Molecularly Imprinted Photo-electrochemical Sensor for Human

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Molecularly Imprinted Photoelectrochemical Sensor for Human Epididymis Protein 4 Based on Polymerized Ionic Liquid Hydrogel and Gold Nanoparticle/ZnCdHgSe QDs Composite Film Caiyun Wang, Xiaoxue Ye, Zhengguo Wang, Tsunghsueh Wu, Yanying Wang, and Chunya Li Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b03486 • Publication Date (Web): 23 Oct 2017 Downloaded from http://pubs.acs.org on October 24, 2017

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Analytical Chemistry

Scheme 1. Schematic illustration for the HE4 imprinted sensor fabrication and the photoelectrochemical responses of AuNPs-ZnCdHgSe QDs nanocomposites. 47x26mm (300 x 300 DPI)

ACS Paragon Plus Environment


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Figure 1. Photographic and SEM images of the poly(ionic liquid) hydrogel film before (a, c) and after (b, d) swelling. 107x96mm (300 x 300 DPI)


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Figure 2. Current - time curve (A) and electrochemical impedance spectroscopy (C) of the bare glassy carbon electrode (a), the AuNPs-ZnCdHgSe QDs/GCE (b), HE4-MIP/AuNPs-ZnCdHgSe QDs/GCE before removing HE4 (c), after removing HE4 (d) and rebinding HE4 (e); Current - time curve (B) and electrochemical impedance spectroscopy (D) of the bare glassy carbon electrode (a'), the AuNPs-ZnCdHgSe QDs/GCE (b'), HE4-NIP/AuNPs-ZnCdHgSe QDs/GCE before washing (c'), after washing (d') and rebinding HE4 (e') 93x72mm (300 x 300 DPI)



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Scanning electron microscope image of the imprinted film before (a) and after (b) removing HE4. 44x16mm (300 x 300 DPI)


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Figure 4. Photocurrent response (A) and calibration curve (B) for HE4 determination at the HE4-MIP/AuNPsZnCdHgSe QDs/GCE based molecularly imprinted sensor; (C) The photocurrent responses of imprinted film electrode towards different interferents; (D) The photocurrent responses of the imprinted sensor towards 1.0 ng mL-1 of HE4 in presence of 50 ng mL-1 of Mb, HSA, AFP, L-Cys, L-His and Gly. (Error bars are standard deviation for three independent determinations.) 93x73mm (300 x 300 DPI)



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Molecularly Imprinted Photoelectrochemical Sensor for Human Epididymis Protein 4 Based on Polymerized Ionic Liquid Hydrogel and Gold Nanoparticle/ZnCdHgSe QDs Composite Film Caiyun Wanga, Xiaoxue Yea, Zhengguo Wangb, Tsunghsueh Wuc, Yanying Wanga,*, Chunya Lia,* a

Key Laboratory of Analytical Chemistry of the State Ethnic Affairs Commission, College of Chemistry and

Materials Science, South-Central University for Nationalities, Wuhan 430074, China b

Institute of Food Science and Engineering Technology, Hezhou University, Hezhou, Guangxi 542899, China

c

Department of Chemistry, University of Wisconsin-Platteville, 1 University Plaza, Platteville, WI 53818-3099,

USA

Corresponding Author *E-mail: [email protected] or [email protected].

1


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ABSTRACT A

novel

ionic

liquid,

3-{[{4-[((carbamoyl)amino)ethylmethacrylate]butyl}

((carbamoyl)amino)ethylmethacrylate]propyl}-1-ethenyl-1H-imidazol-3-ium

bromide

(CCPEimBr) functionalized with vinyl, amino and methacrylate groups, was synthesized and characterized with 1H-NMR, FTIR and HPLC-MS. CCPEimBr was adopted as the functional monomer to prepare a molecularly imprinted polymerized ionic liquid hydrogel film on a glassy carbon electrode surface for human epididymis protein 4 (HE4) sensing. Gold nanoparticles (AuNPs) and ZnCdHgSe QDs were incorporated into the imprinted film as photoelectric active materials. The photocurrent response was measured to investigate the sensing performance of the imprinted sensors towards HE4. The imprinted photoelectrochemical sensor shows excellent selectivity, sensitivity, stability and accuracy for HE4 determination. Experimental conditions including incubation time and pH value for determining HE4 were optimized in this study. The photocurrent variation (∆I) decreased with increasing HE4 concentration (cHE4) and it was linearly proportional to cHE4 varied from 25 pg mL-1 to 4.0 ng mL-1. The detection limit of the imprinted sensor for determining HE4 was estimated to be 15.4 pg mL-1 (S/N = 3). The imprinted photoelectrochemical sensor was used to determine HE4 in human serum samples accurately.

2



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INTRODUCTION In gynecological oncology, ovarian cancer is considered as a life-threatening illness with a mortality rate ranked the highest among all other cancers for females.1 Early diagnosis of ovarian cancer allows patients to be timely treated and is the critical factor to improve patient’s survival chance. Human epididymis protein 4 (HE4), is a secreted glycoprotein with a molecular weight of approximately 25 KD and an isoelectric point (IP) of 4.1. HE4 is also named WFDC2 as its structure has two whey acidic protein (WAP) domains and a “four disulfide bond core”. As a new biomarker, HE4 concentration in blood was commonly used as an index for early diagnosis of ovarian cancer.2-4 The normal concentration of HE4 for a healthy person is less than 3.75 ng mL-1 while its concentration for a patient varies with four stages of ovarian cancer: Early stage I ranging from 3.75 to 6.225 ng mL-1; stage II from 6.25 to 8.725 ng mL-1; stage III from 8.75 to 11.225 ng mL-1; stage IV from 11.25 to >21.25 ng mL-1.5 Up to date, many methods have been developed for HE4 determination including localized surface plasmon resonance (LSPR) biosensor, chemiluminescence

immunoassay,

electronic

sensor

and

electrochemical

immunoassay.6-9 Furthermore, enzyme-linked immunosorbent assay (ELISA), immuno-radiometric assay (IRMA) and enzyme immunoassay (EIA) have also been used to determine HE4.5,10 Although immunoassay exhibits high sensitivity and selectivity, some challenges such as high cost, tedious operation and use of fragile antibodies remain and are waiting to be overcome.11 Therefore, developing another novel method with fast response, high selectivity, sensitivity and accuracy is an urgent need in cancer diagnostics. Recently, photoelectrochemical (PEC) detection by combining the superior 3


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characteristics of the optical assay and the electrochemical sensing has attracted great attention. The completely separation of the detection signals (photocurrent) from the excitation signals (light) offers excellent sensing performance for PEC sensors. Past studies have made improvement in PEC technology platforms and materials.

Shen

et al. demonstrated a biomimetic photoelectrocatalytic platform by introducing metal organic frameworks (MOFs) materials to a semiconductor-based PEC system to achieve biomimetic photoelectrocatalytic conversion of carbon dioxide.12 The recent breakthrough in PEC platform is the replacement of costly conventional light sources such as xenon lamp, metal halide lamp, cathode lamp or laser with low-cost LEDs. Zhang et al. developed a multiplexed photoelectrochemical immunoassay where the signals were induced by flexible paper-based ZnO nanorod light-emitting diodes as the first attempt of using LED in PEC.13 Huang et al. developed a semiconductor/metal-complex hybrid photoelectrocatalytic interface to enhance CO2 adsorption for efficient formate production.14 Semiconductors such as CdS and ZnS quantum dots are commonly used as photoactive materials for photoelectrochemical sensing; however, their wide band gaps require UV excitation wavelength, which often leads to irreversible and deactivating effects on biomolecules. In addition, the low energy conversion efficiency and photobleaching effect also hinder the performance of PEC biosensors. Thus, the characteristics of the high-performing PEC sensors will rely heavily on the optoelectric properties of photoelectric active material.15-19 Semiconductors that can be excited by a light source with a long wavelength (especially visible or near infrared light) and a high photoelectric conversion efficiency will be considered as the desirable photoelectric active elements.20,21 ZnCdHgSe QDs were recently prepared in our lab and have 4



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demonstrated to be a good candidate for photoelectrochemical sensing as they can be excited by white light to generate photocurrent with a high photoelectric conversion efficiency.22 Molecularly imprinting technique was originally proposed by Pauling when artificial antibodies were synthesized by using antigen as template in a polymer matrix.23 Molecular imprinting is a relative simple and low-cost way to produce highly selective method towards the target molecules; thus, it has been widely adopted in sample pretreatment, chromatographic analysis, environmental trace analysis and sensors.24-31 To improve the sensitivity, Li et al. fabricated a MIP-ECL sensors based on signal amplification for selective determination of trace gibberellin A3.32 In recent years, molecular imprinting combined with PEC sensing has been paid much more attention and most applications are focused on small molecule detection. Liu et al. developed a PEC approach for selective analysis of Microcystin-LR based on molecularly imprinted surface, which was functionalized with TiO2@CNTs hybrid nanostructure (denoted as MI-TiO2@CNTs).33 Zhang et al. fabricated a novel PEC biosensor for uric acid detection based on Fe3O4@C nanoparticles and molecularly imprinted TiO2. A review paper concluded that the sensor possess effective photochemical catalysis and molecular recognition ability.34 Using CdTe quantum dots embedded in molecularly imprinted polymer, Wang et al. fabricated a novel microfluidic origami PEC sensor with high selectivity and sensitivity towards S-fenvalerate.16 Sun et al. also developed a molecularly imprinted PEC sensor for pentachlorophenolon by using microfluidic paper as a substrate.35 Thus, molecularly imprinted PEC sensors have been successfully demonstrated for small molecule detection. 5


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Molecularly imprinted PEC sensor is seldom reported for biomacromolecules because three main challenges have been identified in the biomolecule imprinted process. First, the common imprinting technique for small molecules is carried out in organic solvents. But to avoid the denaturing of biomolecules, the imprinting technique for biomolecule has to be in aqueous solution. Second, harsh conditions for initiating the polymerization can cause irreversible structural change of biomolecules, leading to poor selectivity or denature. Finally, the complete removal of target molecules from the polymer matric to create imprinted target-specific sites is difficult to achieve due to the large molecular size and complex structure of the target biomolecules. Therefore, developing a molecularly imprinted process in an aqueous environment with mild polymerization conditions suitable for biomolecules truly has a great impact in moving the molecularly imprinted sensor technology forward and it has been the focus of this study. Ionic liquids (ILs), known as salts with low melting points, commonly consist of relatively large organic cations with a high molecular mass and relatively small inorganic anions. They are also well-known for many beneficial characteristics such as thermal stability, nonvolatile, excellent conductivity and biocompatibility; thus, they have been extensively used in catalysis, green synthesis, energy harvesting and sensors.36-39 ILs can be polymerized as Poly(ionic liquid)s (PILs), which have the anionic and cationic electrolyte groups on the repeating units for good ionic conductivity, biocompatibility and structural stability of the ionic liquids.40,41 Therefore, PILs have been used in chemoresistive sensors, non-enzymatic sensors, immunosensors, fluorescence sensors, etc.42-46 PILs have also attracted great interest in molecularly imprinted sensors since earliest reported imprinted hydrogel sensors 6



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showed good affinity and high selectivity to the target molecules.47-49 Hydrogel is a polymeric network with high water absorption ability, excellent water retention and flexible matrix. It can absorb a large amount of water while maintaining its three-dimensional structure.50 Due to the high water content and soft texture similar to the biological tissue, hydrogel not only provides entrapped proteins with the required micro-water environment but also maintains their biological activity and structural integrity, thus improving the selectivity of molecularly imprinted sensor.51 Herein, a novel CCPEimBr ionic liquid was synthesized and used as a functional monomer to fabricate a molecularly imprinted hydrogel for HE4 with the following key features. First, CCPEimBr ionic liquid contains methacrylate groups, which can be easily polymerized close to room temperature. Second, amino group, carboxyl and imidazolium cation can serve as active sites to interact with HE4 by hydrogen bonding and electrostatic attraction to achieve high selectivity of the HE4 imprinted sensor. Third, polymerized ionic liquid hydrogel can provide an aqueous environment which can maintain the structure and activity of entrapped proteins and thus to achieve high performance in HE4 sensing. Finally, gold nanoparticles (AuNPs) and ZnCdHgSe QDs were incorporated into the imprinted hydrogel as cooperative photoactive elements to create an enhanced photoelectrochemical sensing platform. In this study, the experimental conditions were optimized systematically to develop a sensitive and selective method for HE4 assay. The practical application of this imprinted photoelectrochemical sensor was then evaluated by HE4 assay on clinical serum samples. EXPERIMENTAL SECTION All chemicals and apparatus are detailed in the supporting information. 7


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1-(3-bromopropyl)-3-vinylimidazolium bromine ionic liquid was prepared according to the previous report.52 Its structure was confirmed by 1H NMR shown as Figure S 1 in Supporting Information. Synthesis of CCPEimBr ionic liquid Di-tert-butyl dicarbonate (3.32 g, 0.0152 mol) was dissolved into 20 mL of CHCl3 and was slowly added dropwise to a mixed solution of 1,4-diaminobutane (7.0 g, 0.079 mol) and 45 mL of CHCl3. After being kept in an ice-bath for 7 h, the mixture was warmed to room temperature and stirred for 12 h.

The mixture was

separated by vacuum filtration and the filtrate was washed with NaCl solution (1 mol L-1) many times. The solvent in the filtrate was evaporated to produce tert-butyl-4-aminobutylcarbamate. Then, tert-butyl-4-aminobutylcarbamate (0.77 g, 0.004 mol) and 1-(3-bromopropyl)-3-vinylimidazolium bromine (1.2 g, 0.004 mol) were dissolved in anhydrous acetonitrile (60 mL) under nitrogen atmosphere, and the mixture was stirred at 55 °C for 12 h. Subsequently, the mixture was filtered and the solvent was evaporated. The residue was extracted with a mixture of water-chloroform for several times, the collected portions of aqueous layer were combined and evaporated to yield a yellow oily product. The product was re-dissolved in dichloromethane (8 mL) and was hydrolyzed by adding 2 mL of trifluoroacetic acid. After it was kept at room temperature for 0.5 h, the pH of the solution was adjusted to 10.0 by adding NaOH solution (1 mol L-1). The solvent was evaporated to yield 3-{3-[(4-aminobutyl)amino]propyl}-1-vinylimidazolium bromide ionic liquid (AAPVimBr). After that, 2-isocyanatoethyl methacrylate (1.2715 g, 0.008 mol) was added into a mixture solution of N,N-dimethylformamide (1 ml) and AAPVimBr (2.2 g, 0.007 mol) which were dissolved in dichloromethane (9 mL). 8



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After reacting at 15 °C for 3 h, the solvent was removed and the product was purified by using a neutral alumina column chromatography (Vmethanol:Vdichloromethane = 1:16) to yield CCPEimBr ionic liquid. Synthesis of the AuNPs-ZnCdHgSe QD composites ZnCdHgSe QDs were prepared according to the previous report22 and were characterized by UV-Vis, FTIR, XRD, XPS and TEM. The results are shown as Figure S 2 in Supporting Information. Gold nanoparticles were fabricated by using 1-(3-bromopropyl)-3-vinylimidazolium bromine (5 ml, 0.24 mol L-1) as the functional monomer, HAuCl4 (5 mL, 0.01 mol L-1) as the precursor for gold nanoparticles and freshly prepared NaHB4 solution (5 mL, 0.06 mol L-1) as the reductant. After NaHB4 solution was added to the gold and monomer mixture under rapid stirring, the color of the solution quickly changed from yellow to black. Then, gold nanoparticles (AuNPs) were separated with centrifugation at 8000 rpm and washed with ultrapure water. The morphological image of AuNPs was characterized with TEM, shown in Figure S 3 in Supporting Information. To make the nanocomposite, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (10 mg mL-1), N-hydroxysuccinimide (20 mg mL-1), and ZnCdHgSe QDs (1 mL) were successively added to a readily mixed solution of mercaptoacetic acid (0.01 mol L-1, 20 mL) and AuNPs (20 mg mL-1, 1 mL) to accomplish the integration of ZnCdHgSe QDs and AuNPs. After being kept at 35 °C for 4 h, AuNPs-ZnCdHgSe nanocomposites were purified by centrifugation and thorough washing. AuNPs-ZnCdHgSe nanocomposites were characterized with XPS and SEM, shown as Figure S 4 in Supporting Information. Fabrication of the HE4 imprinted photoelectrochemical sensor 9


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After being polished using alumina slurry, a glassy carbon electrode (GCE) with a diameter of 3 mm was successively treated with ultrasonication in aqueous nitric acid (1:1) solution, ethanol and water to yield a clean electrode surface. Then, 7 µL of AuNPs-ZnCdHgSe QDs composite was coated onto GCE surface and dried at 35 °C to create a modified electrode denoted as AuNPs-ZnCdHgSe QDs/GCE. HE4 solution at the concentration of 0.1 mg mL−1 was prepared by using a phosphate buffer (0.01 mol L-1, pH 7.4). Then, 50 µL of the HE4 solution, 16.8 µL of the

CCPEimBr

ionic

liquid

solution

(0.2

mol

L-1),

80

µL of

N,N'-

methylenebisacrylamide (6 mg mL-1), 80 µL of N,N,N',N'-tetramethylethylenediamine (5 %, w/w), 60 µL of ammonium persulfate (10 %, w/w) solution and 247 µL of Tris-HCl solution (0.1 mol L-1, pH 7.4) were thoroughly homogenized and used as precursor solution to fabricate imprinted sensor. Subsequently, 4 µL of the mixed solution was drop-coated onto an AuNPs-ZnCdHgSe QDs/GCE surface and the polymerization reaction was conducted at 35 °C. The imprinted hydrogel film electrode was successively washed with NaOH solution (3%, w/w), sodium dodecyl sulfate solution (3%, w/w), 0.1 mol L-1 Tris-HCl solution (pH 7.4) and ultrapure water to thoroughly remove template proteins and yield a HE4 molecularly imprinted sensor, which was denoted as HE4-MIP/AuNPs-ZnCdHgSe QDs/GCE. Non-imprinted hydrogel film electrode was fabricated with identical procedure in the absence of HE4 and was denoted as HE4-NIP/AuNPs-ZnCdHgSe QDs/GCE. The fabrication steps and photoelectrochemical sensing mechanism of the HE4 imprinted film electrode were shown in Scheme 1. HE4 rebinding and determination For rebinding the template protein to the imprinted active sites, the imprinted 10



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sensor was incubated in a HE4 solution for 12 min with gentle stirring at room temperature. Then, the imprinted sensor was successively washed with phosphate buffer

and

ultrapure

water

to

remove

nonspecific

adsorbed

HE4.

Photoelectrochemical measurements were conducted in a phosphate buffer (pH 7.0, 0.1 mol L−1) containing 0.2 mol L−1 ascorbic acid. Current-time curve was recorded and used for evaluating the sensing performance of the imprinted sensor towards HE4. RESULTS AND DISSCUSSION CCPEimBr ionic liquid was synthesized according to the schematic shown in Scheme S 1. The ionic liquid was characterized with 1H NMR (D2O) and the spectrum is shown in Figure S 5 (a). The data are consistent with the structure of the target compound and presented as following: δ 8.947 (1H, s), 7.678 (1H, s), 7.484(1H, s), 7.028 (1H, q), 5.98(2H, s), 5.688(1H, d), 5.565 (2H, s), 5.315(1H, d), 4.10 (6H, m), 3.323 (4H, m), 3.215 (2H, t), 3.05 (2H, t), 2.948 (2H, t), 2.015 (2H, m), 1.763 (6H, q), 1.375 (2H, m); 1.282 (2H, m). FTIR spectrum of the CCPEimBr ionic liquid was illustrated in Figure S 5 (b). The characteristic peak at 3361.73 cm-1 corresponds to the stretching vibration of secondary amine. The stretching vibration of C-H for -CH2and -CH3 shows at 2923.50 cm-1. The band at 1709.61 cm-1 is the stretching vibration of C=O, which is conjugated to C=C resulting a shift to a lower wavenumber. Two bands at 1634.55 cm-1 and 1545.76 cm-1 correspond to the skeleton vibration peak of C=C and C=N on the imidazole ring, respectively. The band at 1172.66 cm-1 is the asymmetric stretching vibration of -C-O-C- and is also the characteristic absorption of the ester. As shown in Figure S 5 (c), HPLC-MS spectrum indicates that the m/z of the CCPEimBr ionic liquid is 533.327 in agreement with the theoretical formula weight 11


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(533.64). All results indicate that the target ionic liquid has been successfully synthesized. CCPEimBr ionic liquid was used as the functional monomer to fabricate a HE4 imprinted hydrogel film on an AuNPs-ZnCdHgSe QD/GCE surface in aqueous phase. CCPEimBr ionic liquid containing vinyl group, amino group and imidazolium cation can provide molecularly imprinted active sites complementary to the HE4. In addition, methacrylate group can help to form a hydrogel film, which can offer an aqueous environment that will be very beneficial to maintain the structure and activity of HE4. The formation of hydrogel film on the electrode surface can be verified by comparing the morphological images of a shrunken hydrogel and a swollen hydrogel film, which are shown in Figure 1 (a) and (b), respectively. The transition of the hydrogel film from a shrunken state to a swellen state can be visually observed. A flat film was changed to have rugged surface after it has been incubated into an aqueous solution due to swelling from absorbing a large quantity of water. Scanning electron microscope was used to characterize the microcosmic change of a poly(ionic liquid) hydrogel film before and after swelling and shown as the SEM images in Fig. 1 (c) and (d) respectively. It is obvious to see that the surface of poly(ionic liquid) hydrogel dried under room temperature has many small pores before swelling (c), and the pore size increases after swelling with water and freeze drying (d). Electrochemical characterizations Amperometric current-time curve was used to investigate the changes in photoelectrochemical response of the imprinted (Figure 2 A) and the non-imprinted (Figure 2 B) polymerized ionic liquid hydrogel film electrodes during the stepwise fabrication process. The photocurrent response of the AuNPs-ZnCdHgSe QDs/GCE is 12



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shown in Figure 2 A (b) and 2 B (b'). After CCPEimBr ionic liquid was polymerized onto the AuNPs-ZnCdHgSe QDs/GCE surface, a significant decrease in photocurrent is observed in both the HE4 imprinted (Figure 2 A, c) and the nonimprinted (Figure 2 B, c') hydrogel film electrodes. Such decrease in photocurrent response is due to the pinhole-free polymer on the electrode and the poor conductivity of the ionic liquid with long carbon chain and the HE4 protein. In order to create active sites for the selective recognition of HE4, the imprinted hydrogel film electrode was thoroughly washed to remove the templates. After the removal of HE4 proteins from the hydrogel film, vacant imprinted sites were formed in the polymerized ionic liquid hydrogel film, leading to the enhanced conductivity and thus resulting an obvious increase in the photocurrent response (Figure 2 A, d). During the rebinding process, the imprinted sensor was incubated in a HE4 solution (10 ng mL-1) and then a dramatic decrease in photocurrent is observed, indicating that HE4 molecules were specifically rebound to the imprinted active sites and blocked the mass and electron transfer (Figure 2 A, e). However, in the case of the nonimprinted hydrogel film electrode, the change in photocurrent before washing (c'), after washing (d') and after incubating in HE4 solution (e') is negligible, indicating no imprinted sites were generated in the film and thus showing no apparent change in the film characteristic either in the presence or absence of HE4. The fabrication processes of the imprinted and the nonimprinted hydrogel film electrode were also characterized by electrochemical impedance spectroscopy. The Nyquist plots were shown in Figure 2 C and Figure 2 D, respectively. In a typical Nyquist plot, the diameter of the semicircle from the response corresponds to the charge transfer resistance (Rct) and can be simulated with a combination of simple 13


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circuit elements by using software (ZSimDemo). The unmodified glassy carbon electrode shows an Rct value of 131 Ω (a and a'). After AuNPs-ZnCdHgSe QDs were coated onto the GCE surface, the charge transfer resistance increased to 374.7 Ω (b and b'), manifesting that AuNPs-ZnCdHgSe QDs nanocomposites limit the electron transfer of electroactive probes between the bulk solution and electrode surface. Before (c) and after (d) formation of imprinted active sites and the rebinding of HE4 (e), the electron transfer resistance are 874.6 Ω, 589.2 Ω and 733.7 Ω, respectively. The changes in Rct values are well consistent with the photocurrent responses from the photoelectrochemical characterization. Meanwhile, it is obvious that no significant change in Rct value of K3Fe(CN)6/K4Fe(CN)6 at the non-imprinted hydrogel film electrode before (c') and after (d') being washed and after incubating with HE4 (e'). These results indicate that non-imprinted film electrode has no imprinted site to detect HE4. Morphological characterizations The morphological images of the imprinted hydrogel film before (a) and after (b) removing HE4 were characterized by scanning electron microscope, shown in Figure 3. A polymer film with a relatively compact network structure can be observed before the removal of HE4 templates. After being thoroughly washed, HE4 templates were removed from the polymer hydrogel film to buffer solution and the apparent rough film structure with porous surface in SEM image was attributed to the vacant imprinted active sites to detect HE4 (Figure 3 b). Under a certain degree of crosslinking, the polymerized ionic liquid hydrogel film possesses appropriate rigidity. Therefore, the structure of HE4 and intermolecular interaction between HE4 and the imprinted active sites were well maintained, thus promoting high sensing performance 14



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towards HE4 in selectivity, sensitivity and stability. Experimental condition optimization The incubation time in HE4 solution and the pH of HE4 solution for specific recognition

were

optimized

for

HE4

assay

by

using

the

imprinted

photoelectrochemical sensor. All data were presented in the Supporting Information as Figures S 6 a and b. The optimal conditions for HE4 determination are to incubate the imprinted sensor in a 1.0 ng mL-1 HE4 solution at pH value of 7.4 for 12 min. Analytical characteristics The analytical characteristics of the imprinted sensor were investigated by recording the photocurrent response towards HE4 at different concentrations. As shown in Figure 4 A, the photocurrent response of the imprinted sensor decreases with an increase in HE4 concentration. The photocurrent variation (∆I), which is defined as ∆I = I0 − I, displays a linear relationship to HE4 concentration in the range from 25 pg mL-1 to 4 ng mL-1 (Figure 4 B) where I0 and I are the photocurrent response of the imprinted sensor prior to (I0) and after (I) being incubated in HE4 solution. The linear regression equation can be expressed as following: ∆I (µA) = 0.681c (ng mL-1) + 0.450 (R2 = 0.993).

The detection limit was calculated from the calibration curve

and S/N=3. A HE4 sample at the concentration of 25 pg mL-1 was measured for 20 times and the standard deviation (δ) was calculated to be 0.0035. Then, the detection limit was calculated to be 15.4 pg mL-1. The comparison of the analytical characteristics of the imprinted sensor with the previous reports was listed in Table S 1 and the advantages of the imprinted sensor were also discussed. Selectivity, Stability and reproducibility

15


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The selectivity of the imprinted sensor towards HE4 was studied by using myoglobin (Mb), human serum albumin (HSA), alpha-fetoprotein (AFP), L-cysteine (L-Cys), L-histidine (L-His) and glycine (Gly) as potential interferents. The photocurrent response of the imprinted sensor, which was independently incubated in these substances at the concentration of 1.0 ng mL-1, was measured and compared. From the results shown in Figure 4 C, the photocurrent variation of HE4 is much higher than any of other tested substances, demonstrating good performance in the specific recognition of HE4. Further experiments were carried out to test the selectivity of the HE4 imprinted sensor. The photocurrent responses of the imprinted sensor towards 1.0 ng mL-1 of HE4 in the presence of 50 ng mL-1 of Mb, HSA, AFP, L-Cys, L-His and Gly are shown as Fig. 4 D. It is found that only a negligible current variation can be observed between HE4 and the mixtures (RSD < 5.0 %, n=3), demonstrating a good selectivity of the imprinted sensor. Ruggedness of our sensor was studied. Five imprinted sensors were fabricated independently and tested at the HE4 concentration of 1.0 ng mL-1. A good reproducibility for preparing the imprinted sensors with the relative standard deviation of 4.22% was obtained. Meanwhile, a parallel determination of HE4 (1.0 ng mL-1) from a single imprinted sensor was carried out to evaluate the reproducibility in measurements. The RSD for five determinations from the same sensor is 6.21 %, and the signal variation can be contributed by the leaching of some photoactive materials or deactivation of the imprinted active sites from repetitive washing of the sensor. The stability of the imprinted sensor was also examined by storing the sensor in a refrigerator at ~ 4 oC for two weeks. The photocurrent variation decreased by 6.67 %, meaning that the HE4 imprinted film electrode has good stability. The decrease in 16



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photocurrent may be attributed to the loss of moisture in the imprinted film during the storage, resulting in the structural change of some imprinted sites and leading to a loss of active binding sites. Application The reliability and applicability of the imprinted sensor were evaluated by determining HE4 in the clinical serum samples, which were obtained from Renmin Hospital of Wuhan University. The samples were diluted by phosphate buffer (0.01 mol L -1, pH 7.4) at a ratio of 1:80 (V/V) before any measurements. All clinical human serum samples were also analyzed by chemiluminescent immunoassay (CLIA) method in Renmin Hospital of Wuhan University. The results are summarized in Table S 2. The relative deviation of the test results between the imprinted sensor and CLIA varied from 3.04% to 5.70%, suggesting the comparable performance in HE4 measurement. Meanwhile, the standard addition method was also used to further confirm the accuracy of the imprinted sensor in HE4 determination. From the results obtained in Table S 3, the recoveries are in the range from 94.0% to 102.1%, indicating the imprinted sensor has good accuracy. This study indicates the imprinted sensor is very promising as a diagnostic tool for ovarian cancer. Conclusion A simple molecularly imprinted photoelectrochemical sensor for determination of HE4 as a diagnostic tool for ovarian cancer was developed based on a polymerized ionic liquid hydrogel film incorporated with AuNPs/ZnCdHgSe QDs nanocomposites, the photoactive material,

to generate photocurrent under white-light excitation in the

presence of ascorbic acid. A novel ionic liquid, CCPEimBr, was used as the functional monomer to fabricate a molecularly imprinted hydrogel film, which provide 17


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biocompatible environment for maintaining the structure and activity of HE4 proteins and improving the sensing performances towards HE4. Under the optimized experimental conditions, the developed sensor showed a linear range from 25 pg mL-1 to 4.0 ng mL-1. A detection limit of 15.4 pg mL-1 was obtained, showing a good and practical sensitivity for early detection of the biomarker for ovarian cancer. The developed molecularly imprinted photoelectrochemical sensor platform demonstrates potential applications in biosensing and also provides a new technological platform for the study of other biomarkers. ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] or [email protected]. Notes The authors declare no competing financial interest. ACKNOWLEDGMENT The authors gratefully acknowledge the financial supports from the National Natural Science Foundation of China (No. 21675175 and 21275166), the Natural Science Foundation of Hubei Province, China (No. 2015CFA092). REFERENCES (1) Parkin, D. M.; Bray, F.; Ferlay, J.; Pisani, P. CA: Cancer. J. Clin. 2005, 55, 74-108. 18



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Scheme 1. Schematic illustration for the HE4 imprinted sensor fabrication and the photoelectrochemical

responses

of

AuNPs-ZnCdHgSe

QDs

nanocomposites. Figure 1. Photographic and SEM images of the poly(ionic liquid) hydrogel film before (a, c) and after (b, d) swelling. Figure 2. Current - time curve (A) and electrochemical impedance spectroscopy (C) of the bare glassy carbon electrode (a), the AuNPs-ZnCdHgSe QDs/GCE (b), HE4-MIP/AuNPs-ZnCdHgSe QDs/GCE before removing HE4 (c), after removing HE4 (d) and rebinding HE4 (e); Current - time curve (B) and electrochemical impedance spectroscopy (D) of the bare glassy carbon electrode

(a'),

the

AuNPs-ZnCdHgSe

QDs/GCE

(b'),

HE4-NIP/AuNPs-ZnCdHgSe QDs/GCE before washing (c'), after washing (d') and rebinding HE4 (e') Figure 3. Scanning electron microscope image of the imprinted film before (a) and after (b) removing HE4. Figure 4. Photocurrent response (A) and calibration curve (B) for HE4 determination at

the

HE4-MIP/AuNPs-ZnCdHgSe

QDs/GCE

based

molecularly

imprinted sensor; (C) The photocurrent responses of imprinted film electrode towards different interferents; (D) The photocurrent responses of the imprinted sensor towards 1.0 ng mL-1 of HE4 in presence of 50 ng mL-1 of Mb, HSA, AFP, L-Cys, L-His and Gly. (Error bars are standard deviation for three independent determinations.)

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Molecularly Imprinted Photo-electrochemical Sensor for Human

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