1 µg/mL) with different concentration of MPBA modified on the surface of electrode. (B) The effect of monomers’ molar ratio on the response current of the different modified electrodes. The ratio between two monomers directly affect the quantity of glycoproteins binding with MIP film. As shown in Figure 5B, as the ratio of MAA to VPBA is in the range of 4:1-1:2, the MIP layer exhibits increased HRP adsorption with the highest adsorption at 1:2 ratio, while the NIP layer almost keeps the same. Beyond the 1:2 ratio of MAA to VPBA, the current response drops down, indicating decrease in HRP adsorption. This could be attributed to the reduced number of carboxyl groups available in the imprinted cavities of MIP film for binding with the amino groups of glycoprotein, while the boronic acid binding to cis-diol groups in glycoprotein



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corresponded to saturation. We achieved the highest imprinting factor (IF) of 5.39 at 1:2 ratio of MAA to VPBA, where IF was calculated according to the ratio of the absorbance value of HRP captured by MIP to that captured by NIP. 3.3. Optimization of detection conditions Figure 6 illustrates how the pH of incubation solution and incubation time influence the HRP recognition ability of MIP. As shown in Figure 6A, the oxidation peak current continuously increases in the pH range of 5-8, suggesting that more boronic acid groups in MIP bind with HRP in alkaline aqueous solution. However, at pH > 9, the response current dramatically drops, indicating that the binding pH value was disadvantaged for boronate affinity-based MIP to bind with HRP.

Figure 6. (A) Effects of the pH value of incubation solution for HRP binding with MIP. (B) Effects of the incubation time for HRP binding with MIP. Figure 6B shows that the binding of HRP by MIP increases with increasing the incubation time from 5 min to 45 min. Although NIP shows increased HRP binding in the same time range, the increase is substantially less compared with MIP. Beyond 45 min of incubation, the HRP binding by both MIP and NIP becomes nearly constant. The imprinting factor increases as the incubation time increased from 5 min to 45 min


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but dropped slightly beyond 55 min of incubation time. Therefore, we can infer that the HRP adsorption effect of MIP lasts for only a certain period, and the quantity of HRP bound by MIP can be controlled within this period by adjusting the incubation time. Furthermore, we investigated the effect of washing time on target protein binding and found that the current response decreased with the increase of washing time (Figure S3). When the washing time was 5 min, the imprinting factor reached a highest point and then decreased, thus we obtained the optimal washing time. 3.4. Performance of the MIP sensor 3.4.1. Analytical performance of the MIP-target-SiO2@Au sandwich sensor To evaluate the performance of the BASA, we used HRP as a representative glycoprotein. We studied the electrochemical detection of glycoproteins by SiO2@Au/FcHT/MPBA and conformed under the optimized experimental conditions. After forming the MIP-target-SiO2@Au sandwich with 5 mL of 100 ng/mL HRP solution,

the

current

responses

of

AuNPs-GO/GCE,

NIP-AuNPs-GO/GCE,

MIP-AuNPs-GO/GCE were measured using DPV, the MIP-AuNPs-GO/GCE was incubated in blank HRP-free 0.1 M PBS (pH 7.4) and measured as same. Figure 7A shows the AuNPs-GO/GCE (curve a) producing a weak electrochemical signal in the presence of HRP. The current intensity of NIP-AuNPs-GO/GCE (curve c) via forming the MIP-target-SiO2@Au sandwich is stronger than that of the AuNPs-GO/GCE, because the MPBA and polymer modified electrode surface adsorbed a few HRP. We found that after the boronate affinity imprinting sandwich process, the current



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intensity of MIP-AuNPs-GO/GCE without rebinding of HRP (curve b) was also stronger than that of the AuNPs-GO/GCE, which might be attributed to the 3D cavities of imprinting film adsorbing few electrochemical tracing tags. The current intensity of MIP-AuNPs-GO/GCE rebinding of HRP (curve d) increased greatly when the MIP-target-SiO2@Au sandwich was completely formed. The above results indicate that the electrochemical tracing tag is capable of amplifying the current intensity of electrochemical detection of glycoproteins.

Figure 7. (A) DPV responses of AuNPs-GO/GCE (a), NIP-AuNPs-GO/GCE (c), and MIP-AuNPs-GO/GCE (d) in 0.1 mM PBS (pH 7.4) after incubating in 100 ng/mL HRP solution for 45 min. While the MIP-AuNPs-GO/GCE (b) was processed similarly except for incubating in blank HRP-free 0.1 M PBS (pH 7.4). (B) The DPV curves of the BASA for detection of varying HRP concentrations: (a) 1 pg mL-1, (b) 10 pg mL-1, (c) 100 pg mL-1, (d) 1 ng mL-1, (e) 10 ng mL-1, (f) 100 ng mL-1, (g) 1000


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ng mL-1. (C) The current intensity of HRP detection by MIP and NIP as a function of logarithm of the HRP concentration. (D) Interference test with 1.0 µg/mL of glucose, BSA, AFP, CEA, TB, and HRP in 0.1 mM PBS (pH 7.4) solution. PBS (0.1 mM, pH 7.4) was used as the blank sample. As shown in Figure 7B, the oxidation current increases with increasing HRP concentrations ranging from 1 pg/mL to 1000 ng/mL. Furthermore, Fig. 7C displays the results by plotting the current intensity against the logarithm of HRP concentration, yielding a response curve. The signal of MIP-AuNPs-GO/GCE increases with the logarithm of HRP concentration within the range of 1 pg/mL-1000 ng/mL. Figure 7C also displays the response curve of NIP-AuNPs-GO/GCE as control under otherwise identical detecting conditions. According to the current response curves of MIP-AuNPs-GO/GCE and NIP-AuNPs-GO/GCE, we obtained an imprinting factor of 6.35 for the MIP, thus ascertaining the stronger binding affinity of the MIP towards target glycoprotein. This high selectivity of MIP is obtained through the recognition of target by imprinting cavities in MIP film and the boronate affinity. MIP film has many imprinting cavities with bare boric acid groups, which could adsorb HRP in a large amount. While, most of boric acid groups on NIP modified electrode are covered by NIP polymer, which reduces the absorption of target protein on the surface. Moreover, the fitting linear calibration curve of electrochemical tracing tag against the logarithm of HRP concentration (lgCHRP) can be described by the following equation of I (µA) = 0.262lgCHRP + 0.637 (ng/mL) (R = 0.996) at the HRP concentration range of 1 pg/mL-100 ng/mL and the detection limit of 0.57 pg/mL (S/N=3).



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To examine the selectivity of the BASA, we chose glucose, BSA (nonglycoprotein), AFP (glycoprotein), CEA (glycoprotein), and TB (glycoprotein) as interferants. The concentration of HRP was 1.0 µg/mL as same as that of the interfering proteins and glucose, respectively. Figure 7D clearly shows that all the interferants yield relatively higher signals than that of blank sample, while the current response of target glycoprotein is significantly higher. These results indicate the excellent specificity of the BASA approach. High specificity of the boronate-affinity MIP and 3D cavities of imprinting film, further reinforced by the dual functionalized SiO2@Au NPs, contributes to the success of the BASA. Table 1 compares the performance of our proposed glycoprotein biosensor with other related sensors published earlier. Our BASA based sensor presents a wide linear range, acceptable detection limit, and good selectivity. The wide linear range is attributed to the boronate-affinity MIP, 3D cavities of imprinting film, and the tracing tag SiO2@Au/FcHT/MPBA, accommodating robust binding for glycoprotein — a greater number of recognition sites and large enough signal for electrochemical detection. As for the detection limit, it is lower than most of glycoprotein sensors, though it is not the lowest. Additionally, our MIP sensor showed high selectivity for glycoprotein owing to the boronate affinity imprinting effect. Therefore, we can infer that our proposed approach has excellent prospect to be utilized as the electrochemical sensor of glycoproteins detection. Table 1. Comparison of the proposed approach with other glycoprotein sensors published earlier.


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analytical methoda

linear range

detection limit

ref

SERS

0.1 ng/mL-100 µg/mL

0.1 ng/mL

5

fluorescence

44 µg/mL-440 µg/mL

29 µg/mL

44

NILET

0.18 ng/mL-11.44 ng/mL

0.16 ng/mL

45

PISA

1 pg/mL-100 ng/mL

1 pg/mL

46

ICPMS

0.1 ng/mL-100 ng/mL

0.027 ng/mL

47

QCM

5 ng/mL-200 ng/mL

2 ng/mL

48

photoelectrochemistry

0.5 pg/mL-10 µg/mL

0.13 pg/mL

49

ECL

0.01 ng/mL-10 ng/mL

3.3 pg/mL

50

electrochemistry

0.5 ng/mL-100 ng/mL

0.5 ng/mL

51

electrochemistry

10 µg/mL-300 µg/mL

5 µg/mL

30

electrochemistry

10 pg/mL-10 µg/mL

7.5 pg/mL

31

electrochemistry

1 pg/mL-100 ng/mL

0.57 pg/mL

this work

a

SERS: surface-enhanced Raman scattering; NILET: near-infrared luminescence

energy transfer; PISA: plasmonic immunosandwich assay; ICPMS: inductively coupled plasma mass spectrometry; QCM: quartz crystal microbalance; ECL: electrochemiluminescence. 3.4.2. Reproducibility and stability of the MIP Sensor. We investigated the reproducibility of the BASA based glycoprotein sensor and found that the relative standard deviation (RSD) of the current intensity for the concentration of 1.0 ng/mL HRP was 11.47% (n=12). Such reproducibility is highly acceptable for an electrochemical detection of glycoproteins. Regarding the stability,



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when the sensor was stored at 4 °C, the sensor retained almost 91% of the initial signal for 1 month, presenting excellent stability of the sensor. 3.4.3. Application in real serum samples Furthermore, we evaluated the analytical reliability and clinical potential of the proposed method by analyzing the recovery of HRP, AFP, and CEA at the concentrations of 0, 0.5, 1.0, 5.0, 10.0, and 50.0 ng/mL in the 10-fold diluted healthy human serum samples (Table 2). The obtained recoveries of HRP, AFP, and CEA from human serum ranged from 96.6% to 106.6%, 95.7% to 103.7%, and 96.0% to 100.9%, respectively. The results show acceptable accuracy of the proposed method for the detection of glycoproteins in clinical samples. Table 2. Assay results for the detection of HRP, AFP, and CEA in clinical human serum samples (n = 3) using the proposed method. serum sample 1 2 3 4 5 6

added (ng/mL) 0 0.5 1.0 5.0 10.0 50.0

found (ng/mL) HRP AFP CEA 0.01 0 0.01 0.51 0.48 0.49 0.99 0.97 0.96 4.83 4.98 5.01 10.66 10.37 10.09 48.34 47.86 49.51

recovery (%) HRP AFP 102.0 96.0 99.0 97.0 96.6 99.6 106.6 103.7 96.7 95.7

CEA 98.0 96.0 100.2 100.9 99.0

4. Conclusions This study introduces a new type of boronate affinity sandwich assay for ultrasensitive electrochemical detection of glycoproteins using a multiple signal amplification strategy based on nanotechnology. The introduction of AuNPs-GO on electrode

surface

efficiently

accelerated

electron


transfer

and

enhanced

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electrochemical detection signal. Loading of numerous AuNPs on SiO2 as nanocarriers by two-step gold seeding process of the tracing tag resulted in the desired signal amplification. The formation of the MIP-target-SiO2@Au sandwich, as a result of the capturing of SiO2@Au/FcHT/MPBA by glycoprotein on the imprinting film surface, further facilitated electrochemical detection of glycoprotein. The boronate affinity sandwich assay approach utilized the benefits of boronate affinity binding and shape matching of 3D imprinted cavities for enhancing the sensitivity and selectivity for glycoprotein detection. Importantly, this new BASA based approach demonstrated high sensitive and selective detection of HRP with a detection limit reaching 0.57 pg/mL, guaranting the reliable detection of glycoprotein disease biomarkers for clinical application.

Supporting Information Preparation of Au nanoparticles; preparation of silica nanoparticles; preparation of GO; Results and discussion of electrochemical behavior of MIP film; FT-IR spectra of SiO2@Au and SiO2@Au/FcHT/MPBA; electrochemical behavior of MIP film; washing time effect on adsorption experiments.

Author Information Corresponding authors *E-mail address: [email protected]. *E-mail address: [email protected].



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Tel/Fax: +86-21-54340049. Notes The authors declare no competing financial interest.

Acknowledgements This work was financially supported by the National Natural Science Foundation of China (No. 21405049, No. 21575042, No. 21275054).

References (1) Paleček, E.; Tkáč, J.; Bartošík, M.; Bertók, T. S.; Ostatná, V.; Paleček, J. Electrochemistry of Nonconjugated Proteins and Glycoproteins. Toward Sensors for Biomedicine and Glycomics. Chem. Rev. 2015, 115, 2045-2108. (2) Alley Jr, W. R.; Mann, B. F.; Novotny, M. V. High-Sensitivity Analytical Approaches for the Structural Characterization of Glycoproteins. Chem. Rev. 2013, 113, 2668-2732. (3) Chinen, A. B.; Guan, C. M.; Ferrer, J. R.; Barnaby, S. N.; Merkel, T. J.; Mirkin, C. A. Nanoparticle Probes for the Detection of Cancer Biomarkers, Cells, and Tissues by Fluorescence. Chem. Rev. 2015, 115, 10530-10574. (4) Zhang, W.; Liu, W.; Li, P.; Xiao, H.; Wang, H.; Tang, B. A Fluorescence Nanosensor for Glycoproteins with Activity Based on the Molecularly Imprinted Spatial Structure of the Target and Boronate Affinity. Angew. Chem., Int. Ed. 2014, 53, 12489-12493.


Page 26 of 34

Page 27 of 34

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


(5) Ye, J.; Chen, Y.; Liu, Z. A Boronate Affinity Sandwich Assay: An Appealing Alternative to Immunoassays for the Determination of Glycoproteins. Angew. Chem., Int. Ed. 2014, 53, 10386-10389. (6) Li, L.; Lu, Y.; Bie, Z.; Chen, H. Y.; Liu, Z. Photolithographic Boronate Affinity Molecular Imprinting: A General and Facile Approach for Glycoprotein Imprinting. Angew. Chem., Int. Ed. 2013, 52, 7451-7454. (7) Yoshikawa, M.; Tharpa, K.; Dima, Ş.-O. Molecularly Imprinted Membranes: Past, Present, and Future. Chem. Rev. 2016, 116, 11500-11528. (8) Schirhagl, R. Bioapplications for Molecularly Imprinted Polymers. Anal. Chem. 2013, 86, 250-261. (9) Wackerlig, J.; Schirhagl, R. Applications of Molecularly Imprinted Polymer Nanoparticles and Their Advances Toward Industrial Use: A Review. Anal. Chem. 2015, 88, 250-261. (10) Nematollahzadeh, A.; Lindemann, P.; Sun, W.; Stute, J.; Lütkemeyer, D.; Sellergren, B. Robust and Selective Nano Cavities for Protein Separation: An Interpenetrating Polymer Network Modified Hierarchically Protein Imprinted Hydrogel. J. Chromatogr. A 2014, 1345, 154-163. (11) Xu, S.; Lu, H. Ratiometric Fluorescence and Mesoporous Structure Dual Signal Amplification for Sensitive and Selective Detection of Tnt Based on MIP @ QD Fluorescence Sensors. Chem. Commun. 2015, 51, 3200-3203. (12) Guo, Y.; Wang, R.; Chi, W.; Liu, S.; Shi, H.; Guo, T. One-Step Synthesis of Reactant-Product-Dual-Template Imprinted Capsules as Phosphotriesterase Mimetic



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

Enzymes for Pesticide Elimination. RSC Adv. 2014, 4, 7881-7884. (13) Wang, S.; Ye, J.; Bie, Z.; Liu, Z. Affinity-Tunable Specific Recognition of Glycoproteins via Boronate Affinity-Based Controllable Oriented Surface Imprinting. Chem. Sci. 2014, 5, 1135-1140. (14) Yang, X.; Dong, X.; Zhang, K.; Yang, F.; Guo, Z. A Molecularly Imprinted Polymer as an Antibody Mimic with Affinity for Lysine Acetylated Peptides. J. Mater. Chem. B 2016, 4, 920-928. (15) Lee, M.-H.; Thomas, J. L.; Lai, M.-Y.; Shih, C.-P.; Lin, H.-Y. Microcontact Imprinting of Algae for Biofuel Systems: The Effects of the Polymer Concentration. Langmuir 2014, 30, 14014-14020. (16) Zhang, Y.; Deng, C.; Liu, S.; Wu, J.; Chen, Z.; Li, C.; Lu, W. Active Targeting of Tumors through Conformational Epitope Imprinting. Angew. Chem., Int. Ed. 2015, 54, 5157-5160. (17) Cai, D.; Ren, L.; Zhao, H.; Xu, C.; Zhang, L.; Yu, Y.; Wang, H.; Lan, Y.; Roberts, M. F.; Chuang, J. H. A Molecular-Imprint Nanosensor for Ultrasensitive Detection of Proteins. Nat. nanotechnol. 2010, 5, 597-601. (18) Shen, X.; Zhou, T.; Ye, L. Molecular Imprinting of Protein in Pickering Emulsion. Chem. Commun. 2012, 48, 8198-8200. (19) Chen, L.; Wang, X.; Lu, W.; Wu, X.; Li, J. Molecular Imprinting: Perspectives and Applications. Chem. Soc. Rev. 2016, 45, 2137-2211. (20) Li, D.; Chen, Y.; Liu, Z. Boronate Affinity Materials for Separation and Molecular Recognition: Structure, Properties and Applications. Chem. Soc. Rev. 2015,


Page 28 of 34

Page 29 of 34

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


44, 8097-8123. (21) Bi, X.; Liu, Z. Facile Preparation of Glycoprotein-Imprinted 96-Well Microplates for Enzyme-Linked Immunosorbent Assay by Boronate Affinity-Based Oriented Surface Imprinting. Anal. Chem. 2013, 86, 959-966. (22) Bi, X.; Liu, Z. Enzyme Activity Assay of Glycoprotein Enzymes Based on A Boronate Affinity Molecularly Imprinted 96-Well Microplate. Anal. Chem. 2014, 86, 12382-12389. (23) Wang, S.; Yin, D.; Wang, W.; Shen, X.; Zhu, J.-J.; Chen, H.-Y.; Liu, Z. Targeting and Imaging of Cancer Cells via Monosaccharide-Imprinted Fluorescent Nanoparticles. Sci. Rep. 2016, DOI: 10.1038/srep22757. (24) Shinde, S.; El-Schich, Z.; Malakpour, A.; Wan, W.; Dizeyi, N.; Mohammadi, R.; Rurack, K.; Gjörloff Wingren, A.; Sellergren, B. r. Sialic Acid-Imprinted Fluorescent Core-Shell Particles for Selective Labeling of Cell Surface Glycans. J. Am. Chem. Soc. 2015, 137, 13908-13912. (25) Wang, D.; Gan, N.; Zhang, H.; Li, T.; Qiao, L.; Cao, Y.; Su, X.; Jiang, S. Simultaneous

Electrochemical

Immunoassay

Using

Graphene-Au

Grafted

Recombinant Apoferritin-Encoded Metallic Labels as Signal Tags and Dual-Template Magnetic Molecular Imprinted Polymer as Capture Probes. Biosens. Bioelectron. 2015, 65, 78-82. (26) Liu, J.; Yin, D.; Wang, S.; Chen, H. Y.; Liu, Z. Probing Low-Copy-Number Proteins in A Single Living Cell. Angew. Chem., Int. Ed. 2016, 55, 13215-13218. (27) Bie, Z.; Chen, Y.; Ye, J.; Wang, S.; Liu, Z. Boronate-Affinity Glycan-Oriented



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

Surface Imprinting: A New Strategy to Mimic Lectins for the Recognition of An Intact Glycoprotein and Its Characteristic Fragments. Angew. Chem., Int. Ed. 2015, 54, 10211-10215. (28) Lin, Z.; Xia, Z.; Zheng, J.; Zheng, D.; Zhang, L.; Yang, H.; Chen, G. Synthesis of Uniformly Sized Molecularly Imprinted Polymer-Coated Silica Nanoparticles for Selective Recognition and Enrichment of Lysozyme. J. Mater. Chem. 2012, 22, 17914-17922. (29) Li, Y.; Bin, Q.; Lin, Z.; Chen, Y.; Yang, H.; Cai, Z.; Chen, G. Synthesis and Characterization of Vinyl-Functionalized Magnetic Nanofibers for Protein Imprinting. Chem. Commun. 2015, 51, 202-205. (30) Li, Y.; Hong, M.; Bin, Q.; Lin, Z.; Cai, Z.; Chen, G. Novel Composites of Multifunctional Fe3O4@Au Nanofibers for Highly Efficient Glycoprotein Imprinting. J. Mater. Chem. B. 2013, 1, 1044-1051. (31) Wang, X.; Dong, J.; Ming, H.; Ai, S. Sensing of Glycoprotein via A Biomimetic Sensor Based on Molecularly Imprinted Polymers and Graphene-Au Nanoparticles. Analyst 2013, 138, 1219-1225. (32) Ignacio-de Leon, P. A. A.; Zharov, I. SiO2@Au Core-Shell Nanospheres Self-Assemble to Form Colloidal Crystals That Can Be Sintered and Surface Modified to Produce pH-Controlled Membranes. Langmuir 2013, 29, 3749-3756. (33) Fan, H.; Wang, X.; Jiao, F.; Zhang, F.; Wang, Q.; He, P.; Fang, Y. Scanning Electrochemical Microscopy of DNA Hybridization on DNA Microarrays Enhanced by HRP-Modified SiO2 Nanoparticles. Anal. Chem. 2013, 85, 6511-6517.


Page 30 of 34

Page 31 of 34

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


(34) Wang, Q.; Lei, J.; Deng, S.; Zhang, L.; Ju, H. Graphene-Supported Ferric Porphyrin as A Peroxidase Mimic for Electrochemical DNA Biosensing. Chem. Commun. 2013, 49, 916-918. (35) Wu, X.; Chai, Y.; Zhang, P.; Yuan, R. An Electrochemical Biosensor for Sensitive Detection of MicroRNA-155: Combining Target Recycling with Cascade Catalysis for Signal Amplification. ACS Appl. Mater. Interfaces. 2014, 7, 713-720. (36) Qiu, J.-D.; Xiong, M.; Liang, R.-P.; Peng, H.-P.; Liu, F. Synthesis and Characterization of Ferrocene Modified Fe3O4@Au Magnetic Nanoparticles and Its Application. Biosens. Bioelectron. 2009, 24, 2649-2653. (37) Usta, D. D.; Salimi, K.; Pinar, A.; Coban, I. l.; Tekinay, T.; Tuncel, A. A Boronate Affinity-Assisted SERS Tag Equipped with a Sandwich System for Detection of Glycated Hemoglobin in the Hemolysate of Human Erythrocytes. ACS Appl. Mater. Interfaces. 2016, 8, 11934-11944. (38) Bi, X.; Li, X.; Chen, D.; Du, X. Sensitive Glycoprotein Sandwich Assays by the Synergistic Effect of In Situ Generation of Raman Probes and Plasmonic Coupling of Ag Core-Au Satellite Nanostructures. ACS Appl. Mater. Interfaces. 2016, 8, 10683-10689. (39) Liu, J.; Qu, Y.; Yang, K.; Wu, Q.; Shan, Y.; Zhang, L.; Liang, Z.; Zhang, Y. Monodisperse Boronate Polymeric Particles Synthesized by A Precipitation Polymerization Strategy: Particle Formation and Glycoprotein Response from the Standpoint of the Flory-Huggins Model. ACS Appl. Mater. Interfaces. 2014, 6, 2059-2066.



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

(40) Liu, L.; Zhong, T.; Xu, Q.; Chen, Y. Efficient Molecular Imprinting Strategy for Quantitative Targeted Proteomics of Human Transferrin Receptor in Depleted Human Serum. Anal. Chem. 2015, 87, 10910-10919. (41) Pan, X.; Chen, Y.; Zhao, P.; Li, D.; Liu, Z. Highly Efficient Solid-Phase Labeling of Saccharides within Boronic Acid Functionalized Mesoporous Silica Nanoparticles. Angew. Chem., Int. Ed. 2015, 54, 6173-6176. (42) Bi, X.; Du, X.; Jiang, J.; Huang, X. Facile and Sensitive Glucose Sandwich Assay Using in Situ-Generated Raman Reporters. Anal. Chem. 2015, 87, 2016-2021. (43) Bi, X.; Li, D.; Liu, Z. Pattern Recognition of Monosaccharides via A Virtual Lectin Array Constructed by Boronate Affinity-Based pH-Featured Encoding. Anal. Chem. 2015, 87, 4442-4447. (44) Guo, T.; Deng, Q.; Fang, G.; Yun, Y.; Hu, Y.; Wang, S. A Double Responsive Smart Upconversion Fluorescence Sensing Material for Glycoprotein. Biosens. Bioelectron. 2016, 85, 596-602. (45) Chen, H.; Guan, Y.; Wang, S.; Ji, Y.; Gong, M.; Wang, L. Turn-on Detection of A Cancer Marker Based on Near-Infrared Luminescence Energy Transfer from NaYF4: Yb, Tm/NaGdF4 Core-Shell Upconverting Nanoparticles to Gold Nanorods. Langmuir 2014, 30, 13085-13091. (46) Tu, X.; Muhammad, P.; Liu, J.; Ma, Y.; Wang, S.; Yin, D.; Liu, Z. Molecularly Imprinted Polymer-Based Plasmonic Immunosandwich Assay for Fast and Ultrasensitive Determination of Trace Glycoproteins in Complex Samples. Anal. Chem. 2016, 88, 12363-12370.


Page 32 of 34

Page 33 of 34

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


(47) Peng, H.; Jiao, Y.; Xiao, X.; Chen, B.; He, M.; Liu, Z.; Zhang, X.; Hu, B. Magnetic Quantitative Analysis for Multiplex Glycoprotein with Polymer-Based Elemental Tags. J. Anal. At. Spectrom. 2014, 29, 1112-1119. (48) Lu, C.-H.; Zhang, Y.; Tang, S.-F.; Fang, Z.-B.; Yang, H.-H.; Chen, X.; Chen, G.-N. Sensing HIV Related Protein Using Epitope Imprinted Hydrophilic Polymer Coated Quartz Crystal Microbalance. Biosens. Bioelectron. 2012, 31, 439-444. (49) Li, Y.-J.; Ma, M.-J.; Zhu, J.-J. Dual-Signal Amplification Strategy for Ultrasensitive Photoelectrochemical Immunosensing of α-Fetoprotein. Anal. Chem. 2012, 84, 10492-10499. (50) Cao, Y.; Yuan, R.; Chai, Y.; Mao, L.; Yang, X.; Yuan, S.; Yuan, Y.; Liao, Y. A Solid-State Electrochemiluminescence Immunosensor Based on MWCNTs-Nafion and

Ru(bpy)32+/Nano-Pt

Nanocomposites

for

Detection

of

α-Fetoprotein.

Electroanalysis 2011, 23, 1418-1426. (51) Santos, A.; Carvalho, F. C.; Roque-Barreira, M.-C.; Bueno, P. R. Impedance-Derived Electrochemical Capacitance Spectroscopy for the Evaluation of Lectin–Glycoprotein Binding Affinity. Biosens. Bioelectron. 2014, 62, 102-105. (52) Luo, J.; Huang, J.; Cong, J.; Wei, W.; Liu, X. Double Recognition and Selective Extraction of Glycoprotein Based on the Molecular Imprinted Graphene Oxide and Boronate Affinity. ACS Appl. Mater. Interfaces. 2017, 9, 7735-7744.



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Graphical Abstract


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Ultrasensitive Electrochemical Detection of Glycoprotein Based on

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