Smart CuS Nanoparticles as Peroxidase Mimetics for the Design of

May 3, 2016 - Nowadays, label-free immunoassay has attracted extensive interest because of the advantages of no label step during the measurements, lo...
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Smart CuS Nanoparticles as Peroxidase Mimetics for the Design of Novel Label-Free Chemiluminescent Immunoassay Zhanjun Yang,* Yue Cao, Juan Li, Mimi Lu, Zhikang Jiang, and Xiaoya Hu* School of Chemistry and Chemical Engineering, Yangzhou University, Yangzhou 225002, P.R. China S Supporting Information *

ABSTRACT: In the present work, a novel label-free chemiluminescent (CL) immunoassay method was designed by employing smart CuS nanoparticles (CuSNPs) as peroxidase mimetics. The CuSNPs were synthesized through a simple coprecipitation method, and showed high catalytic activity and stability. This efficient label-free CL immunoassay could be easily achieved through a simple strategy. First, CuSNPs dispersed in chitosan were modified on the epoxyfunctionalized glass slide to form a solid CL signal interface. Streptavidin was then used to functionalize CuSNPs to capture the biotinylated antibody, further producing a sensing interface. After online incubation with antigen molecules, the formed antibody−antigen complex on the biosensing substrate could prevent the diffusion channel of CL substrate toward the signal interface, and restrained the mimic enzyme-catalyzed CL reaction, finally resulting in the decrease of CL signals of the assay system. Compared to the label-based CL immunoassay, the proposed label-free assay mode is more simple, cheap and fast. Using a model analyte alpha-fetoprotein, the label-free CL immunoassay method had a linear range of 0.1−60 ng/mL and a low detection limit of 0.07 ng/mL. Moreover, the peroxidase mimetic-based label-free CL immunoassay system showed good specificity, acceptable repeatability, and good accuracy. The study provided a promising strategy for the development of highly efficient label-free CL immunoassay system. KEYWORDS: peroxidase mimetics, CuS nanoparticles, label-free, chemiluminescence, immunoassay, tumor markers

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INTRODUCTION Since Albrecht first described the chemiluminescence (CL) from the emission of luminol in 1928,1 CL detection as a versatile analytical technique has been widely exploited in the immunoassay areas because of its very high sensitivity, wide range of linearity, simple instrument, and fitness for miniaturization.2−4 For traditional CL immunoassay method, horseradish peroxidase (HRP) probe was extensively used to catalyze luminol (or isoluminol)-H2O2 CL system to achieve highly sensitive CL detection. Unfortunately, the natural enzyme bears some serious shortcomings such as the poor stability, limited sources, temperature-sensitive property as well as easy denaturation by environment changes.5−7 Owning to the limits of the natural enzyme, a variety of peroxidase mimetics including hemin,8 hemeatin,9 hemoglobin,10 cyclodextrin,11 and porphyrin12,13 have been explored in recent years. Since Manea’s group first proposed the concept of “nanozyme”,14 these nanozymes with intrinsic peroxidase-like activity have attracted considerable attention. Various types of nanozymes such as ceria nanoparticles (NPs),15 carbon NPs,16 Pt NPs,17,18 Fe3O4 magnetic NPs,19,20 and gold NPs21,22 have been reported for the biosensing applications. As a remarkable semiconductor material, copper monosulfide (CuS) has recently attracted growing attention in many fields because of the prominent physical and chemical properties.23−26 Up to © XXXX American Chemical Society

now, CuSNPs have been used as excellent peroxidase mimetics for colorimetric studies.27−29 To the best of our knowledge, CuSNPs have been not reported as peroxidase mimetics to construct immunoassay system so far. Nowadays, label-free immunoassay has attracted extensive interest because of the advantages of no label step during the measurements, low detection cost, simple manipulation and rapid assay speed.30,31 However, the present CL immunoassay system, either competitive or “sandwich” assay, often uses signal probes to label antibody and further to measure target analyte concentration.32−36 The labeling probes on the secondary antibody will cause more complicated, timeconsuming, and high-cost operations.37,38 Moreover, the labeling process usually damages the bioactivity of antibody molecules, which will result in the unsatisfied calibration range and limit of detection.39,40 Therefore, the practical applications of this CL immunoassay mode is extremely restricted in clinical detection. In the previous work, we proposed a first nonlabel CL immunosensing method by the simultaneous immobilization of HPR and capture antibodies on an interface.41 But this label-free method requires natural enzyme probes and addiReceived: February 28, 2016 Accepted: April 25, 2016

A

DOI: 10.1021/acsami.6b02481 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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of solutions into the assay system was carried out by a multiposition valve containing one outlet and five inlets. The CL signals produced in the flow cell, which was placed on the photomultiplier (PMT), were collected through PTM operated at −600 V. An IFFM software package which ran under Windows 2003 performed the instrument control and data record. An IFFM-E luminescent analyzer produced by Remex Analytical Instrument Co. Ltd. (Xi’an, China) was used to perform the flowthrough CL measurements. The electrochemiluminescence immunoassay of serum samples was carried out by a Roche Elecsys 2010 immunoassay analyzer (from Roche Diagnostics GmbH). Transmission electron micrographs (TEM) was obtained by a Philips Tecnai12 electron microscope (Holland) with an acceleration voltage of 120 kV, and scanning electron micrographs (SEM) was obtained by a Hitachi S-4800 (Japan) scanning electron microscope with an acceleration voltage of 15 kV. X-ray photoelectron spectroscopic (XPS) spectrum was obtained with an ESCALAB 250Xi spectrometer (USA). Fourier transform infrared (FTIR) spectrum measurements were performed by a Tensor 27 spectrophotometer (Bruker Co., Germany). The static water contact angle measurements were obtained by a Rame-Hart-100 contact angle meter at 25 °C. Electrochemical impedance spectroscopy (EIS) dada were recorded in KCl solution (0.1 M) comprising 5 mM of K3[Fe(CN)6]/ K4[Fe(CN)6] with an Autolab/PGSTAT30 (The Netherlands). The amplitude of the applied sine wave potential was located at 5 mV. The impedance measurements were performed with a frequency ranging from 0.05 to 10 kHz at a 190 mV bias potential. Synthesis of CuSNPs. CuSNPs were chemically synthesized according to a coprecipitation method in liquid phase at room temperature (RT). In brief, Cu(Ac)2 and Na2S were first dissolved in water/ethanol (3:1, V/V) solution, respectively. After stirring for 10 min, the two solutions were mixed together under vigorous stirring for 30 min at RT. The pH of the solution was kept at 4.0−5.0 and then black precipitate was obtained. The precipitate was dried under a vacuum after washing with ethanol three times. Finally, black particles of CuSNPs were obtained for the next use. Preparation of the Label-Free Immunosensor. A glass slide with 2.1 cm length, 0.4 cm width, and 0.1 cm height was modified with abundant epoxy groups as the following process. It was soaked in the piranha solution (H2SO4/30% H2O2 with 7:3 v/v) for 12 h, washed carefully with distilled water, and finally dried under a nitrogen stream. The pretreated slide was dipped in 1% GPTMS toluene solution for the silylanization reaction overnight at RT.42 Afterward, the glass substrate was successfully activated with epoxy group after rinsing for three times with pure toluene and ethanol (removing physical absorption of GPTMS), and dried using a steam of nitrogen. Subsequently, 2.0 mg of CuSNPs was first dispersed in 1.0 mL of distilled water, and then CuSNPs aqueous solution was mixed with 1.0 wt % chitosan solution in a volume ratio of 1:1 with the assistance of sonication. The resulting suspension (20 μL) was dropped on the silylanized glass slide for the reaction at RT until the membrane was formed. The epoxy group on the glass slide could react with aminogroup in chitosan, leading to a firm and stable composite film. Then 50 μg/mL of streptavidin (20 μL) was dropped on the membrane of CuSNPs/chitosan for 30 min at RT, followed by storage in a refrigerator overnight at 4 °C. After being washed three times using PBST, the streptavidin/CuSNPs-chitosan composite was obtained. The capture antibody was modified on the glass substrate by dropping 1 μg/mL of biotinylated AFP antibodies (20 μL) on the streptavidin-coated surface and reacted for 3h at RT. After the antibody-loaded substrate was carefully rinsed three times using PBST, and blocked using blocking buffer at 4 °C for 12 h, the label-free immunosensor was finally obtained. The antibody-immobilized glass slide was fixed on the inner side of Teflon cover (4.0 cm length, 2.5 cm width, and 0.8 cm height). The total thickness for the modified glass slide and membrane was close to 1.1 mm. The volume of the microreactor was close to 80 μL (2.1 cm length, 0.4 cm width, and 0.09 cm height). The prepared immunosensor was kept in PBS (0.01 M, pH 7.4) at 4 °C before use.

tional support materials, which leads to the increase in the analysis cost. In addition, the HRP is easy to lose the bioactivity during the CL detection and storage processes. Consequently, it is yet highly desired to explore a low-cost and reliable labelfree approach for highly efficient CL immunoassay. In the present research, an innovative label-free CL immunosensing method was constructed by use of the biofunctional CuSNPs, which here acted as both peroxidase mimetics and immobilization support materials. The CuSNPs with high catalytic activity and stability were synthesized by a facile coprecipitation method. The designed label-free strategy was preformed based on the following process. CuSNPs were first dispersed in chitosan solution, and dropped on the epoxysilanized glass slide to form a solid CL signal interface. Then streptavidin was immobilized on the signal interface to capture the biotinylated antibody and to further form a sensing interface. After different concentration of antigens flowed into the immunoreactor for online incubation, the formed proteins complex on the sensing substrate could prevent the diffusion path of CL substrate toward the signal interface, and therefore restricted the mimic enzyme-catalyzed CL reaction. According to the linear decrease in CL intensity along with increasing amount of antigens, a CL immunoassay strategy without any label could be achieved to rapidly determine tumor markers. The present work proposed a promising idea to develop of highly efficient nonlabel CL immunoassay technique for clinical diseases diagnosis.

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EXPERIMENTAL SECTION

Materials and Reagents. An alpha-fetoprotein (named as AFP) ELISA reagent kit that contains different concentrations of standard solutions of AFP (0−500 ng/mL) and biotinylated mouse monoclonal AFP antibodies (1.0 μg/mL) was purchased from CanAg Diognostics. Electrochemiluminescent immunoassay reagent kit, as reference method for measurement of target AFP, was supplied by Roche Diagnostics GmbH (Germany). Streptavidin, 3-gycidoxypropyltrimethoxysilane named as GPTMS (98%), bovine serum albumin (named as BSA) and chitosan were bought from Sigma-Aldrich Chemical Co. (St. Louis, MO). Sodium sulfide (Na2S), cupric acetate [Cu(Ac)2] and 30% of hydrogen peroxide (H2O2) were supplied by Sinopharm Chemical Reagent Co. Ltd. (China). Luminol and p-iodophenol (PIP) were purchased from Acros (Belgium) and Alfa Aesar Ltd. (China), respectively, and their stock solutions (0.01 M) were first dissolved in 0.1 M NaOH (100 mL). The mixed stock solution of luminol and PIP was diluted in Tris-HCl buffer (0.1 M, pH 8.5) before use, and the CL substrate solution consists of 5 mM of luminol, 0.6 mM of PIP and 4.0 of mM H2O2. Coupling buffer used for the immobilization of streptavidin and antibodies was 0.01 M of phosphate buffer solution (PBS, pH 7.4). Blocking buffer was prepared by spiking 1% BSA in PBS, which was used to block the residual reactive sites. Wash buffer used to minimize nonspecific adsorption was 0.01 M of PBS (pH 7.4), comprising 0.05% Tween-20 (PBST). Clinical human serum samples used in the experiment were supplied by Jiangsu Institute of Cancer Research. All of reagents used are of analytical grade and used in the experiment as received. All water used in the label-free immunoassay was distilled water. Apparatus. The proposed flow-through CL label-free immunosensing system was presented in Figure S1. The flow device with 2.1 cm length, 0.4 cm width, and 0.09 cm height (a cell volume of 80 μL) was composed of three components including a Teflon cover with inlet and outlet (4.0 cm length, 2.5 cm width, and 0.8 cm height), a silicon slice rubber spacer with 2.0 mm thickness, and a transparent plexiglass slice with 0.5 cm thickness. All fluids were carried out using a multichannel bidirectional peristaltic pump through 0.8 mm i.d.Teflon tube and 1.0 mm i.d. silicon rubber tube, which connected all components of the immunoassay system. The transfer of different kind B

DOI: 10.1021/acsami.6b02481 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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Scheme 1. Schematic Exhibition of Fabrication Process of the Immunosensor and Label-Free CL Immunoassay Protocol

Label-Free CL Assay Protocol. A detailed description of the proposed flow-through label-free CL system is displayed in Scheme 1 and Table 1. Eighty microliters of AFP standard solutions or serum

Table 1. Details of the Proposed Label-Free CL Immunoassay Based on CuSNPs as Peroxidase Mimetics step no.

valve position

1

1

2

1

3

2

4

3

5

1

6

1

step introduce 80 μL sample into the flow device stop flow and incubation at room temperature wash the flow device with PBST at a flow rate of 1.0 mL/min introduce 80 μL CL substrate into the flow device and stop flow to collect data introduce PBS to wash flow device at 1.0 mL/min and renew the immunosensor ready for the next assay cycle

starting time (min:second) 00:00 00:30 25:30 27:30

Figure 1. Feasibility of the developed label-free CL immunosensing system based on CuSNPs as peroxidase mimetics (three systems were incubated with 0, 50, and 100 ng/mL AFP; PMT, −600; n = 5 for each point; incubation time, 25 min; CL reaction time, 300 s).

33:00 35:00

samples were first carried into the flow cell and stopped flow for incubation for 25 min at RT. Then PBST was injected to the flow device at an optimal flow rate of 1.0 mL/min to wash the immunosensor. Subsequently, the CL substrate flowed to the immune microreactor. The resulting CL signals were measured after the CuSNP-catalyzed CL reaction continued for 300 s under stop-flow conditions. It only took 35 min to finish the whole analysis process from sample introduction to signals collection.

system shows an intense CL signal in the presence of 0 ng/mL of AFP (from the mimic enzyme-catalyzed CL reaction), there are also no obvious changes of CL intensity with the increasing amount of AFP. In comparison above-mentioned systems, the anti-AFP+CuSNPs+CS system displays a intense CL signal at the concentration of 0 ng/mL AFP. At the same time, its CL intensity distinctly decreased after the system was incubated with 50 and 100 ng/mL AFP. On the basis of the above fact, it can be verified that the immunocomplex formed on the sensing interface, from the specific immune reaction of anti-AFP and AFP, can efficiently prevent the diffusion route of CL substrate toward the signal interface. Accordingly, the mimic enzymatic CL reaction is further inhibited, thereby resulting in the decrease in CL signal. Therefore, the CuSNPs-based label-free strategy is feasible for CL immunoassay of proteins. Characterizations of Peroxidase Mimetics of CuSNPs. CuSNPs in this work were simply synthesized for constructing label-free CL immunoassay method. As shown in Figure 2A, the TEM image of the as-prepared CuSNPs shows irregular particles shaped morphology. The typical XRD pattern of CuSNPs samples is shown in Figure 2B, and the synthesized product only displays the XRD peaks characteristic of CuS, suggesting that CuS with pure hexagonal phase (JCPDS card

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RESULTS AND DISCUSSION Principle of the Label-Free CL Immunosensing System. Herein, a new label-free strategy based on CuSNPs as peroxidase mimetics was developed for cheap, fast, and convenient CL immunoassay (seen in Scheme 1). To verify the principle of this label-free CL immunoassay, we investigated the changes of CL signal of different systems after the incubation with different concentrations of AFP (Figure 1). To keep the same assay conditions, the interface of three systems was coated with streptavidin, and blocked with blocking solution. The antiAFP+CS (CL substrate, luminol-H2O2−PIP) system shows a weak CL signal in the presence of AFP of 0 ng/mL, and this comes from the reaction of luminol-H2O2−PIP. In addition, its CL intensity dose not obviously change upon incubation with different concentration of AFP. Although the CuSNPs+CS C

DOI: 10.1021/acsami.6b02481 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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Figure 2. (A) TEM and (B) XRD spectra of the prepared CuSNPs, and (C) UV−vis spectra of the (a) TMB (0.50 mM)-H2O2 (100 mM) solution and (b)TMB (0.50 mM)-H2O2 (100 mM)-CuSNPs (10 μg/mL) solution, inset: photographs of the (1) TMB-H2O2 solution and (2) TMB-H2O2CuSNPs solution.

Figure 3. SEM images of (a) CuSNPs/chitosan, (b) streptavidin/CuSNPs/chitosan, and (c) biotinylated anti-AFP/streptavidin/CuSNPs/chitosan modified glass slides.

Figure 4. (A) XPS of N 1s peak and (B) FT-IR spectra of (a) CuSNPs/chitosan, (b) streptavidin/CuSNPs/chitosan, and (c) biotinylated anti-AFP/ streptavidin/CuSNPs/chitosan modified glass slides.

CuSNPs/chitosan (Figure 3b), the aggregations of the protein macromolecule could be clearly observed. After biotinylated anti-AFP was immobilized on the composite film, the SEM image of biotinylated anti-AFP/streptavidin/CuSNPs/chitosan (Figure 3c) clearly shows distinct surface morphology, suggesting the successful immobilization of anti-AFP on the CuSNPs/chitosan composite. Moreover, biotinylated AFP antibody almost covered the surface of the composite, which ensures that the formed AFP immunocomplex can efficiently prevent the diffusion route of CL substrate toward the signal interface. XPS spectra and FT-IR spectrum were further used to examine the preparation of the immunosensor (shown in Figure 4). As seem from Figure 4A, the XPS spectrum of the CuSNPs/chitosan composite has a N 1s peak at 399.9 eV (curve a), suggesting the existence of chitosan in the film. After streptavidin was coated on the CuSNPs/chitosan, N 1s peak increased clearly (curve b), verifying this successful immobilization of streptavidin on the film. The N 1s peak at 399.9 eV further greatly increased after loading biotinylated antibodies on the film, which confirms the successful modification of more protein molecules. As shown in Figure 4B, the FTIR spectrum of streptavidin-coated CuSNPs-chitosan film (curve b) has two

no. 78−0877) was successfully prepared. In addition, the peroxidase mimicking activity of resultant CuSNPs are examined using TMB and H2O2 as the peroxidase substrate (Figure 2C). The reaction process was investigated with UV− vis spectroscopy at 652 nm corresponding to oxidized TMB in the kinetic mode. The absorbance of TMB-H2O2 solution (curve a) shows no obvious change with the increasing reaction time. After CuSNPs were added into this solution, the absorbance of TMB-H2O2-CuSNP solution quickly increased and reached an equilibrium at a time of approximate 700 s. Moreover, the color of TMB-H2O2 solution changed from colorless to blue upon addition of CuSNPs (inset photographs in Figure 2C). These results demonstrated the excellent peroxidase mimicking ability of the as-prepared CuSNPs. Characterizations of the Fabricated Non-Label Immunosensor. The nonlabel CL immunosensor was prepared by the modification of the capture antibody in the CuSNPs/ chitosan composite through the biotin−streptavidin system. SEM as an effective tool was used for characterizing surface morphology of CuSNPs/chitosan film and resultant label-free immunosensor. Figure 3a displays the SEM image of the CuSNPs/chitosan film modified on epoxy-activated glass substrate. As seen From the SEM image of streptavidin/ D

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ACS Applied Materials & Interfaces absorption peaks in response to amide I and II bands at 1648 and 1532 cm−1. Furthermore, two peaks at 1656 and 1531 cm−1 were also observed from the FTIR spectrum of antibody-loaded substrate, and the absorption peak intensity greatly increased after the modification of biotinylated antibody (curve c). These further confirm the successful immobilizations of streptavidin and biotinylated anti-AFP on the CuSNPs-chitosan film. EIS measured in 0.1 M of KCl comprising 5 mM of K3[Fe(CN)6]/K4[Fe(CN)6] was used to investigate the preparation process of the label-free immunosensor. In a typical Nyqusit plot of impedance spectra, its electron transfer resistance (Rct) was equal to its semicircle diameter. As seen from Figure S2, the pretreated electrode shows a very small semicircle (curve a), whereas the Rct of the CuSNPs/chitosan coated electrode greatly increased to 254 Ω (curve b), indicating that the CuSNPs/chitosan film was modified on the electrode surface. After streptavidin was trapped in on composite film, the Rct of streptavidin/CuSNPs/chitosan/GCE increased to 376 Ω (curve c). When the biotinylated anti-AFP were immobilized on the composite film through the biotionavidin system, its Rct further increased to 465 Ω (curve d), indicating the successful immobilization of antibody in the composite structure. After the residual reactive sites of the resulting immunosensor was blocked using BSA molecules, leading to the increase of Rct value (560 Ω, curve e). Finally, the fabricated label-free immunosensor was incubated with AFP antigen, its displays the maximum Rct of 684 Ω (curve f). This result indicates that the immunocomplex layers on the interface remarkably prevent the diffusion of ferricyanide probe to the surface of the electrode, which is beneficial to the design of the CL label-free immunoassay. The contact angle measurements of the substrate were also used for characterizing the fabrication of CL immunosensor, and could be seen from Figure S3. Compared with the contact angle of the bare glass slide (58°), piranha-soaked glass substrate has a smaller contact angle of 36.4°, indicating the activation of massive hydroxyl group on the glass substrate. The glass slide was then silylanized by GPTMS, a bigger contact angle (49.0°) was obtained because of the epoxy groups produced on the substrate. The CuSNPs/chitosan filmdecorated substrate displayed the increased value of contact angle (52.1°), suggesting the formation of the stable signal interface. After streptavidin was immobilized on the CuSNPs/ chitosan film, its contact angle dramatically decreased to 31.9° because of the high hydrophilic interface. The biotinylated AFP antibody was finally immobilized on the glass substrate, and the interface demonstrated the smallest contact angle (15.0°), demonstrating the successful immobilization of the biotinylated antibodies. Kinetic Characteristics and Incubation Time. A static method was used to investigate the kinetic behavior of CuSNPs-catalyzed CL reaction produced on the signal interface. The label-free immunosensor was first incubated with AFP sample at RT for 25 min under stop flow. After PBST was injected into flow device to wash the system, CL substrate was introduced to the flow device. As shown from Figure 5A, the mimic enzymatic CL reaction occurred immediately, and the CL signal rapidly enhanced with the increase of the reaction time. Its intensity could increase to a maximum CL value within 300 s. Despite a long time exposure of CuSNPs on H2O2 environment, the CL intensity showed no obvious decrease after 300 s, suggesting high catalytic activity and stability of the peroxidase mimetics. Thinking over the satisfactory sensitivity

Figure 5. (A) Kinetic curve of CL reaction at 50 ng/mL AFP and (B) effect of the incubation time on CL intensity at 50 ng/mL AFP (PMT: −600; n = 5 for each point; incubation time for A, 25 min; CL reaction time for B, 300 s).

and assay time, the CuSNPs-catalyzed CL signal was measured at 300 s. The influence of incubation time on CL signals was studied at 50 ng/mL AFP concentration. As showed in Figure 5B, the CL signal quickly declined with the prolonged incubation time, and tended to a relatively constant value within 25 min, showing a saturated reaction between capture antibodies and target analytes on the sensing interface. The incubation process of the developed method is much simpler, rapider, and convenient compared to the traditional multiwall plate-based ELISA43 and CL immunoassay method.44 So as to acquire the optimal sensitivity, 25 min, therefore, was used as the selected incubation time in this immunoassay. Dose−Response Curves for AFP. Under the selected condition, the resultant CL intensity of the label-free immunoassay system decreased along with the increase of the amount of AFP in the sample solution. As seen from Figure 6,

Figure 6. Dose−response curve for AFP, inset: calibration curve for AFP (n = 5 for each point).

the proposed label-free CL immunosensing method demonstrated a linear relationship between the CL intensity and target AFP concentration. The linear range was 0.1 to 60 ng/mL, and the correlation coefficient was 0.9980. The obtained linear regression equation was I = 8822.06−128.58 [AFP]. The limit of detection at signal-to-noise ratio of 3 was calculated to be 0.07 ng/mL, and this value was much lower than that reported by label CL immunoassay method.34,45−48 In addition, the commercial AFP ELISA kit (from CanAg Diognostics), used in this work, gives a linear range of 0.5−500 ng/mL and a detection limit of 0.5 ng/mL. Although the linear range of our method is narrower than that of commercial ELISA, the detection limit obtain by our method is much lower than that of commercial ELISA. Moreover, after the concentration of E

DOI: 10.1021/acsami.6b02481 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces AFP was more than 60 ng/mL, only a facile step of sample dilution was needed. More importantly, this label-free immunoassay method showed obvious superiority such as simple operation, low cost, fast assay rate, and high sensitivity. Specificity, Reproducibility and Stability of the Developed Label-Free CL Immunosensor. In order to evaluate the specificity of this label-free immunosensor, the effect of six kinds of interfering antigens including 50 ng/mL AFP, 50 ng/mL CEA, 150 ng/mL IgG, 150 ng/mL BSA, 50 ng/mL CA125, and 150 ng/mL CHIL-4 toward CL intensity of the proposed method were examined in Figure 7. After the

Figure 8. CL responses of the label-free biosensor after the storage of 0, 5, 10, 20, and 30 days at a concentration of 50 ng/mL AFP (n = 5 for each point).

Table 2. Detection Results of Clinical Serum Samples Using Proposed and Reference Methods (n = 5)

Figure 7. CL responses of the proposed label-free biosensor to 50 ng/ mL AFP, 50 ng/mL CEA, 150 ng/mL IgG, 150 ng/mL BSA, 50 ng/ mL CA125, and 150 ng/mL CHIL-4 (n = 5 for each point).

sample

proposed method (ng/ mL)

reference method (ng/ mL)

relative error (%)

1 2 3 4 5

1.03 1.69 4.43 14.18 643.3

1.08 1.55 4.85 15.22 618.3

−4.6 9.0 −8.7 −6.8 4.0

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CONCLUSIONS In this research, we develop a highly efficient label-free CL immunosensing system based on CuS nanoparticles as peroxidase mimetics. The resultant CuSNPs and label-free immunosensor are characterized with various methods. The synthesized CuSNPs shows high catalytic activity and stability. This novel strategy can be easily achieved by immobilization of the capture antibodies on CuS nanoparticles through biotin− avidin system. The formed immmunocomplex on the sensing substrate can prevent the diffusion of CL substrate toward signal interface, and restrains the CuSNPs mimetics-catalyzed CL reaction, and finally results in the decease of CL signal. The proposed label-free immunoassay method is simple, cheap, and fast and has high sensitivity, good selectivity, and acceptable reproducibility. This study proposes a new idea to develop the potential label-free CL immunoassay for the diagnosis of clinical diseases.

label-free immunosensor was incubated with these antigens, the presence of AFP leaded to an obvious decrease in CL intensity because the AFP immunocomplex was formed on the sensing surface. However, other interfering antigens showed no obvious effect on CL intensity of the present immunoassay system compared with the blank value. The above results indicate that the fabricated label-free immune sensor possess high selectivity in the determination of AFP. The reproducibility was discussed by use of the intra- and interassay coefficients of variation (CVs). These CVs were preformed five times at 1.0 and 50 ng/mL of AFP. The obtained values of intra- and interassay CVs for the label-free CL immunosensor were 5.7 and 8.6% and 5.9 and 9.0%, respectively, which indicated an accepted reproducibility. After the immunosensor was kept at 4 °C in 0.01 M of PBS (pH 7.4) for not less than 30 days, and it could remain about 96% of its initial response (shown in Figure 8), demonstrating acceptable storage stability. Determination of AFP in Practical Samples. The potential application of the nonlabel CL immunosensing method was investigated by assaying human serum samples with the proposed method and the reference method. The latter is the electrochemiluminescence immunoassay (ECLIA), and done by Jiangsu Institute of Cancer Prevention and Cure. In case the level of serum tumor marker was beyond the calibration range, the samples needed an appropriate dilution using 0.01 M of PBS (pH 7.4). The AFP concentration in human serum samples was obtained through five measurements. As shown in Table 2, the obtained results demonstrated an acceptable relative errors of less than 9.0% between these two methods, suggesting the satisfactory accuracy of the developed nonlabel CL immunosensing system in detection of real clinical samples.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.6b02481. Scheme of the label-free chemiluminescent immunoassay for AFP, EIS, and contact angles for the fabrication process of the label-free immunosensor (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Phone/fax: +86-514-87972034. *E-mail: [email protected]. Phone/fax: +86-514-87972034. Notes

The authors declare no competing financial interest. F

DOI: 10.1021/acsami.6b02481 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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Catalase and Peroxidase Activities of Ferritin-Platinum Nanoparticles. Biomaterials 2011, 32, 1611−1618. (19) Dong, Y. L.; Zhang, H. G.; Rahman, Z. U.; Su, L.; Chen, X. J.; Hu, J.; Chen, X. G. Graphene Oxide-Fe3O4 Magnetic Nanocomposites with Peroxidase-Like Activity for Colorimetric Detection of Glucose. Nanoscale 2012, 4, 3969−3976. (20) Guan, G. J.; Yang, L.; Mei, Q. S.; Zhang, K.; Zhang, Z. P.; Han, M. Y. Chemiluminescence Switching on Peroxidase-Like Fe3O4 Nanoparticles for Selective Detection and Simultaneous Determination of Various Pesticides. Anal. Chem. 2012, 84, 9492−9497. (21) Burda, C.; Chen, X.; Narayanan, R.; El-Sayed, M. A. Chemistry and Properties of Nanocrystals of Different Shapes. Chem. Rev. 2005, 105, 1025−1102. (22) Jiang, X.; Sun, C. J.; Guo, Y.; Nie, G. J.; Xu, L. Peroxidase-Like Activity of Apoferritin Aaired Gold Clusters for Glucose Detection. Biosens. Bioelectron. 2015, 64, 165−170. (23) Chung, J. S.; Sohn, H. J. Electrochemical Behaviors of CuS as a Cathode Material for Lithium Secondary Batteries. J. Power Sources 2002, 108, 226−231. (24) Zhu, Y. D.; Peng, J.; Jiang, L. P.; Zhu, J. J. Fluorescent Immunosensor Based on CuS Nanoparticles for Sensitive Detection of Cancer. Analyst 2014, 139, 649−655. (25) Zhang, X. J.; Wang, G. F.; Gu, A. X.; Wei, Y.; Fang, B. CuS Nanotubes for Ultrasensitive Nonenzymatic Glucose Sensors. Chem. Commun. 2008, 45, 5945−5947. (26) Zhang, S. S.; Zhong, H.; Ding, C. F. Ultrasensitive Flow Injection Chemiluminescence Detection of DNA Hybridization Using Signal DNA Probe Modified with Au and CuS Nanoparticles. Anal. Chem. 2008, 80, 7206−7212. (27) Nie, G. D.; Zhang, L.; Lu, X. F.; Bian, X. J.; Sun, W. N.; Wang, C. A One-pot and in Situ Synthesis of CuS-Graphene Nanosheet Composites with Enhanced Peroxidase-Like Catalytic Activity. Dalton trans. 2013, 42, 14006−14013. (28) He, W. W.; Jia, H. M.; Li, X. X.; Lei, Y.; Li, J.; Zhao, H. X.; Mi, L. W.; Zhang, L. Z.; Zheng, Z. Understanding the Formation of CuS Concave Superstructures with Peroxidase-Like Activity. Nanoscale 2012, 4, 3501−3506. (29) Dutta, A. K.; Das, S.; Samanta, S.; Samanta, P. K.; Adhikary, B.; Biswas, P. CuS Nanoparticles as a Mimic Peroxidase for Colorimetric Estimation of Human Blood Glucose Level. Talanta 2013, 107, 361− 367. (30) Wei, Q.; Zhao, Y. F.; Xu, C. X.; Wu, D.; Cai, Y. Y.; He, J.; Li, H.; Du, B.; Yang, M. H. Nanoporous Gold Film Based Immunosensor for Label-Free Detection of Cancer Biomarker. Biosens. Bioelectron. 2011, 26, 3714−3718. (31) Du, Y.; Li, B. L.; Wei, H.; Wang, Y. L.; Wang, E. K. Multifunctional Label-Free Electrochemical Biosensor Based on an Integrated Aptamer. Anal. Chem. 2008, 80, 5110−5117. (32) Ouyang, H.; Wang, L. M.; Yang, S. J.; Wang, W. W.; Wang, L.; Liu, F. Q.; Fu, Z. F. Chemiluminescence Reaction Kinetics-Resolved Multianalyte Immunoassay Strategy Using a Bispecific Monoclonal Antibody as the Unique Recognition Reagent. Anal. Chem. 2015, 87, 2952−2958. (33) Liu, H.; Fu, Z. F.; Yang, Z. J.; Yan, F.; Ju, H. X. SamplingResolution Strategy for One-Way Multiplexed Immunoassay with Sequential Chemiluminescent Detection. Anal. Chem. 2008, 80, 5654− 5659. (34) Wu, Y. F.; Zhuang, Y. F.; Liu, S. Q.; He, L. Phenylboronic Acid Immunoaffinity Reactor Coupled with Flow Injection Chemiluminescnece for Determination of α-Fetoprotein. Anal. Chim. Acta 2008, 630, 186−193. (35) Yuan, L.; Xu, L. L.; Liu, S. Q. Integrated Tyramide and Polymerization-Assisted Signal Amplification for a Highly-Sensitive Immunoassay. Anal. Chem. 2012, 84, 10737−10744. (36) Wu, W. H.; Li, J.; Pan, D.; Li, J.; Song, S. P.; Rong, M.; Li, Z. X.; Gao, J. M.; Lu, J. X. Gold Nanoparticle-Based Enzyme-Linked Antibody-Aptamer Sandwich Assay for Detection of Salmonella Typhimurium. ACS Appl. Mater. Interfaces 2014, 6, 16974−16981.

ACKNOWLEDGMENTS The funding of this work was provided by National Natural Science Foundation of China (21575125, 21475116, and 21275124), University Natural Science Foundation of Jiangsu Province (13KJB150039), Priority Academic Program Development of Jiangsu Higher Education Institution (PAPD), and the Open Research Fund of State Key Laboratory of Analytical Chemistry for Life Science (SKLACLS1410). In addition, much thanks to The Testing Center of Yangzhou University for all the characterizations.

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REFERENCES

(1) Albrecht, H. O. Chemiluminescence of Aminophthalic Hydrazide. Z. Phys. Chem. 1928, 136, 321−330. (2) Adam, W.; Kazakov, D. V.; Kazakov, V. P. Singlet-Oxygen Chemiluminescence in Peroxide Reactions. Chem. Rev. 2005, 105, 3371−3387. (3) Zhao, L. X.; Sun, L.; Chu, X. G. Chemiluminescence Immunoassay. TrAC, Trends Anal. Chem. 2009, 28, 404−415. (4) Chen, F. M.; Mao, S. F.; Zeng, H. L.; Xue, S. H.; Yang, J. M.; Nakajima, H.; Lin, J. M.; Uchiyama, K. Inkjet Nanoinjection for HighThoughput Chemiluminescence Immunoassay on Multicapillary Glass Plate. Anal. Chem. 2013, 85, 7413−7418. (5) Nelson, D. L.; Cox, M. M. Lehninger Principles of Biochemistry, 4th ed.; W. H. Freeman & Co Ltd.: New York, 2005; Chapter 6. (6) Shoji, E.; Freund, M. S. Potentiometric Sensors Based on the Inductive Effect on the pKa of Poly(aniline): A Nonenzymatic Glucose Sensor. J. Am. Chem. Soc. 2001, 123, 3383−3384. (7) Wei, H.; Wang, E. K. Fe3O4 Magnetic Nanoparticles as Peroxidase Mimetics and Their Applications in H2O2 and Glucose Detection. Anal. Chem. 2008, 80, 2250−2254. (8) Lin, X. X.; Chen, Q. S.; Liu, W.; Li, H. F.; Lin, J. M. A Portable Microchip for Ultrasensitive and High-throughput Assay of Thrombin by Rolling Circle Amplification and Hemin/G-quadruplex System. Biosens. Bioelectron. 2014, 56, 71−76. (9) Zhang, G.; Dasgupta, P. K. Hematin as a Peroxidase Substitute in Hydrogen Peroxide Determinations. Anal. Chem. 1992, 64, 517−522. (10) Souza, T. T. L.; Moraes, M. L.; Ferreira, M. Use of Hemoglobin as Alternative to Peroxidases in Cholesterol Amperometric Biosensors. Sens. Actuators, B 2013, 178, 101−106. (11) Liu, Z. H.; Cai, R. X.; Mao, L. Y.; Huang, H. P.; Ma, W. H. Highly Sensitive Spectrofluorimetric Determination of Hydrogen Peroxide with β-Cyclodextrin-Hemin as Catalyst. Analyst 1999, 124, 173−176. (12) Bonar-law, R. P.; Sanders, J. K. M. Polyol Recognition by a Steroid-Capped Porphyrin. Enhancement and Modulation of Misfit Guest Binding by Added water or Methanol. J. Am. Chem. Soc. 1995, 117, 259−271. (13) Xu, J.; Wu, J.; Zong, C.; Ju, H. X.; Yan, F. Manganese PorphyrindsDNA Complex: A Mimicking Enzyme for Highly Efficient Bioanalysis. Anal. Chem. 2013, 85, 3374−3379. (14) Manea, F.; Houillon, F. B.; Pasquato, L.; Scrimin, P. Nanozymes: Gold-Nanoparticle-Based Transphosphorylation Catalyses. Angew. Chem., Int. Ed. 2004, 43, 6165−6169. (15) Jiao, X.; Song, H. J.; Zhao, H. H.; Bai, W.; Zhang, L. C.; Lv, Y. Well-Redispersed Ceria Nanoparticles: Promising Peroxidase Mimetics for H2O2 and Glucose Detection. Anal. Methods 2012, 4, 3261−3267. (16) Li, R. M.; Zhen, M. M.; Guan, M. R.; Chen, D. Q.; Zhang, G. Q.; Ge, J. C.; Gong, P.; Wang, C. R.; Shu, C. Y. A Novel Glucose Colorimetric Sensor Based on Intrinsic Peroxidase-Like Activity of C60-Carboxyfullerenes. Biosens. Bioelectron. 2013, 47, 502−507. (17) Gao, Z. Q.; Xu, M. D.; Hou, L.; Chen, G. N.; Tang, D. P. Irregular-Shaped Platinum Nanoparticles as Peroxidase Mimics for Highly Efficient Colorimetric Immunoassay. Anal. Chim. Acta 2013, 776, 79−86. (18) Fan, J.; Yin, J. J.; Ning, B.; Wu, X. C.; Hu, Y.; Ferrari, M.; Anderson, G. J.; Wei, J. Y.; Zhao, Y. L.; Nie, G. J. Direct Evidence for G

DOI: 10.1021/acsami.6b02481 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

Research Article

ACS Applied Materials & Interfaces (37) Zhao, Y. F.; Wei, Q.; Xu, C. X.; Li, H.; Wu, D.; Cai, Y. Y.; Mao, K. X.; Cui, Z. T.; Du, B. Label-Free Electrochemical Immunoassay for Sensitive Detection of Kanamycin. Sens. Actuators, B 2011, 155, 618− 625. (38) Okuno, J.; Maehashi, K.; Kerman, K.; Takamura, Y.; Matsumoto, K.; Tamiya, E. Label-Free Immunosensor for ProstateSpecific Antigen Based on Single-Walled Carbon Nanotube ArrayModified Microelectrodes. Biosens. Bioelectron. 2007, 22, 2377−2381. (39) Jia, X. L.; Liu, Z. M.; Liu, N.; Ma, Z. F. A Label-Free Immunosensor Based on Graphene Nanocomposites for Simultaneous Multiplexed Electrochemical Determination of Tumor Markers. Biosens. Bioelectron. 2014, 53, 160−166. (40) Qi, Y. Y.; Li, B. X. A Sensitive, Label-Free, Aptamer-Based Biosensor Using a Gold Nanoparticle-Initiated Chemiluminescence System. Chem. - Eur. J. 2011, 17, 1642−1648. (41) Yang, Z. J.; Cao, Y.; Li, J.; Wang, J. T.; Du, D.; Hu, X. Y.; Lin, Y. H. A New Label-Free Strategy for a Highly Efficient Chemiluminescence Immunoassay. Chem. Commun. 2015, 51, 14443−14446. (42) Yang, Z. J.; Shen, J.; Li, J.; Zhu, J.; Hu, X. Y. An Ultrasensitive Streptavidin-Functionalized Carbon Nanotubes Platform for Chemiluminescent Immunoassay. Anal. Chim. Acta 2013, 774, 85−91. (43) Darwish, I. A.; Wani, T. A.; Alanazi, A. M.; Hamidaddin, M. A.; Zargar, S. Kinetic-Exclusion Analysis-Based Immunosensors Versus Enzyme-Linked Immunosorbent Assays for Measurement of Cancer Markers in Biological Specimens. Talanta 2013, 111, 13−19. (44) Fu, Z. F.; Liu, H.; Ju, H. X. Flow-Through Multianalyte Chemiluminescent Immunosensing System with Designed Substrate Zone-Resolved Technique for Sequential Detecion of Tumor Markers. Anal. Chem. 2006, 78, 6999−7005. (45) Yang, Z. J.; Fu, Z. F.; Yan, F.; Liu, H.; Ju, H. X. A Chemiluminescent Immunosensor Based on Antibody Immobilized Carboxylic Resin Beads Coupled with Micro-bubble Accelerated Immunoreaction for Fast Flow-Injection Immunoassay. Biosens. Bioelectron. 2008, 24, 35−40. (46) Fu, Z. F.; Yan, F.; Liu, H.; Lin, J. H.; Ju, H. X. A ChannelResolved Approach Coupled with Magnet-Captured Technique for Multianalyte Chemiluminescent Immunoassay. Biosens. Bioelectron. 2008, 23, 1422−1428. (47) Yang, Z. J.; Liu, H.; Zong, C.; Yan, F.; Ju, H. X. Automated Support-Resolution Strategy for a One-Way Chemiluminescent Multiplex Immunoassay. Anal. Chem. 2009, 81, 5484−5489. (48) Wu, Y. F.; Liu, S. Q. Immunosensing System for α-Fetoprotein Through Boronate Immunoaffinity Column in Combination with Flow Injection Chemiluminescence. Analyst 2009, 134, 230−235.

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DOI: 10.1021/acsami.6b02481 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX