General Strategy to Fabricate Electrochemiluminescence Sandwich

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A General Strategy to Fabricate Electrochemiluminescence Sandwich-type Nano-immunosensors Using CdTe@ZnS Quantum Dots as Luminescent Labels and Fe3O4@SiO2 Nanoparticles as Magnetic Separable Scaffolds Xin Zhang, and Shou-Nian Ding ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.5b00242 • Publication Date (Web): 22 Jan 2016 Downloaded from http://pubs.acs.org on January 26, 2016

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A General Strategy to Fabricate Electrochemiluminescence Sandwich-type Nano-immunosensors Using CdTe@ZnS Quantum Dots as Luminescent Labels and Fe3O4@SiO2 Nanoparticles as Magnetic Separable Scaffolds

Xin Zhang and Shou-Nian Ding*

School of Chemistry and Chemical Engineering, Southeast University, Nanjing 211189, China. *Corresponding authors. (S.-N. Ding) Fax: (+86) 25-52090621. E-mail: [email protected].

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ABSTRACT: This work aims to develop universal sandwich-type electrochemiluminescence (ECL) nano-immunosensors for quantitative detection of biomarkers. A series of low-toxic CdTe@ZnS QDs with different core sizes have been synthesized via hydrothermal method and characterized by UV-vis spectra, photoluminescence spectra, TEM, EDS and XRD. Especially, the ECL behaviors of CdTe@ZnS QDs have been investigated carefully. The CdTe@ZnS QDs with the highest ECL quantum yields have been chosen as ECL labels for conjugation with secondary antibodies. The QDs-labelled antibodies have been characterized by agarose gel electrophoresis. Meanwhile, Fe3O4@SiO2 magnetic nanoparticles were utilized as nano-carriers for the immobilization of primary antibodies due to magnetic separation ability, large specific surface area, and ease of amination for biofunctionalization. Successful fabrication of the nano-immunosensor was confirmed by SEM and Electrochemical impedance spectroscopy. Carcinoembryonic antigen was detected as a model to prove the feasibility of the above strategy. Under the optimal conditions, the proposed nano-immunosensor exhibited a wide linear range of 0.01 to 125 ng mL-1 for carcinoembryonic antigen determination with a low detection limit of 3.0 pg mL-1 (S/N=3). Moreover, the nano-immunosensor also displayed excellent selectivity, good stability and acceptable reproducibility, indicating its potential applications in clinical diagnostics and immune research. Keywords: CdTe@ZnS QDs, magnetic nanoparticles, carcinoembryonic antigen, nano-immunosensor, electrochemiluminescence


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Accurate detection of tumor biomarker is of significance in predicting tumor and early diagnosis1. Carcinoembryonic antigen (CEA) is one of the most common tumor biomarkers for colorectal, pancreatic, and gastric carcinomas2. CEA concentration in serum could be used as diagnostic criteria to evaluate benign or malignant tumor. Therefore, many researchers have focused on the detection methods of CEA over years. Up to now, various types of immunoassays have been developed and reported, including enzyme-linked immunosorbent assay3, colorimetric immunoassay4, fluorescence immunoassay5, surface plasmon resonance immunoassay6, mass spectrometric immunoassay7, electrochemiluminescence (ECL) immunoassay8, electrochemical immunoassay9, and so on. As compared to other counterparts, ECL immunoassay has gained increasing attention owing to its high sensitivity, simple instrumentation, and ease of automation. For a sandwich-type ECL immunoassay, design of the label conjugated with secondary antibodies (Ab2) for signal detection is always the focus we concerned. Common luminophore-coreactant systems applied in ECL-based immunoassay mainly include luminol-H2O2, Ru(bpy)32+-tripropylamine, and semiconductor quantum dots (QDs)-S2O82-. Recently, many ECL immunoassays based on luminol-H2O2 system adopt the ways of using peroxidase or employing precious metal nanoparticles to conjugate with Ab210-12. ECL signal could be amplified through H2O2 decomposition catalyzed by peroxidase or metal nanoparticles. But there are some shortcomings in these designs, such as rigorous catalytic condition of enzyme, expensive cost and tedious synthesis of precious metal nanoparticles. By comparison, QDs-labeled biomolecules might be an interesting candidate for immunoassay, owing to the unique advantages of QDs like low cost, good biocompatibility, simple synthesis process and superior optical property13. Among them, core-shell (ZnS shell) QDs are a kind of low-toxic label which could keep the bioactivity of antibodies. For QDs-based ECL immunoassay, how to enhance luminescence of the QDs for signal amplification is extremely important. With this in mind, we decided to prepare a series of typeⅠ core-shell QDs (CdTe@ZnS) with different core sizes to compare their ECL for seeking the best ECL emitter. To the best of our knowledge, there is no


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report about the influence of core size on the ECL behaviors of core-shell QDs. And the combination of low-toxic core-shell QDs with immunoassay is meaningful. The support for immobilizing primary antibodies (Ab1) is another factor influencing the sensibility of the immunosensor. More recently, magnetic nanomaterials have attracted tremendous interests for their excellent magnetic manipulation and separation ability. Magnetic-supported design has been widely popularized in bioanalytical studies, encompassing microRNA recognition14, t-DNA detection15, separation and detection of bacteria16, immunoassay5,17,18, and so on. As a kind of good biological carrier, Fe3O4 nanoparticles have some attractive features, such as large specific surface area for loading more biomolecules and good magnetic performance for facilitating simple separation. In biological detection, it is of importance to avoid nonspecific adsorption, and the separation ability of Fe3O4 nanoparticles could meet this requirement. However, Fe3O4 nanoparticles are easily oxidized in air, which leads to the loss of magnetism and the low solubility in water. Therefore, silica coating is often applied to stabilize the Fe3O4 nanoparticles. In addition, Fe3O4@SiO2 could be functionalized easily, which may promote the biocompatibility. Inspired by these merits, Fe3O4@SiO2 was selected as the support for immobilizing Ab1 in our immunosensor. Herein, we report a novel ECL immunosensor for CEA detection based on CdTe@ZnS QDs as luminescent labels and Fe3O4@SiO2 nanoparticles as magnetic separable matrices. A schematic diagram of the construction process of the ECL immunosensor is shown in Scheme 1. Initially, five core-shell CdTe@ZnS QDs samples with different core sizes were prepared and characterized. By comparing their ECL, CdTe@ZnS-5 (QD650nm) with the highest luminescence was chosen as signal label for immunoassay. Then, QD650nm was further conjugated with Ab2 using EDC as activator, and successful conjugation was confirmed by running agarose gels. Next, amino-modified Fe3O4@SiO2 nanoparticles were prepared by APTES to improve feasibility of biofunctionalization. And then Ab1 was immobilized on the aminated-Fe3O4@SiO2 through cross-linking with glutaraldehyde (GA). After that, Bovine serum albumin (BSA) was used to block the nonspecific binding sites. Finally,


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a sandwich-type immunosensor was fabricated via incubating Ab1-immoblized Fe3O4@SiO2 with Ag and QD650nm-labeled Ab2 successively. As a result, the ECL immunosensor displayed excellent selectivity, good stability and acceptable reproducibility for CEA detection, exhibiting potential applications in clinical diagnostics.

Scheme 1. Schematic illustration of preparation process of QDs-labeled Ab2 (A) and fabrication process of proposed ECL nano-immunosensor (B).

EXPERIMENTAL SECTION Reagents and chemicals. Carcinoembryonic antigen (CEA) and anti-CEA antibodies (Ab1 and Ab2) were purchased from Biocell Biotechnology Co., Ltd. BSA was obtained from Sangon Biotech Co., Ltd. Phosphate buffer solution (PBS, pH 7.4) was freshly prepared before use. All other reagents were of analytical grade and were used without further purification. Double distilled water was used throughout.


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Apparatus. All electrochemical and ECL experiments were carried out in a conventional three-electrode cell with a modified glassy carbon working electrode (GCE, diameter 3 mm), a Pt wire counter electrode and a saturated calomel electrode (SCE) as reference electrode. The ECL intensities were measured with MPI-ECL Analyzer (Xi’an Remax Electronic High-Tech Ltd). ECL spectrum was recorded by PG2000-pro scientific class spectrometer (Shanghai, China). Electrochemical impedance spectroscopy (EIS) was studied on a Versa STAT 3 electrochemical workstation (Princeton) with the frequency range from 0.01 Hz to 100,000 Hz and signal amplitude of 5 mV. Synthesis of water-soluble CdTe cores and CdTe@ZnS QDs. CdTe QDs capped with MPA were synthesized by hydrothermal method according to the literature19. By controlling the heating time, different sizes of CdTe QDs were attained. The purified CdTe QDs with different sizes were used as cores to grow ZnS layers directly. And CdTe@ZnS core-shell QDs were prepared as reported before with slight modifications20-22. Details regarding the synthesis are shown in the Supporting Information. Standard procedures for ECL detection of QDs. A droplet of CdTe QDs (five different sizes) or corresponding CdTe@ZnS QDs dispersion with the same amount was dropped on GCE, and dried in air at room temperature to modify the electrode. ECL measurements were carried out in 0.1 M PBS (pH 7.4) containing 0.05 M K2S2O8. The applied working potential ranged from 0 to -1.6 V, and the scan rate was 100 mV/s. Bioconjugation of CdTe/ZnS QDs with Ab2. The water-soluble CdTe/ZnS QDs with fluorescence emission wavelength of 650nm (QD650nm) were further conjugated with Ab2 via similar procedures in previous reports23,24. At first, 75 µL of freshly prepared EDC solution (4.2 mg/mL) was added into 500 µL of QDs (OD599nm ~ 0.1) to activate the carboxylic acid groups on QD surface. Then 1 mL of Ab2 (10 µg/mL) was mixed with above solution. The mixture was shaken gently at room temperature in the dark


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for 2 h. To remove unconjugated QDs and byproduct, the resulting product was purified by ultrafiltration using a 50 KD filter (3,000 g for 10 min). It is notable that too high QDs concentration or too much EDC may cause aggregate formation. The final complex was kept at 4℃ for future use. Preparation of Fe3O4@SiO2/Ab1. Fe3O4 nanoparticles were synthesized using a one-pot hydrothermal method according to the literature with a slight modification25. The modification lies in that the heating time in autoclave was extended to 12 h. To avoid aggregation of magnetic nanoparticles and improve feasibility of conjugation with Ab1, silanization and amination of Fe3O4 were arranged before conjugation. These steps were carried out according to the reported method without any change26. Subsequently, Ab1 was immobilized on the aminated Fe3O4@SiO2 through cross-linking by GA. 10 mg of aminated Fe3O4@SiO2 was dissolved in 7.5 mL water, and then mixed with 2.5 mL of GA solution (2.5 wt%). The mixture was stirred at room temperature for 6 h. To remove the unreacted GA, several washes with water by magnetic separation were carried out. The obtained product was re-dispersed in 5 mL of PBS (0.01 M, pH 7.4). After that, 500 µL of Ab1 (10 µg/mL) was added into 500 µL of as-prepared aldehyde-functionalized Fe3O4@SiO2, and shaken gently at 37℃ for 1 h4,27. To remove unconjugated Ab1, the product was washed by PBS for several times and separated by a magnet. Finally, the Ab1-conjugated Fe3O4@SiO2 was incubated in a PBS solution containing BSA (100 µL, 1.0 wt%) to block the nonspecific binding sites. After washing, the product was re-dispersed in 500 µL PBS and stored at 4℃ for future use. Fabrication of the immunosensor. The fabrication process of the immunosensor is displayed as Scheme 1B. 50 µL of above Fe3O4@SiO2/Ab1 solution was added into 50 µL PBS solution containing various concentrations of CEA antigen (Ag). The mixture was shaken gently at 37 ℃ for 1h. After the incubation, formed antigen-antibody complex was separated with the help of magnet, and washed with PBS carefully to remove physically adsorbed antigen. And then the resultant immunosensor was further incubated with QD650nm-labeled Ab2 for 1 h to construct


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sandwich-type immunosensor. Subsequently, the immnosensor was washed with PBS to remove unconjugated QD650nm-labeled Ab2. Ultimately, the immunosensor was re-dispersed in 100 µL PBS solution and stored at 4℃. Measurement procedure. A typical ECL detection of CEA was conducted as follow. 7.2 µL of above sandwich-type immunosensors with various concentrations of CEA was dropped on GCE and dried in air at room temperature to modify the electrode. 4 mL of 0.1 M PBS solution (pH 7.4) containing 0.05 M K2S2O8 was added into the detector cell. In the detection process, the voltage of the photomultiplier tube (PMT) was set at 700 V. The applied potential and scan rate were the same as above.

RESULTS AND DISCUSSION Characterization of CdTe cores and CdTe@ZnS QDs. Table 1 shows the respective evolutions of absorption and emission spectra of CdTe and CdTe@ZnS QDs. The size of CdTe core is controlled by varying reaction time. It can be seen with the increase of QD size, both UV-vis absorption band and the FL peak shifted to longer wavelength, owing to the well-known quantum size effect28. After the growth of ZnS shell, small bathochromic shifts of the excitonic peak could be observed in absorption and emission spectra. This observation is attributed to a partial leakage of the exciton into the shell material, similar with other typeⅠ core-shell nanocrystals29. Detailed spectra are shown in Figure S1. Table 1. The absorption wavelengths in UV-vis spectra and emission wavelengths in fluorescence spectra of CdTe QDs and CdTe@ZnS QDs, and the diameters of cores. Samples (core

Absorption peak,

Emission peak,

core sizea

Samples (core-shell

Absorption peak,

Emission peak,

QDs)

λabs (nm)

λem (nm)

(nm)

QDs)

λabs (nm)

λem (nm)

CdTe-1

461

518

0.84

CdTe@ZnS-1

473

532

CdTe-2

519

543

2.79

CdTe@ZnS-2

531

562

CdTe-3

547

581

3.21

CdTe@ZnS-3

554

593

CdTe-4

567

614

3.40

CdTe@ZnS-4

584

629

CdTe-5

589

637

3.57

CdTe@ZnS-5

599

650

a

Calculated diameter according to the empirical formula.


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The morphologies of CdTe-5 and CdTe@ZnS-5 are shown in Figure 1A and B. It can be observed that CdTe cores (λem=637 nm) were mostly monodispersed and of spherical shape with the average diameter of ca. 3.50 nm, close to the value of 3.57 nm resulting from the empirical formula30. Following the growth of ZnS shell on the CdTe core, the size of the core-shell QD increased to 4.21 nm, which implies a ZnS shell grew in situ on the CdTe core.

Figure 1. TEM images of CdTe-5 QDs (A) and CdTe@ZnS-5 QDs (B). Inset (A) and (B): HRTEM of CdTe QDs and CdTe@ZnS QDs, respectively. The EDS spectrum in Figure S2A shows the peaks corresponding to Cd, Te, Zn, S, C and O in the as-prepared MPA capped CdTe@ZnS sample. The appearances of the sulfur peak at 2.3 keV and the zinc peak at 8.6 keV demonstrate the existence of ZnS shell, and further verify the formation of core-shell QDs. X-ray powder diffraction (XRD) can offer useful information on the crystal structure for qualitative analysis. The XRD pattern of CdTe sample (Figure S2B) shows characteristic zinc blend planes of 111, 220, and 311 located at 24.04°, 40.48°, 46.81°, consistent with the standard values of CdTe (JCPDS No. 15-0770). After the growth of ZnS shell, the peaks shifted to higher angles at 25.40°, 42.24°, 50.74°, inclining to ZnS cubic structure peaks (JCPDS No.05-0566). These results also substantiate core-shell CdTe@ZnS QDs were successfully prepared. ECL behavior of CdTe@ZnS QDs/GCE. Figure 2A shows the ECL intensity increased with the increasing CdTe size in the range of 0.8-3.5 nm (calculated according to empirical formula). Previous studies28,31 have elucidated QDs size deeply


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affected their ECL behavior. With the increment of size, band gap of QD decreases. Two critical factors of ECL intensity, the quantum efficiency of producing excited-state QDs and the luminescent quantum efficiency of excited-state QDs related to band gap, would increase. Finally, the ECL intensity of CdTe increases as particle size becomes bigger. It also could be found the ECL onset potentials positively shifted from -1.30V to -1.14V with the particle size increased (Figure S3A), meaning that the injection of electrons to the bigger QDs became easier.

Figure 2. (A) ECL intensity comparison of CdTe QDs with different sizes before and after ZnS shell growth. The voltage of the photomultiplier tube (PMT) was set at 400V. (B) FL (a) and ECL (b) spectra of CdTe@ZnS-5. After ZnS shell growth on five different-sized CdTe cores, it could be observed that each ECL intensity increased in a certain degree (Figure 2A; Figure S3B). Taking CdTe@ZnS-5 for example, the ECL intensity was twice as high as that of corresponding CdTe core. Control experiments prove the enhancement of ECL intensity stems from shell growth on the core surface rather than a simple mix of CdTe and ZnS (Figure S4). Shell growth reduces the number of surface dangling bonds, which could act as trap states for charge carriers and thereby reduce the luminescent quantum efficiency. And finally the highly-passivated surface state results in the increase of ECL intensity. The passivation effect of ZnS shell also could be confirmed by comparing PL and ECL spectra. It is believed that PL spectrum mainly reflects the bandgap of nanocrystals and ECL spectrum represents surface state transitions. Highly-passivated nanocrystals without deep surface trap often show


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an overlap in their PL and ECL spectra, due to the bandgap engineered model of ECL32-34. In Figure 2B, the ECL spectrum shows a main peak at 660nm, very close to that of PL spectrum, indicating the surfaces have been largely passivated. As illustrated in Figure S5, upon successive potential scanning for 18 cycles, the ECL signal of CdTe@ZnS-5/GCE was not only high but also stable, suggesting CdTe@ZnS-5 (QD650nm) could be selected as the ECL emitter. Characterization of QDs-Ab probe. Running agarose gels could offer the evidence of the conjugation of QD650nm with Ab2. We ran samples with 2 µL of glycerol on a 1.0% agarose gel at 100V in TAE buffer. As shown in Figure 3, QD650nm-Ab2 conjugate ran slower than either free QD650nm or QD650nm activated by EDC, attributed to the larger size after conjugation. The distinct migration difference confirms successful conjugation of QD650nm with Ab2.

Figure 3. Agarose gel electrophoresis imaging of the conjugation of QD650nm with Ab2: (1) unconjugated QD650nm, (2) QD650nm activated by EDC, and (3) purified QD650nm-Ab2 conjugates. Characterization of Fe3O4@SiO2/Ab1. TEM images of Fe3O4@SiO2 (Figure 4A and B) offer the information on the size and structure of the nanoparticles. They appeared to be uniform in size and well-dispersed on the grid surface. The SiO2 shell was coated on the Fe3O4 core successfully, and the Fe3O4@SiO2 nanoparticles were spherical-shaped with an average diameter of 247 nm (core 171 nm, shell 38 nm).


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Figure 4. TEM images of Fe3O4@SiO2 nanoparticles (A) and the magnification (B); SEM images of amino-functionalized Fe3O4@SiO2 (C) and Fe3O4@SiO2/Ab1 (D) on the electrode; Particle size distributions of amino-functionalized Fe3O4@SiO2 (E) and Fe3O4@SiO2/Ab1 (F). FT-IR spectra (Figure S6) confirm successful amino modification on the surface of Fe3O4@SiO2 nanoparticles. Before modified with APTES, the absorption bands at 1092 cm-1 and 799 cm-1 are assigned to the asymmetric and symmetric stretching vibrations of Si-O-Si in the silica shell, respectively. The band at 945 cm-1 corresponds to Si-OH stretching vibration on the surface, and the Fe-O stretching


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vibration of Fe3O4 core appeared at 564 cm-1. The Fe-O-Si stretching vibration at 473 cm-1 demonstrates Fe3O4 core has been encapsulated by silica shell. After amino modification by APTES, two new bands at 2917 cm-1 and 2847 cm-1 appeared and they are ascribed to the characteristic absorption of C-H stretching vibration of propyl group. The broad band at 3439 cm-1 should be attributed to N-H stretching, which is partly overlapped by O-H stretching vibration from silanol groups and adsorbed water. The results corroborate that amino functionalization of silica shell by APTES was successful. The morphologies of amino-functionalized Fe3O4@SiO2 and Fe3O4@SiO2/Ab1 were

observed

by

SEM.

High-resolution

SEM

image

of

obtained

amino-functionalized Fe3O4@SiO2 clearly show the nanoparticle surface was a little rough (Figure 4C). The roughness is ascribed to uneven growth on the silica surface during the process of amination in aqueous phase. After antibodies were immobilized on the microspheres, the surface became rougher, which may attributed to the formation of protein

layer (Figure 4D). Meanwhile, amino-functionalized

Fe3O4@SiO2 nanoparticles displayed an average diameter of 410 nm (Figure 4E). After antibodies were immobilized on the microspheres, particle sizes increased to 460±15 nm (Figure 4F), further indicating Ab1 have been conjugated. EIS monitoring of immunosensor fabrication. Figure 5 displays the Nyquist plot of impedance of each step in the process of immunosensor fabrication, and the general Randle’s equivalent circuit is shown in the inset. It is well established that, Ret could be used as the suitable signal for sensing the interfacial properties of the prepared immunosenor during the assembly procedures35. The EIS of bare electrode displayed an almost straight line, suggesting a mass diffusion limiting process (curve a). For Ab1/Fe3O4@SiO2 modified electrode, it exhibited a relatively low interfacial electron-transfer resistance (Ret=572Ω), as Ab1/Fe3O4@SiO2 layer obstructed the diffusion of redox probe to the electrode surface (curve b). After the magnetic bionanoparticles were blocked by BSA, the Ret value was distinctly higher (846Ω, curve c). The reason should be protein layer further hinder the diffusion of redox


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probe. Similarly, continuous increases of Ret value were obtained after the magnetic nanoparticles were incubated in Ag and QD650nm-labeled Ab2 (Ret=1650Ω, curve d; Ret=2269Ω, curve e), reflecting Ag and QDs-labeled Ab2 were successfully captured. EIS and following ECL results confirm that the immunosensor was successfully fabricated.

Figure 5. EIS of different modified electrodes in a solution of 0.1 M KCl containing 5 mM [Fe(CN)6]2-/[Fe(CN)6]3-: (a) bare GCE; (b) Ab1/Fe3O4@SiO2/GCE; (c) BSA/Ab1/Fe3O4@SiO2/GCE;

(d)

Ag/BSA/Ab1/Fe3O4@SiO2/GCE;

(e)

QD650nm-labeled Ab2/Ag/BSA/Ab1/Fe3O4@SiO2/GCE. Inset: the equivalent circuit applied to fit the impedance data. Rs, solution resistance; CPE, constant phase angle element; Ret, electron-transfer resistance; Zw, Warburg diffusion resistance. Detection of CEA (Ag). To obtain better performance for CEA detection, a series of optimization experiments (pH value, addition amount of QD650nm during labeling process, modified amount of sandwich nano-composite on electrode and incubation time) were conducted based on sandwiched immunoassay toward 50 ng/mL CEA (see the Supporting information for details; Figure S7). Under the optimum conditions, the proposed immunosensor was applied to detect CEA. As expected, based on a sandwich mechanism, the ECL intensity increased with the increasing of the CEA concentration (Figure 6). As shown in the inset, the calibration plot displayed a good


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linear relationship between ECL change and the concentration of CEA in the range of 0.01-125 ng mL-1. The linear regression equation could be expressed as ΔI=1.02 c+1.924 (c, ng mL-1) with the correlation coefficient of 0.997. ΔI represents the degree of ECL intensity change,ΔI=(I-I0)/I0, where I and I0 are the ECL intensity in the presence and absence of CEA, respectively. And a detection limit of 3.0 pg mL-1 was estimated according to the signal-to-noise ratio of three. The results suggest this developed immunosensor is applicable to quantitative detection of CEA. Moreover, we made a comparative work on the performance of proposed immunosensor in CEA detection with other methods (shown in Table S1). It could be seen that our immunosensor presents a relatively wide linear range with a low detection limit.

Figure 6. ECL profiles of the immunosensor in the presence of different concentration of CEA (ng mL-1) in PBS (0.1 M, pH 7.4) containing 0.05 M K2S2O8. The voltage of the PMT was set at 700 V. Inset: Calibration curve for CEA detection. Specificity, stability and reproducibility of the ECL immunosensor. The specificity of immunosensor for CEA detection was tested via interference experiment by incubating the immunosensor with other tumor markers (human IgG and AFP) or protein (BSA). The assay was carried out under the same experimental procedures


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and the ratio between the target analyte and the interfering agent was 1:10. As shown in Figure 7A, when the immunosensor was incubated with interfering agent only, ECL intensity was as weak as the blank sample. When CEA coexisted with these interfering agents, ECL intensity increased greatly, and no significant difference (RSD = 3.34%) was found in comparison with that of CEA only. The results indicate the selectivity of the immunosensor was good. Stability is also a very important factor for assessing immunosensor. As shown in Figure 7B, the relatively stable ECL curves could be obtained at each concentration. The stability of proposed immunosensor was also examined by checking ECL response periodically (Figure S8). The response of the immunosensor retained about 89.4% of its initial value after stored at 4℃ in the dark for one week. The results suggest the stability of the immunosensor was satisfactory. To monitor the reproducibility of the immunoassay, five immunosensors were modified on the same electrode to detect 125 ng mL-1 CEA, respectively. The results are shown in Figure 7C. The RSD of the five independent assay results was about 3.19%, indicating the reproducibility of the immunosensor was acceptable.

Figure 7. (A) Specificity investigations of the immunosensor for CEA detection: (a) blank (0 ng mL-1 target), (b) Human IgG (250 ng mL-1), (c) AFP (250 ng mL-1), (d) BSA (250 ng mL-1), (e) CEA (25 ng mL-1), (f) CEA (25 ng mL-1) + Human IgG (250 ng mL-1), (g) CEA (25 ng mL-1) + AFP (250 ng mL-1), (h) CEA (25 ng mL-1) + BSA (250 ng mL-1). (B) The stability of proposed immunosensor for the detection of various CEA concentrations. (C) The study on the reproduction of the immunosensor. Error bar = RSD (n=3). Preliminary analysis of real samples. To evaluate the reliability and potential


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application of the proposed immunosensor, recovery tests were conducted in human serum according to standard addition method. The human serum samples were obtained from Nanjing Zhongda Hospital. As shown in Table S2, 5 ng mL-1, 10 ng mL-1 and 20 ng mL-1 of CEA were added into the serum samples, respectively. The recovery ranged from 95.66% to 104.18%, and the RSD was in the range from 2.37% to 4.63%. The results demonstrate the immunosensor is feasible to detect CEA concentration in human serum.

CONCLUSIONS In summary, the well-dispersed Fe3O4@SiO2 nanoparticles have been prepared to act as magnetic separable nano-carriers to conjugate with the primary antibodies after amination. Meanwhile, an enhanced ECL has been obtained from core-shell CdTe@ZnS QDs. The ECL spectrum is consistent with its FL spectrum. The prepared CdTe@ZnS-5 QDs have been used as ECL probes to attach to the secondary antibodies.

Thus

a

general

strategy

to

fabricate

ECL

sandwich-type

nano-immunosensors has been realized. The whole immuno-reactions have taken place in homogenous aqueous solutions and the separation is facile with the aid of an external magnet, which not only saves the detection time but also makes the detection process simple. The ECL detection via simply casting the resulted sandwich-type bioconjugates onto electrode surface ensures the high sensitivity due to the absence of background. As a result, the nano-immunosensor successfully achieved the detection of CEA with desirable sensitivity, stability, reproducibility and selectivity, suggesting that it possesses potential applications in clinical diagnostics or immune research. Furthermore, the strategy can also achieve multicomponent analysis by conjugating Fe3O4@SiO2 nanoparticles with different primary antibodies and the corresponding secondary antibodies with multi-color QDs. The detection of each component could be realized by measuring ECL signals on PMT in sequence or on CCD simultaneously. This is another work we are going to accomplish in the near future.


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ACKNOWLEDGEMENTS This work was supported by the National Natural Science Foundation of China (21575022, 21535003), the National High Technology Research and Development Program (“863” Program) of China (2015AA020502), the Open Research Fund of Key Laboratory of Energy Thermal Conversion and Control of Ministry of Education, Southeast University, and the Fundamental Research Funds for

the Central

Universities.

Supporting Information Available The following file is available free of charge. Supporting Information.docx. This Supporting Information document contains preparation details, optimal experimental parameters, UV-vis absorption and fluorescence emission spectra, EDS and XRD patterns, ECL behaviors, control experiments, stability studies, FT-IR spectra, comparative work, and recovery tests in real samples.

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For TOC only


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General Strategy to Fabricate Electrochemiluminescence Sandwich

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