Ultrasensitive Electrochemical Detection of Glycoprotein Based onBoronate Affinity Sandwich Assay and Signal Amplification withFunctionalized SiO2@Au NanocompositesMin 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

*S Supporting Information

ABSTRACT: Herein we propose a multiple signal amplificationstrategy designed for ultrasensitive electrochemical detection ofglycoproteins. This approach introduces a new type of boronate-affinity sandwich assay (BASA), which was fabricated by using goldnanoparticles combined with reduced graphene oxide (AuNPs-GO)to modify sensing surface for accelerating electron transfer, thecomposite of molecularly imprinted polymer (MIP) including 4-vinylphenylboronic acid (VPBA) for specific capturing glycopro-teins, and SiO2 nanoparticles carried gold nanoparticles (SiO2@Au)labeled with 6-ferrocenylhexanethiol (FcHT) and 4-mercaptophe-nylboronic acid (MPBA) (SiO2@Au/FcHT/MPBA) as tracing tagfor binding glycoprotein and generating electrochemical signal. As asandwich-type sensing, the SiO2@Au/FcHT/MPBA was capturedby glycoprotein on the surface of imprinting film for further electrochemical detection in 0.1 M PBS (pH 7.4). Using horseradishperoxidase (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 amplificationapproach based on boronate-affinity molecularly imprinted polymer and SiO2@Au/FcHT/MPBA, exhibiting greatly enhancedsensitivity for glycoprotein detection. Furthermore, the newly constructed BASA based glycoprotein sensor demonstrated HRPdetection in real sample, such as human serum, suggesting its promising prospects in clinical diagnostics.

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

1. INTRODUCTION

Glycoproteins, widely distributed in the cytosolic proteins andmembrane-bound proteins, participate in various biologicalprocesses such as molecular recognition, cell signaling, cellularcomponent, immune response, enzymatic reaction, etc.1,2

Protein glycosylation is the most common post-translationalmodification in higher organisms including human, and thefunctionality of glycoproteins is associated with manysignificant metabolic progresses of life. Moreover, glycoproteinsare vital marking composition in various diseases and clinicaldiagnostics.3 Until now, quantities of glycoproteins have beenused as conventional disease biomarkers for early detection ofpathological processes, while increasing glycoproteins havebeen proposed as potential biomarkers.4,5 Those clinicaldiagnostics approaches using glycoproteins as disease bio-markers are novel, interesting, and promising for the detectionof diseases such as diabetes, rheumatoid arthritis, cardiovasculardisease, 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 specificitybinding of antibodies.6 However, these biomolecules are related

to obvious disadvantages, such as poor stability, difficultpurification, and high cost. Therefore, developing efficientand reliable approaches for sensitive and selective glycoproteindetection in clinical diagnostics is of great importance.Considering the importance of selectivity and sensitivity

toward the efficiency of detection approach, molecularimprinting, an important technology to create economicaland stable synthetic receptors with antibody-like bindingproperties or enzyme-like catalytic activities, can play significantrole.7−9 Because of the ease of preparation, stability at harshconditions, and selective template recognition, MIPs offernumerous applications such as separation,10 molecularsensing,11 and catalysis.12 However, molecular imprinting ofbiological macromolecules, especially proteins, is challengingbecause of conformational changes in proteins under harshimprinting conditions and difficulty in removing target proteinfrom the imprinted cavities. To overcome these issues,

Received: January 10, 2017Accepted: April 10, 2017Published: April 10, 2017

Research Article

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© 2017 American Chemical Society 13855 DOI: 10.1021/acsami.7b00444ACS Appl. Mater. Interfaces 2017, 9, 13855−13864

researchers have proposed a variety of approaches includingsurface imprinting,13 hierarchical imprinting,14 microcontactimprinting,15 epitope imprinting,16 and nanotechnology-basedimprinting,17 Pickering emulsion imprinting and so on.18−20

Boronate affinity-based molecular imprinting is a brand new,facile, and reliable approach for imprinting glycopro-teins.6,13,21−23,27,52 Boronate affinity has attracted tremendousattention due to its unique reversible covalent binding withcompounds containing cis-diol such as sugars, small biologicalmolecules, ribonucleotides, and glycoproteins.21 The boronate-affinity MIP exhibits several excellent features including pHbased tunable affinity (e.g., pH ≥5 binding, whereas pH <3release), 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 constructingantibody-free, enzyme-free immunoassay, an appealing alter-native of antibodies for immunoassay.23−25 Liu’s groupdeveloped a variety of boronate affinity-based MIP grafted ondifferent substrates via oriented surface imprinting and furtheremployed them to fabricate highly selective and sensitiveglycoproteins assay, and they first termed the BASA.5,21−23,26,27

Chen’s group combined surface imprinting with nanomaterialsto construct nanostructured imprinted materials that exhibiteda high surface-to-volume ratio, convenient template removal,and large binding capacity required for glycoproteinsanalysis.28−30 Ai’s group prepared boronate affinity-basedsurface imprinted polymer using electrochemical polymer-ization combined with nanomaterials for signal amplificationand obtained a highly sensitive glycoprotein sensor.31

In this work, we first proposed a new ultrasensitiveelectrochemical assay for glycoprotein detection based ondual functionalized SiO2@AuNPs and boronate affinity-basedoriented surface imprinting. Scheme 1 illustrates the principleof the proposed approach. As shown in Scheme 1A, the signalamplification tracing tag SiO2@Au/FcHT/MPBA was preparedby an appropriate in situ synthesis of AuNPs on SiO2nanoparticle surface and subsequent functionalization ofnanoparticles by FcHT and MPBA, which were used forgenerating electrochemical signal and binding glycoprotein,respectively. Scheme 1B illustrates the preparation of boronateaffinity-based oriented surface imprinting film. The AuNPs-GO

was immobilized on the glassy carbon electrode (GCE) surfaceusing chitosan, and MPBA on AuNPs-GO was used to capturetarget glycoprotein, and then fabricate the MIP film. Scheme1C illustrates the fabrication of MIP-target-SiO2@Au sandwichfabrication and the electrochemical detection process ofglycoproteins. Using HRP as a representative target, weinvestigated the effects of imprinting conditions and charac-terized the properties and performance of the prepared MIP.This approach further combined the AuNPs-GO acceleratedelectron transfer with the glycoprotein binding (with boronicacid, imprinting cavities, and SiO2@Au/FcHT/MPBA) togenerate a multiple signal amplification strategy for ultra-sensitive electrochemical sensing approach. This novel BASA-based glycoprotein sensor exhibited excellent selectivity,sensitivity, stability, and reusability, showing potential applica-tions in clinical diagnostics.

2. EXPERIMENTAL SECTION2.1. 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 azobis(isobutyronitrile)(AIBN) were purchased from Sinopharm Chemical Reagent Co.Ltd. (Shanghai, China). Horseradish peroxidase, thrombin (TB), 3-aminopropyltrimethoxysilane (APTMS), ethylene glycol dimethacry-late (EGDMA), 4-mercaptophenylboronic acid, 4-vinylphenylboronicacid, and 6-ferrocenylhexanethiol were purchased from Sigma-Aldrich(USA). Bovine serum albumin (BSA) was obtained from SangonBiotechnology Inc. (Shanghai, China). Human α-fetoprotein (AFP)and carcinoembryonic antigen (CEA) were purchased from ShuangliuZhenglong Biochem Laboratory (Chengdu, China). Other chemicalsused were of analytical grade and were purchased from SinopharmGroup Chemical Regent Co., Ltd. (Shanghai, China). MAA wasdistilled under reduced pressure to remove the polymerizationinhibitor. Human blood serum samples were collected from a localpathology laboratory and stored at 4 °C. All other solvents were ofanalytical reagent grade and used as received. Phosphate buffersolution (PBS, 0.1 M, pH 7.4), prepared with ultrapure water from aMillipore Milli-Q system, was employed as the supporting electrolyte.

2.2. Instruments. Fourier transform infrared (FT-IR) spectro-scopic characterization were performed using NEXUS 670 FT-IRspectrometer (Nicolet, USA). Surface morphological images and

Scheme 1. Step-by-Step Illustration of the Proposed Approach: (A) Preparation of SiO2@Au/FcHT/MPBA;(B) PreparationProcess of the Boronate Affinity-Based Glycoprotein-Imprinted Electrode; (C) Fabrication of Boronate Affinity Sandwich Assayand the Electrochemical Detection of Glycoproteins Procedure

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energy-dispersive spectroscopy (EDS) spectra were obtained by aHITACHI 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 IIIaMultiMode AFM microscope in tapping mode. Electrochemicalmeasurements were carried out using a CHI 820b electrochemicalworkstation (CH Instruments Co., Shanghai, China). A conventionalthree-electrode system was used with a modified or unmodified glasscarbon electrode (3 mm in diameter) as working electrode, whereasAg/AgCl electrode with saturated KCl solution as reference electrodeand platinum wire as auxiliary electrode in a 10 mL of glass cellcontaining electrolytic solution.2.3. Preparation of Tracing Tag SiO2@Au/FcHT/MPBA.

Scheme 1A schematically describes the preparation of tracing tagSiO2@Au/FcHT/MPBA. The Au NPs and SiO2 nanoparticles wereprepared according to a previous literature with some modifica-tions,32,33 and the details are given in the Supporting Information. Thepreshrunk SiO2 nanaoparticles were aminated prior to Au-coating.After dispersing 0.05 g of SiO2 nanoparticles in 50 mL of absoluteethanol by ultrasonication, 0.05 mL of APTMS was added, and themixture was then vigorously stirred under N2(g) overnight at roomtemperature. The aminated particles were purified by repeatedcentrifugation and resuspension in ethanol and then twice in waterand were finally dispersed in 50 mL of ethanol. In 45 mL of the gold-seed solution, 5 mL of aminated nanoparticles were uniformlyimmersed with stirring for 10 h. The faintly pink color of the solutiondisappeared gradually as the Au nanoparticles started adsorbing ontothe surface of SiO2 NPs. The purple nanoparticles were then collectedvia centrifugation, washed by sonication in deionized water at leastthree 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 then50 mL of an Au-plating solution (0.1% w/w HAuCl4 in 0.40 mMNH2OH·HCl) was added into the flask with continuous stirring for 30min. The colloidal solution color turned a deep purple as Au shellswere chemically deposited onto the surface of the silica nanoparticles.The SiO2@Au NPs were collected by centrifugation and resuspensionrepetitively with deionized water and ethanol at least three times. Themodified nanoparticles were finally dispersed in 5.0 mL of ethanol.

The tracing tag SiO2@Au/FcHT/MPBA was prepared by mixing1.5 mL of 1.0 mg/mL SiO2@Au NPs in ethanol with 0.5 mL of 6.0mM FcHT and 2.0 mM MPBA in ethanol with shaking for 24 h undernitrogen atmosphere. The final product was rinsed with copiousamounts of hexane, ethanol, and water and finally suspended in 1.5 mLof ultrapure water.

2.4. Fabrication of the Boronate Affinity-Based MIP-Modified Electrodes. AuNPs-GO was prepared according to apreviously reported method with some modification.34,35 Thepreparation of GO is provided in the Supporting Information. Afterthe GO was dried in vacuum, 10 mg of GO was dispersed in 100 mLof aqueous solution containing 0.25 mM trisodium citrate bysonicating for 1 h. Next, 1.0 mL of 1% HAuCl4·4H2O were addedto the suspension with gentle stirring for 10 min. After cooling to 0 °C,3.0 mL of 0.1 M NaBH4 solution was slowly added to the mixture withstirring. After stirring for 2 h, the black solid was collected viacentrifugation at 10 000 rpm, washed thrice by sonication in ultrapurewater, and then dried in air overnight at 80 °C.

Prior to modification, the GCE (3 mm in diameter) wassuccessively polished to a mirror finish using 1.0 and 0.05 μm aluminaslurry. After the electrode was rinsed thoroughly with ultrapure waterand dried under nitrogen flow, 5 μL chitosan solution (1%) was firstdropped on the pretreated GCE electrode and dried in air for 1 h.Then 5 μL dispersion of AuNPs-GO composite was cast onto itssurface and dried in air. Next, the modified electrode was immersed inan ethanol solution containing 200 μM MPBA at room temperaturefor 12 h. Next, the modified electrode was washed with ethanol andwater to remove residual reagents and was dried in air. To form a thintemplate layer, the boronic acid-functionalized electrodes wereimmersed into a solution, containing 0.1 mg/mL HRP and 0.1 Mphosphate buffer (pH 8.0) for 30 min, followed by rinsing with 0.1 Mphosphate buffer (pH 8.0). The template-anchored electrodes wereimmersed into a prepolymer solution mixture containing 2.0 mMVPBA, 1.0 mM MAA, 5.0 mM AIBN and 1.25 mM EGDMA at roomtemperature for 12 h. Finally, the electrodes were rinsed with 0.1 MHCl containing 10% SDS (w/v) to remove the template. Forcomparison, nonimprinted polymer (NIP) covered electrodes wereprepared following the same processing procedure except immobiliz-ing the template onto the boronic acid-functionalized electrodes.

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 EDSspectra of SiO2@Au/FcHT/MPBA.

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2.5. Electrochemical Detection Procedure of the BoronateAffinity Sandwich Assay. HRP solutions (5 μL) of certainconcentrations containing 100 mM phosphate buffer (pH 7.4) wasadded onto each MIP-modified electrode, and each one was thenincubated for 45 min in a humidity chamber. Next, the MIP-modifiedelectrodes were washed with acetonitrile−water solution (30:70, v/v)for 5 min. Captured glycoprotein on the MIP-modified electrode wasincubated with 5 μL of SiO2@Au/FcHT/MPBA for 10 min. Theresulting electrodes were gently washed with 30:70 v/v acetonitrile-10mM phosphate buffer (pH 9.0) for 5 min, dried and then detected bythe differential pulse voltammetry (DPV) in 0.1 M PBS (pH 7.4). Thepulse amplitude, pulse period, and pulse width of DPV were set at 50mV, 0.2 s, and 50 ms, respectively.

3. RESULTS AND DISCUSSION

3.1. Preparation and Characterization of the TracingTag SiO2@Au/FcHT/MPBA. The FcHT and MPBA, self-assembled on the surface of SiO2@Au NPs via Au-thiolbonding, formed two kinds of capacities for the fabrication ofboronate affinity sandwich assay.36−38 To confirm successfulsynthesis of the SiO2@Au/FcHT/MPBA, we took SEM andTEM micrographs to characterize SiO2 NPs, SiO2@Au NPs,and SiO2@Au/FcHT/MPBA (Figure 1). As shown in Figure1A, the pure SiO2 NPs exhibited uniform size distribution andsmooth surfaces with an average diameter of about 200 nm.The AuNP-coated SiO2 NPs showed many small-sized AuNPsscattered on the surface of SiO2 NPs (Figure 1B), indicating thesuccessful formation of SiO2@Au NPs. When compared withthe functionalized SiO2@Au NPs image (Figure 1C), weobserved that the SiO2@Au/FcHT/MPBA exhibited relativelyblurred edges and partially melted AuNPs, indicating successfulmodification. Energy-dispersive spectroscopy analysis (Figure1D) of the SiO2@Au/FcHT/MPBA clearly revealed theirelemental composition Si and Au signals confirmed the SiO2@Au NPs, whereas the Fe and C signals proved the presence ofFcHT and MPBA on the Au surface. To further verifysuccessful surface modification with FcHT and MPBA, werecorded the FT-IR spectra of the nanoparticles. The FT-IRspectrum of SiO2@Au NPs and SiO2@Au/FcHT/MPBA areprovided in the Figure S1. We found asymmetric andsymmetric stretching vibrations of the υa (CH2) and υs(CH2) of FcHT at 2927 and 2855 cm−1, respectively.39 Incomparison with the FT-IR spectrum of SiO2@Au NPs, theintensities of υa (CH2) and υs (CH2) of the SiO2@Au/FcHT/MPBA FT-IR spectrum were significantly strengthened, whilethese peaks were invisible in the FT-IR spectrum of SiO2@AuNPs. The other two peaks associated with the ferrocene moietywere clearly observed at 1393 cm−1 (υ, C−C) and 819 cm−1 (δ,C−H). The variation of the intensities was directly related tothe mole fraction of the FcHT, so the changes observed clearlyindicated that the surface of SiO2@Au NPs was successfullymodified with FcHT. Furthermore, the successful functionaliza-tion of MPBA was evidenced by the presence of the B−Ostretching band 1347 cm−1 of aryl-boroxines, and the boardband at 3217 cm−1 indicated the presence of free hydroxylfunctional groups of boronic acid on the surface of SiO2@AuNPs. The FT-IR results confirmed the successful modificationof FcHT and MPBA on SiO2@Au NPs, which could bind withglycoproteins and generate electrochemical signal.The sensitivity of BASA might be affected by the ratio

between FcHT and MPBA, which in turn could affect thequantity of the electrochemical tracing tag binding with HRPand the strength of electrochemical signal. Figure 2 shows thatthe current response reaches a maximum when the ratio of

FcHT and MPBA is 3:1. However, when the ratio betweenFcHT and MPBA increases from 3:1 to 5:1, the intensity ofcurrent decreases. The reason could be the reduction of boricacid groups on SiO2@Au NPs, resulting in the relative decreasein the quantity of the electrochemical tracing tag binding withHRP. The results show that 3:1 is an appropriate ratio forFcHT and MPBA to fabricate nanoprobe SiO2@Au/FcHT/MPBA.

3.2. Preparation and Characterization of the Boro-nate-Affinity Molecularly Imprinted Film. 3.2.1. Character-ization of MIP Film. The boronate-affinity molecularlyimprinted film was characterized by FT-IR spectroscopytaken in KBr pressed pellets. Figure 3 presents the FT-IR

spectra of NIP, MIP after removal of the template, and MIPprior to washing. The peak at approximately 1722 cm−1 can beattributed to the stretching of CO, whereas the peak at 1160cm−1 can be ascribed to the characteristic of C−O stretching.40

The existence of these two peaks provides the obvious evidenceof carboxyl group in all the three polymers. However, the peakintensity of MIP prior to washing is the strongest, indicating theimprinting of glycoprotein in the polymer. Furthermore, thebands at 3429 and 1451 cm−1 in unwashed MIP can beattributed to the N−H stretching of the amino group in HRP,and as evident in Figure 3, these bands were relatively weak inthe spectra of NIP and MIP after removal of the template. TheFT-IR results confirm the successful imprinting of glycoproteintemplate in the polymer. Moreover, the MIP film exhibitedexcellent adsorption performance toward glycoprotein, as

Figure 2. Influence of the ratio of FcHT and MPBA in SiO2@Au/FcHT/MPBA on the intensity of current.

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

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evidenced by the electrochemical behavior of MIP film (SectionS2).Figure 4 shows AFM images of NIP film (Figure 4A) and

MIP film (Figure 4B) in 2 × 2 μm2 scanned area after removingthe template, which suggest a successful deposition of thepolymer film on the AuNPs-GO surface. Obviously, the surfaceof MIP film presents a relatively rough morphology with a largenumber of cavities whose dimensions are in the range of 25 ± 7nm across and 1.5 ± 0.7 nm deep (Figure 4B), whereas thesurface of NIP film is relatively smooth with a large number ofsmall cavities whose dimensions are in the range of 12 ± 6 nmacross and 0.7 ± 0.3 nm deep (Figure 4A). The morphologiesof MIP and NIP sufficiently differ from one to another and MIPsurface shows the cavities in the expected size range as ref 27

reported that the size of HRP is 2.05 nm, while NIP surfacecompletely absence those features. Hence, both FT-IR resultsand AFM images strongly corroborate successful synthesis ofMIP film.

3.2.2. Fabrication of the MIP Film. We investigated theelements of boronate affinity imprinting film, composed of the4-mercaptophenylboronic acid modified electrode surface andMIP film prepared from two polymerizable monomers MAAand MPBA,41−43 using HRP as a representative target anddemonstrated adsorption behavior in terms of electrochemicaldetection of the target protein.In boronate affinity-based surface imprinting, the perform-

ance of imprinted polymer is significantly influenced bythickness and chemical properties of the imprinting layer. To

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

Figure 5. (A) Current intensity of the boronate affinity sandwich assay for HRP (1 μg/mL) with different concentration of MPBA modified on thesurface of electrode. (B) Effect of monomers’ molar ratio on the response current of the different modified electrodes.

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obtain excellent binding properties of imprinted cavities towardtarget glycoprotein, imprinting layer should be of appropriatethickness and hydrophilic, thus preventing nonspecificadsorption toward proteins. Unlike the previous approachesof boronate affinity-based surface imprinting where VPBA hasbeen used as the only monomer, our study has used self-copolymerization of VPBA and MAA in water to form theimprinting layer. There are two reasons for adding MAA in thisapproach. First, the imprinting layer formed by self-polymer-ization of VPBA alone exhibits relatively poor hydrophilicity,while the presence of MAA as a comonomer significantlyimproves hydrophilicity by reducing nonspecific adsorption.Second, the carboxyl group on MAA can interact with theamino acid residual of proteins via hydrogen bonding. Thus, the

presence of methacrylic acid can improve the affinity ofimprinted cavities toward target glycoprotein.One of the important factors influencing the adsorption of

HRP onto the MIP surface and in turn the current response, isthe concentration of MPBA used for the modification of thesurface of electrode. Figure 5A clearly shows that the modifiedelectrode with MPBA works better than unmodified electrode.The MIP layer exhibits increased HRP adsorption as the MPBAconcentration increases from 10 to 200 μM and then graduallydecreases. However, the excess MPBA might be preventingelectron transfer, resulting the decrease in HRP adsorptionbeyond 200 μM of MPBA.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

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 withMIP.

Figure 7. (A) DPV responses of (a) AuNPs-GO/GCE, (c) NIP-AuNPs-GO/GCE, and (d) MIP-AuNPs-GO/GCE in 0.1 mM PBS (pH 7.4) afterincubating in 100 ng/mL HRP solution for 45 min. (b) MIP-AuNPs-GO/GCE was processed similarly except for incubating in blank HRP-free 0.1M PBS (pH 7.4). (B) 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) 1ng mL−1, (e) 10 ng mL−1, (f) 100 ng mL−1, (g) 1000 ng mL−1. (C) Current intensity of HRP detection by MIP and NIP as a function of logarithmof 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.

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MIP layer exhibits increased HRP adsorption with the highestadsorption at 1:2 ratio, whereas the NIP layer almost keeps thesame. Beyond the 1:2 ratio of MAA to VPBA, the currentresponse drops down, indicating decrease in HRP adsorption.This could be attributed to the reduced number of carboxylgroups available in the imprinted cavities of MIP film forbinding with the amino groups of glycoprotein, whereas theboronic acid binding to cis-diol groups in glycoproteincorresponded to saturation. We achieved the highest imprintingfactor (IF) of 5.39 at 1:2 ratio of MAA to VPBA, where IF wascalculated according to the ratio of the absorbance value ofHRP captured by MIP to that captured by NIP.3.3. Optimization of Detection Conditions. Figure 6

illustrates how the pH of incubation solution and incubationtime influence the HRP recognition ability of MIP. As shown inFigure 6A, the oxidation peak current continuously increases inthe pH range of 5−8, suggesting that more boronic acid groupsin MIP bind with HRP in alkaline aqueous solution. However,at pH >9, the response current dramatically drops, indicatingthat the binding pH value was disadvantaged for boronateaffinity-based MIP to bind with HRP.Figure 6B shows that the binding of HRP by MIP increases

with increasing the incubation time from 5 to 45 min. AlthoughNIP shows increased HRP binding in the same time range, theincrease is substantially less compared with MIP. Beyond 45min of incubation, the HRP binding by both MIP and NIPbecomes nearly constant. The imprinting factor increases as theincubation time increased from 5 to 45 min but droppedslightly beyond 55 min of incubation time. Therefore, we caninfer that the HRP adsorption effect of MIP lasts for only acertain period, and the quantity of HRP bound by MIP can becontrolled 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 responsedecreased with the increase in washing time (Figure S3). Whenthe washing time was 5 min, the imprinting factor reached ahighest point and then decreased, thus we obtained the optimalwashing 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 arepresentative glycoprotein. We studied the electrochemicaldetection of glycoproteins by SiO2@Au/FcHT/MPBA andconformed under the optimized experimental conditions. Afterforming the MIP-target-SiO2@Au sandwich with 5 mL of 100ng/mL HRP solution, the current responses of AuNPs-GO/GCE, NIP-AuNPs-GO/GCE, MIP-AuNPs-GO/GCE weremeasured using DPV, the MIP-AuNPs-GO/GCE was in-cubated in blank HRP-free 0.1 M PBS (pH 7.4) and measuredas same. Figure 7A shows the AuNPs-GO/GCE (curve a)producing a weak electrochemical signal in the presence ofHRP. The current intensity of NIP-AuNPs-GO/GCE (curve c)via forming the MIP-target-SiO2@Au sandwich is stronger thanthat of the AuNPs-GO/GCE, because the MPBA and polymermodified electrode surface adsorbed a few HRP. We found thatafter the boronate affinity imprinting sandwich process, thecurrent intensity of MIP-AuNPs-GO/GCE without rebindingof HRP (curve b) was also stronger than that of the AuNPs-GO/GCE, which might be attributed to the 3D cavities ofimprinting film adsorbing few electrochemical tracing tags. Thecurrent intensity of MIP-AuNPs-GO/GCE rebinding of HRP(curve d) increased greatly when the MIP-target-SiO2@Ausandwich was completely formed. The above results indicate

that the electrochemical tracing tag is capable of amplifying thecurrent intensity of electrochemical detection of glycoproteins.As shown in Figure 7B, the oxidation current increases with

increasing HRP concentrations ranging from 1 pg/mL to 1000ng/mL. Furthermore, Figure 7C displays the results by plottingthe current intensity against the logarithm of HRP concen-tration, yielding a response curve. The signal of MIP-AuNPs-GO/GCE increases with the logarithm of HRP concentrationwithin the range of 1 pg/mL-1000 ng/mL. Figure 7C alsodisplays the response curve of NIP-AuNPs-GO/GCE as controlunder otherwise identical detecting conditions. According tothe current response curves of MIP-AuNPs-GO/GCE andNIP-AuNPs-GO/GCE, we obtained an imprinting factor of6.35 for the MIP, thus ascertaining the stronger binding affinityof the MIP toward target glycoprotein. This high selectivity ofMIP is obtained through the recognition of target by imprintingcavities in MIP film and the boronate affinity. The MIP film hasmany imprinting cavities with bare boric acid groups, whichcould adsorb HRP in a large amount, whereas most boric acidgroups on the NIP-modified electrode are covered by NIPpolymer, which reduces the absorption of the target protein onthe surface. Moreover, the fitting linear calibration curve ofelectrochemical tracing tag against the logarithm of HRPconcentration (lgCHRP) can be described by the followingequation 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).To examine the selectivity of the BASA, we chose glucose,

BSA (nonglycoprotein), AFP (glycoprotein), CEA (glycopro-tein), and TB (glycoprotein) as interferants. The concentrationof HRP was 1.0 μg/mL as same as that of the interferingproteins and glucose, respectively. Figure 7D clearly shows thatall the interferants yield relatively higher signals than that ofblank sample, while the current response of target glycoproteinis significantly higher. These results indicate the excellentspecificity of the BASA approach. High specificity of theboronate-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 publishedearlier. Our BASA-based sensor presents a wide linear range,acceptable detection limit, and good selectivity. The wide linearrange is attributed to the boronate-affinity MIP, 3D cavities ofimprinting film, and the tracing tag SiO2@Au/FcHT/MPBA,accommodating robust binding for glycoprotein a greaternumber of recognition sites and large enough signal forelectrochemical detection. As for the detection limit, it is lowerthan that for most glycoprotein sensors, though it is not thelowest. Additionally, our MIP sensor showed high selectivity forglycoprotein because of the boronate affinity imprinting effect.Therefore, we can infer that our proposed approach hasexcellent prospects to be utilized as the electrochemical sensorof glycoproteins detection.

3.4.2. Reproducibility and Stability of the MIP Sensor. Weinvestigated the reproducibility of the BASA based glycoproteinsensor and found that the relative standard deviation (RSD) ofthe current intensity for the concentration of 1.0 ng/mL HRPwas 11.47% (n = 12). Such reproducibility is highly acceptablefor an electrochemical detection of glycoproteins. Regardingthe stability, when the sensor was stored at 4 °C, the sensorretained almost 91% of the initial signal for 1 month, presentingexcellent stability of the sensor.

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3.4.3. Application in Real Serum Samples. Furthermore, weevaluated the analytical reliability and clinical potential of theproposed method by analyzing the recovery of HRP, AFP, andCEA 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 (Table2). The obtained recoveries of HRP, AFP, and CEA from

human serum ranged from 96.6 to 106.6%, 95.7 to 103.7%, and96.0 to 100.9%, respectively. The results show acceptableaccuracy of the proposed method for the detection ofglycoproteins in clinical samples.

4. CONCLUSIONSThis study introduces a new type of boronate affinity sandwichassay for ultrasensitive electrochemical detection of glycopro-teins using a multiple signal amplification strategy based onnanotechnology. The introduction of AuNPs-GO on electrodesurface efficiently accelerated electron transfer and enhancedelectrochemical detection signal. Loading of numerous AuNPson SiO2 as nanocarriers by two-step gold seeding process of thetracing tag resulted in the desired signal amplification. Theformation of the MIP-target-SiO2@Au sandwich, as a result ofthe capturing of SiO2@Au/FcHT/MPBA by glycoprotein onthe imprinting film surface, further facilitated electrochemicaldetection of glycoprotein. The boronate affinity sandwich assay

approach utilized the benefits of boronate affinity binding andshape matching of 3D imprinted cavities for enhancing thesensitivity and selectivity for glycoprotein detection. Impor-tantly, this new BASA-based approach demonstrated highsensitive and selective detection of HRP with a detection limitreaching 0.57 pg/mL, guaranting the reliable detection ofglycoprotein disease biomarkers for clinical application.

■ ASSOCIATED CONTENT*S Supporting InformationThe Supporting Information is available free of charge on theACS Publications website at DOI: 10.1021/acsami.7b00444.

Preparation of Au nanoparticles; preparation of silicananoparticles; preparation of GO; results and discussionof electrochemical behavior of MIP film; FT-IR spectraof SiO2@Au and SiO2@Au/FcHT/MPBA; electro-chemical behavior of MIP film; washing time effect onadsorption experiments (PDF)

■ AUTHOR INFORMATIONCorresponding Authors*E-mail: fzhang@chem.ecnu.edu.cn.*E-mail: pghe@chem.ecnu.edu.cn. Tel/Fax: +86-21-54340049.ORCIDFan Zhang: 0000-0002-0821-6741NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSThis work was financially supported by the National NaturalScience Foundation of China (21405049, 21575042,21275054).

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Table 1. Comparison of the Proposed Approach with OtherGlycoprotein Sensors Published Earlier

analytical methoda linear rangedetectionlimit ref

SERS 0.1 ng/mL−100 μg/mL 0.1 ng/mL 5fluorescence 44 μg/mL−440 μg/mL 29 μg/mL 44NILET 0.18 ng/mL−11.44 ng/

mL0.16 ng/mL 45

PISA 1 pg/mL−100 ng/mL 1 pg/mL 46ICPMS 0.1 ng/mL−100 ng/mL 0.027 ng/

mL47

QCM 5 ng/mL−200 ng/mL 2 ng/mL 48photoelectrochemistry 0.5 pg/mL−10 μg/mL 0.13 pg/mL 49ECL 0.01 ng/mL−10 ng/mL 3.3 pg/mL 50electrochemistry 0.5 ng/mL−100 ng/mL 0.5 ng/mL 51electrochemistry 10 μg/mL−300 μg/mL 5 μg/mL 30electrochemistry 10 pg/mL−10 μg/mL 7.5 pg/mL 31electrochemistry 1 pg/mL−100 ng/mL 0.57 pg/mL this work

aSERS, surface-enhanced Raman scattering; NILET, near-infraredluminescence energy transfer; PISA, plasmonic immunosandwichassay; ICPMS, inductively coupled plasma mass spectrometry; QCM,quartz crystal microbalance; ECL, electrochemiluminescence.

Table 2. Assay Results for the Detection of HRP, AFP, andCEA in Clinical Human Serum Samples (n = 3) Using theProposed Method

found (ng/mL) recovery (%)

serumsample

added(ng/mL) HRP AFP CEA HRP AFP CEA

1 0 0.01 0 0.012 0.5 0.51 0.48 0.49 102.0 96.0 98.03 1.0 0.99 0.97 0.96 99.0 97.0 96.04 5.0 4.83 4.98 5.01 96.6 99.6 100.25 10.0 10.66 10.37 10.09 106.6 103.7 100.96 50.0 48.34 47.86 49.51 96.7 95.7 99.0

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