Quantum Dots Based Potential-Resolution Dual-Targets

Article pubs.acs.org/ac

Quantum Dots Based Potential-Resolution Dual-Targets Electrochemiluminescent Immunosensor for Subtype of Tumor Marker and Its Serological Evaluation Xuan Liu,*,† Hui Jiang,*,‡ Yuan Fang,† Wei Zhao,† Nianyue Wang,† and Guizhen Zang† †

Department of Clinical Laboratory, Second Affiliated Hospital of Southeast University, Nanjing 210003, People’s Republic of China State Key Laboratory of Bioelectronics, Southeast University, Nanjing 210096, People’s Republic of China

‡

Downloaded by NANYANG TECHNOLOGICAL UNIV on August 26, 2015 | http://pubs.acs.org Publication Date (Web): August 25, 2015 | doi: 10.1021/acs.analchem.5b02660

S Supporting Information *

ABSTRACT: The identification of subtypes of known tumor markers is of great importance for clinical diagnosis but still a great challenge in novel detection methodologies with simple operation and acceptable sensitivity. This work for the first time reported a quantum dots (QDs) based potential-resolved electrochemiluminescent (ECL) immunosensor to realize simultaneous detection of dual targets. Because of different surface microstructures, dimercaptosuccinic acid stabilized CdTe (DMSA-CdTe) QDs and TiO2 nanoparticles-glutathione stabilized CdTe (TiO2-GSH-CdTe) QDs composites showed a large difference of ECL peak potential (∼360 mV), which provided an access for potential-resolution detection. The ECL emission on indium tin oxide electrodes showed consistent strength during the cyclic scan, and intensity data were collected at −0.89 V and −1.25 V (vs Ag/AgCl) for DMSA-CdTe QDs and TiO2-GSH-CdTe QDs composites, respectively. The interface modification procedures of immunosensor construction were characterized by atomic force microscopy. The portion of Lens culinaris lectin affiliated isoform of alpha fetoprotein (AFP), AFP-L3%, in total AFP, is recently a novel criteria showing even higher sensitivity and specificity than AFP at the early stage of cancer. Combined with the enzyme cyclic amplification strategy, linear ranges for AFP-L3 and AFP dual-targets detection were 3.24 pg mL−1−32.4 ng mL−1 and 1.0 pg mL−1−20 ng mL−1, with limits of detection of 3.24 pg mL−1 and 1.0 pg mL−1, respectively. Compared with clinical detection data, the calculated portion of AFP-L3% by as-prepared immunosensor showed acceptable accuracy. These results open a new avenue for facile and rapid multiple-components detection based on the nano-ECL technique and provide a new clinical diagnosis platform for HCC.

H

relatively tedious and not always reliable for serum samples with low total AFP concentrations because of limitations in instrument sensitivity.11 In addition, AFP-L3% test in clinical laboratory involves two separate analysis procedures of AFP and AFP-L3, with relative high economic and human costs. Thus, novel methodologies for AFP-L3% test with high sensitivity and low cost, especially simultaneous detection of AFP and AFP-L3 were of great significance. Electrochemiluminescent (ECL) technique based on quantum dots (QDs) has been extensively used for biosensing since the first ECL sensor using CdSe QDs was proposed as an ECL emitter in 2004.12 The advantages of nano-ECL system possessed the following features: (1) the ECL emission potentials could be tuned by surface structures;13 (2) the ECL systems were mild with physiological pH, assisted by various coreactants;14−17 (3) the multiple surface structures provided approaches of further functionalization for bioanal-

epatocellular carcinoma (HCC) is the third most common cause of cancer related death.1 In recent years, screening programs for high-risk patients have gained popularity;2 however, a few patients have been diagnosed with small-sized HCCs (3 cm or less in diameter),3 and more than two-thirds of patients diagnosed with advanced disease have a poor outcome.4 Surveillance of populations at risk based on serum biomarkers might detect tumors at an early stage, when potentially curative therapy in the form of liver transplantation, surgical resection, or tumor ablation can be implemented, leading to a significant reduction in overall mortality.5 Beyond alpha fetoprotein (AFP), the most commonly used serum biomarker for HCC, recent advances have led to the introduction of a more sensitive biomarker, Lens culinaris agglutinin-reactive fraction of AFP (AFP-L3), which showed higher sensitivity and specificity to HCC than AFP at an early stage.6,7 Noteworthy, the portion of AFP-L3 in total AFP (AFP-L3%) is an independent predictor factor, and the patients with negative AFP predictive value in serum, while showing AFP-L3% ≥ 10%, should be highly suspected as HCC.8 However, the measurement of AFP-L3%, which is determined by a reference lectin-affinity electrophoresis assay,9,10 was © XXXX American Chemical Society

Received: March 30, 2015 Accepted: August 20, 2015

A

DOI: 10.1021/acs.analchem.5b02660 Anal. Chem. XXXX, XXX, XXX−XXX

Article


Analytical Chemistry ysis.18 Until now, IIB-VIA type QDs based ECL biosensors for detection of proteins,19,20 cells,21,22 and nucleic acids14 showed high sensitivity, making rapid, sensitive and in vitro detection realizable. In addition, TiO2 nanostructures and QDs composites also acted as an ECL emitter,16,23 showing good stability and lower toxicity for biosensing. Recently, QDs based ECL resonance energy transfer systems with QDs as donor and Au nanoparticles (NPs)24 or luminol25 as acceptor were reported, exhibiting high sensitivity with limits of detection (LODs) of 13 nM for p53 ssDNA and 1.4 fM for thrombin, respectively. QDs-ECL aptasensors for carcinoembryonic antigen26 and adenosine triphosphate27 showed LODs of 3.8 pg mL−1 and 5 pmol L−1, respectively. For clinical purpose, multiple-components detection always plays an important role for laboratory diagnosis.28,29 Until now, QDs-based photoluminescent (PL)30,31 and electrochemical32,33 multiple-components detection strategies have been frequently reported. Hogan’s pioneer works34,35 realized resolution of three ECL peaks from Ru and Ir complexes, by both potential- and wavelength-resolution in acetonitrile. Jiang’s group36 reported a potential-resolution based ECL system in the water phase for simultaneous detection of two proteins on the cell surface, using Ru(bpy)32+/luminol as ECL luminophores. In addition, based on ECL potential-resolution of Ru(bpy)32+/carbon nanodots as luminophores, combining with microfluidics devices and a switch for potential control, multiplexed targets were detected simultaneously by switchover of the detection potential at +1.2 V and −1.2 V.37 These pioneer works provided a huge impulse for multiplexed target detection based on the ECL potential-resolution strategy. However, although because of extremely high sensitivity, the ECL potential-resolution strategy based methodologies for multiple-components detection still showed drawbacks, such as relatively weak ECL intensity in the water phase, instability caused by cross talk of the ECL luminophores, coexistence of strong oxidant and reductant as coreactants in the detection systems, and so on. In our previous work, 2,3-dimercaptosuccinic acid-stabilized CdTe (DMSA-CdTe) QDs exhibited a relatively low emission potential, centered at −0.89 V (vs Ag/ AgCl),13 which was assisted by self-produced coreactant H2O2 from reduction of dissolved oxygen. In combination with commonly used monomercapto stabilized CdTe QDs, such as glutathione stabilized-CdTe (GSH-CdTe) QDs as ECL luminophor (peak potential around −1.25 V), a facile and mild nano-ECL system could be constructed for dual targets simultaneous detection. As homogeneous luminophores, the DMSA- and GSH-CdTe QDs underwent a similar luminescent mechanism in the water phase, in which both of the two ECL emissions were assisted by the only coreactant of self-produced H2O2, leading to a mild and simplified nano-ECL system. Herein, a QDs based dual-targets ECL immunosensor was constructed, for simultaneous detection the model molecules of AFP and its AFP-L3 isoform. AFP-L3%, a novel biomarker for HCC laboratory diagnosis, was calculated with simplification by simultaneous detection of AFP and AFP-L3. By composite with TiO2 NPs, the ECL intensity of GSH-CdTe QDs could be largely increased, making the quantity of CdTe QDs largely decreased. The ECL potential-resolution strategy was realized using two kinds of QDs as luminophores, which occurred in the water phase, with only one coreactant in a single cathodic cyclic voltammetric (CV) scan, showing relatively high ECL intensities. Two separate interfaces of indium tin oxide (ITO) electrodes were modified to specifically recognize AFP

and AFP-L3, respectively. By making use of special resolution due to the unique property of ITO electrodes, the detection strategy was largely simplified without combination of microfluidics devices or other techniques. The sandwich immunorecognition of the immobilized anti-AFP(AFP-L3)/AFP(AFPL3)/anti-AFP(AFP-L3)-horseradish peroxidase (HRP) produced an HRP-activated surface for enzymatic cycle amplification. In the presence of substrate (hydroquinone, HQ), ECL emission of QDs would be quenched due to the consumption of self-produced coreactant H2O2 in the HRP-catalyzed oxidation process of HQ. 38 The ECL signal change corresponding to the immuno-recognition led to high sensitivity and a wide detection range. This work will pave a new avenue for multiple-components ECL biosensing and extend the application of QDs in clinical fields.

■

EXPERIMENTAL SECTION Reagents. Cadmium chloride hemi(pentahydrate) (CdCl2· 2.5H2O), HQ, and 25% glutaraldehyde aqueous solution were purchased from Alfa Aesar Ltd., and bovine serum albumin (BSA) was purchased from Boster Bioengineering Ltd. Co. (China). DMSA, GSH, TiO2 NPs (anatase, 20−30 nm in diameter), chitosan (from crab shells) and Tris-base (reagent grade) were purchased from Sigma Chemical Co. (St. Louis, MO). AFP and AFP-L3 ELISA kits were purchased from AMEKO Ltd. (U.S.). Other reagents were of analytical grade and used as received. The Tris-HCl buffer of 0.1 M containing 0.1 M KNO3 was adjusted to pH 7.4 by adding 1 M HCl. The clinical serum samples were obtained from the Second Affiliated Hospital of Southeast University (Nanjing, China). The ultrapure water (18.2 MΩ, Milli-Q, Millipore) was used throughout the experiments. Apparatus. The electrochemical and ECL measurements were carried out on an MPI-E multifunctional electrochemical and chemiluminescent analytical system (Xi’an Remex Analytical Instrument Ltd. Co., China) at room temperature with a configuration consisted of ITO electrodes (1.0 cm × 0.5 cm, 30 Ω, Nanbo Display Parts Ltd., Shenzhen, China) as the working electrode, a platinum wire as the counter electrode, and a Ag/AgCl (filled with saturated KCl) as the reference electrode. During measurements, the CV technique was used with cathodic potentials ranging from 0 to −1.3 V at a scan rate of 100 mV/s, and the ECL emission signal was recorded in the meantime. The ECL intensities were detected at −0.89 V (for DMSA-CdTe QDs) and −1.25 V (for TiO2-GSH-CdTe QDs composites). The emission window was placed in front of the photomultiplier tube (PMT, detection range from 300 to 650 nm) biased at −600 V. The ECL spectrum for TiO2-GSHCdTe QDs composites was obtained by collecting the ECL intensity data at −1.25 V during cyclic potential sweep with 6 pieces of short-pass filters (2 mm in thickness) at 650, 630, 610, 600, 580, 550 nm, respectively. The transparent efficiency of all filters is around 88%. The PL experiments were performed on an RF-5301 PC fluorometer (Shimadzu Co., Japan). The UV−vis absorption spectrum was obtained on a Shimadzu UV-4100 UV−vis-NIR photospectrometer (Shimadzu Co., Japan). The atomic force microscopy (AFM) images were obtained from Molecular Imaging Pico SPM (U.S.). The transmission electron microscopy (TEM) images were obtained from a JEOL JEM2100 TEM, with an acceleration voltage of 200 kV. FT-IR spectra were recorded on a Nicolet 400 FT-IR spectrometer (Madison, WI). The electrochemical impedance spectroscopies B


Article


Analytical Chemistry (EIS) measurements were carried out on a PGSTAT30/FRA2 system (Autolab, Netherlands). As a control, the reference values of AFP and AFP-L3 concentrations in clinical human serum samples were obtained from an Architect i2000 chemiluminescent immunology analyzer (Abbott Laboratories, U.S.). Synthesis of DMSA-CdTe and GSH-CdTe QDs. The synthesis of DMSA-CdTe and GSH-CdTe QDs were referred to a green one-pot method reported previously.39 Briefly, the electrogenerated Te precursor was produced on a CHI 660B workstation (Austin, TX) by using a Te electrode (99.99% purity) as a working electrode at −1.02 V (vs Ag/AgCl) in the electrolyte, and the mole ratios of Cd2+/DMSA and Cd2+/GSH were 1:1.25 and 1:2.5, respectively. The electrolysis processes proceeded under thoroughly stirred and N2 atmosphere, with a final electric quantity of 0.5 C for the 0.6 mM Cd2+ precursor. The mixtures were maintained at 80 °C for 20 h to obtain the DMSA-CdTe QDs and 8 h to obtain the red emitted GSHCdTe QDs, respectively. The QDs are purified and concentrated with an ultrafilter (Millipore, molecular weight cutoff 10 kDa). The as-prepared QDs were stored at 4 °C before use. Construction of Immunosensor. First, the DMSA-CdTe QDs solution (35 μM, 1.0 mL) was mixed with the isometric volume of isopropyl alcohol and centrifuged at 6000 r/min for 3 min. The precipitation was washed twice using a 1:1 (v/v) mixture of water and isopropyl alcohol and dissolved in 20 μL of ultrapure water, which was then dropped on an ITO electrode surface. TiO2-GSH-CdTe QDs composites were prepared by continuous ultrasonication of 20 μL of 1 mg mL−1 TiO2 NPs suspension and 3 μL of 15 μM GSH-CdTe QDs solution. The composites were then dropped on another ITO electrode. DMSA-CdTe QDs and TiO2-GSH-CdTe QDs composites possess the advantage of excellent absorption performance, thus could adhere to the ITO surface by physical absorption. After drying in air, 10 μL of 0.025% chitosan solution was coated on the QD films (Scheme 1A) to consolidate the QD film as well as for covalent binding of streptavidin. The formed chitosan film was activated by 20 μL of 0.1% glutaraldehyde (prepared in 0.1 M Tris-HCl, pH 8) for 2 h. The electrode was then rinsed with the washing solution (in the ELISA kits) to remove the excess glutaraldehyde and incubated with 15 μL of streptavidin (1 mg mL−1 in standard diluted buffer of the ELISA kits) for 60 min at room temperature and overnight at 4 °C (Scheme 1B). The resulting surface was slowly washed with streams of washing solution. Then biotinylated anti-AFP and biotinylated anti-AFP-L3 were linked to the electrode surface at 37 °C for 1 h, via the high affinity between streptavidin and biotin (Scheme 1C). The naked area of chitosan film was then blocked with 20 μL of 5% BSA solution for 1 h at 37 °C to obtain the dual targets immunosensor (Scheme 1D). Assay Procedures. AFP and AFP-L3 solutions of 20 μL with different concentrations as the incubation solution were dropped on respective specific surfaces of the immunosensor and incubated at 37 °C for 1 h, during which the antigens in the incubation solution react with the binding sites of immobilized antibodies (Scheme 1D). Then 30 μL of HRP-anti-AFP and HRP-anti-AFP-L3 were dropped on the surfaces of the immunosensor to form a sandwich immuno-structure (Scheme 1E). After washed with washing solution, the ECL intensities of immunosensors were recorded in 0.1 M pH 7.4 Tris-HCl buffer

Scheme 1. Layer by Layer Modification and Detection Procedures of Dual Target ECL Immunosensor for AFP and AFP-L3a

a (A) DMSA-CdTe QDs (left)/TiO2-GSH-CdTe QDs composites (right) and chitosan film coated on two ITO electrodes respectively. (B) Streptavidin covalent binding on chitosan film. (C) Biotinylated anti-AFP and biotinylated anti-AFP-L3 linked to the electrode surface via affinity between streptavidin and biotin. (D) Immuno-reaction between antigens and mobilized antibodies after BSA blocking. (E) Sandwich immuno-structure of HRP labeled antibody-antigenbiotinylated antibody on immunosensor and detection procedures of analyte (green arrow, luminenscence; gray arrow, quenching).

containing 0.1 mM HQ to produce the detection signal corresponding to the concentration of analytes (Scheme 1E).

■

RESULTS AND DISCUSSION Characterization of the As-Prepared DMSA- and GSHCdTe QDs. According to high-resolution TEM images (Figure S1 in Supporting Information), the DMSA-CdTe QDs and GSH-CdTe QDs both showed relatively uniform size, ∼1.5 ± 0.5 nm and 3 ± 0.5 nm in diameter, respectively. The formation of DMSA- and GSH-CdTe QDs could be confirmed by the FTIR spectra, using QDs powder acquired by centrifugation of the QDs solutions. In FT-IR spectrum of DMSA-CdTe QDs,13 the stretch vibration peaks of S−H bond at 2566.3 and 2533.4 cm−1 disappeared compared with DMSA, while the asymmetric vibration peak of COO− at 1614.2 cm−1 still exist, indicating the formation of S−Cd bonds between DMSA and CdTe core. Similarly, compared to the standard FT-IR spectrum of GSH, the FT-IR spectrum of GSH-CdTe QDs (Figure S2 in Supporting Information) showed one peak at 1630 cm−1 attributed to the asymmetric vibration peak of COO−, without the 2500−2600 cm−1 peak for stretch vibration peaks of the S− H bond. These results confirmed that the S−H groups of DMSA and GSH molecules linked to CdTe QDs with a stable structure. ECL and Electrochemical Behaviors of DMSA-CdTe QDs and TiO2-GSH-CdTe QDs Composites. To obtain matched intensities, dosages of two kinds of QDs were optimized. As shown in Figure S3 (Supporting Information), the TiO2-GSH-CdTe QDs composites showed much stronger ECL strength than that of DMSA-CdTe QDs. The obtained DMSA-CdTe QDs solutions were uniformly concentrated to 20 μL before use. A volume of 1 mL of obtained DMSA-CdTe C


Article


Analytical Chemistry

cathodic scan.16 OH• could be considered as a crucial parameter for ECL emission of CdTe QDs. The ECL procedures for GSH-CdTe QDs could be expressed as following equations:

QDs showed almost the same ECL intensity as 1.2 mL of QDs. Thus, 1 mL of obtained DMSA-CdTe QDs was selected in the following experiments. Although 3 μL of obtained GSH-CdTe QDs in 20 μL of 1 mg mL−1 TiO2 composite still showed obviously stronger ECL intensity than that of DMSA-CdTe QDs, lowering the dosage of GSH-CdTe QDs could largely decrease the stability of the ECL signal. Considering the ECL signals from 3 μL of GSH-CdTe QDs in 20 μL of 1 mg mL−1 TiO2 NPs composites and 1.0 mL of obtained DMSA-CdTe QDs were almost a match to each other for simultaneous detection, thus, 1.0 mL of obtained 35 μM DMSA-CdTe QDs and 3 μL of 15 μM GSH-CdTe QDs with 20 μL of 1 mg mL−1 TiO2 NPs composites were used throughout the experiments. In air-saturated pH 7.4 Tris-HCl buffer, the simultaneous scan of dual ITO electrodes modified with DMSA-CdTe QDs and TiO2-GSH-CdTe QDs composites showed two emission peaks at −0.90 V and −1.21 V, respectively, with almost comparative intensities (curve a, Figure 1A). Under the same parameters,

GSH‐CdTe + e− → GSH‐CdTe−•

(5)

O2 + 2e− + 2H 2O → H 2O2 + 2OH−

(6)

2GSH‐CdTe•− + H 2O2 → 2OH− + 2GSH‐CdTe*

(7)

•

In the presence of TiO2 NPs, OH radicals significantly enhance ECL through the following reactions: TiO2 + e− → TiO2•−

(8)

TiO2•− + H 2O2 → TiO2 + OH• + OH−

(9)

GSH‐CdTe + OH• → GSH‐CdTe•+ + OH− GSH‐CdTe

•−

+ GSH‐CdTe

•+

→ GSH‐CdTe*

(10) (11)

Then, GSH‐CdTe* → GSH‐CdTe + hv

Figure 1B showed the electrochemical curves of the QDs modified ITO electrodes. The current of combined QDs modified ITO electrodes at −1.3 V (2.91 × 10−3 A, curve a) was nearly the simple addition of the currents on two individual electrode (2.54 × 10−3 A, curves b and c). In addition, the simultaneous scanning of two QDs modified ITO electrodes showed almost stable ECL emissions during 10 continuous CV scans (insert of Figure 1A). Characterization of TiO2-GSH-CdTe Composites. The formation of composites could be verified according to the TEM images of TiO2 NPs (Figure 2A) and TiO2-GSH-CdTe

Figure 1. ECL (A) and electrochemical (B) curves of simultaneous scan of DMSA-CdTe QDs and TiO2-GSH-CdTe QDs composites (a), individual scan of DMSA-CdTe QDs (b), and TiO2-GSH-CdTe QDs composites (c) modified ITO electrodes in air-saturated pH 7.4 TrisHCl buffer. Insert: Continuous cyclic simultaneous scans of DMSACdTe QDs and TiO2-GSH-CdTe QDs composite modified ITO electrodes. Scan rate: 0.1 V s−1.

ECL emission of DMSA-CdTe QDs and TiO2-GSH-CdTe QDs composites modified ITO electrodes showed peak potentials at −0.89 V (curve b, Figure 1A) and −1.25 V (curve c, Figure 1A) during individual scan, showing almost no overlap at their respective peak potentials. The H2O2 produced from reduction of dissolved oxygen worked as a coreactant for DMSA-CdTe QDs ECL emission. TiO2-GSH-CdTe QDs composites showed enhanced ECL intensity compared to GSH-CdTe QDs (not shown), leading to a lowered quantity of Cd contained QDs and a lower toxic ECL system. The emission processes involved in a series of transient radicals. The electron injected radical DMSA-CdTe•− was produced by electric excitation during the cathodic scan and disappeared after reaction with the coreactant of H2O2 to produce DMSA-CdTe*, the CdTe QDs at the excited stage, which gave ECL emission when returned back to the ground state. The ECL procedures of DMSA-CdTe QDs could be expressed as following:13 DMSA‐CdTe + e− → DMSA‐CdTe•−

(1)

O2 + 2e− + 2H 2O → H 2O2 + 2OH−

(2)

Figure 2. TEM images of TiO2 NPs (A) and TiO2-GSH-CdTe composites (B) on the ITO electrode surface.

QDs composites (Figure 2B). The aggregation area should be attributed to the encompassing of GSH-CdTe QDs to TiO2 NPs. The UV−vis (curve a, Figure 3) and PL (curve b, Figure 3) spectra of the as-prepared GSH-CdTe QDs showed an absorption band with an inflection point at 590 nm and a strong emission peak at 628 nm, respectively, which almost overlap with each other. The fitting curve of the ECL spectrum of TiO2-GSH-CdTe QDs composites showed a peak wavelength of 617 nm (curve c, Figure 3), close to PL peak of GSHCdTe QDs, confirming that the ECL luminophor of composites was GSH-CdTe QDs. AFM and EIS Characterization of Immunosensor Construction. The morphological changes of ITO electrodes modified with different assemblies at each step of immunosensor fabrication were investigated by AFM. The AFM image of TiO2-GSH-CdTe QDs composites/chitosan/streptavidin film (Figure 4A) on the ITO substrate showed an undulate height of ∼43 nm. After biotinylated anti-AFP-L3 was bound to the ITO surface by affinity between biotin−streptavidin, the

2DMSA‐CdTe•− + H 2O2 → 2OH− + 2DMSA‐CdTe* (3)

DMSA‐CdTe* → DMSA‐CdTe + hv

(12)

(4)

For TiO2-GSH-CdTe QDs composites, TiO2 NPs acted as a catalyst, which assisted the production of OH• during the D


Article



Figure 3. UV−vis (a, solid line) and PL (b, dash line) spectra of asprepared GSH-CdTe QDs. Fitting curve of ECL spectrum of TiO2GSH-CdTe QDs composites (c, dashed dotted line).

Figure 5. EIS of bare (a), QDs/chitosan (b), QDs/chitosan/ streptavidin (c), QDs/chitosan/streptavidin/biotin-anti-AFP (d), QDs/chitosan/streptavidin/biotin-anti-AFP/BSA (e), and QDs/chitosan/streptavidin/biotin-anti-AFP/BSA/AFP/anti-AFP-HRP (f) modified ITO electrodes in 1 mM [Fe(CN)6]3‑/4‑ containing 0.1 M KCl.

electrode interface, resulting in the increasing impedance of the electrode. AFP/AFP-L3 Dual Targets Detection. Two individual ITO electrodes with respect to specific surfaces for AFP and AFP-L3 were tightened back-to-back as a working electrode in the dual-targets immunosensor. Compared to the QDs modified ITO electrodes, the more negative ECL emission potential of the immunosensor at −0.97 V and −1.28 V (Figure 6A−C) should result from the increased impedance. The

Figure 4. AFM images of TiO2-GSH-CdTe QDs composites/ chitosan/streptavidin film (A), A+biotinylated anti-AFP-L3 film (B), B+BSA film (C), C+AFP-L3 antigen film (D), and D+HRP-anti-AFPL3 film (E) on the ITO electrode surface.

average height increased largely to ∼600 nm, and the aggregation of biotinylated anti-AFP-L3 could be observed (Figure 4B). After blocking with BSA, the surface morphology showed much more coverage of protein than that before blocking, whereas the undulation became more smooth (Figure 4C). After immuno-reactions of AFP-L3 antigen (Figure 4D) and HRP-anti-AFP-L3 (Figure 4E), the height increased by ∼600 and 750 nm, respectively. The significant changes in surface morphology after immunoreaction confirm the specific conjugation between antigen and antibody. The EIS of the resulting ITO electrodes could further confirm the modification processes (Figure 5). The diameter of the semicircle is equal to the electron-transfer resistance, Ret. The bare (curve a) and DMSA-CdTe QDs/chitosan (curve b) modified ITO electrodes showed a relatively small Ret. Then, the proteins of streptavidin (curve c), biotin-anti-AFP (curve d), BSA (curve e), and AFP/anti-AFP-HRP (curve f) could all resist the electron-transfer kinetics of the redox probe at the

Figure 6. Cyclic ECL curves of immunosensor for AFP-L3 detection (A) at concentrations of 0.00324 (a), 0.324 (b), and 32.4 (c) ng mL−1; AFP detection (B) at concentrations of 0.001 (a), 0.01 (b), and 20 (c) ng mL−1. Simultaneous detection (C) at concentrations of AFP-L3 0.324 ng mL−1/AFP 0.1 ng mL−1 (a) and AFP-L3 3.24 ng mL−1/AFP 1 ng mL−1 (b). Linear calibration plots for AFP-L3 (D) and AFP (E) detection.

detection procedures could be expressed as Scheme 1E. All of the AFP and AFP-L3 specific sensing interfaces were fabricated under the same conditions and tightened back-to-back during the codetection, and it was obvious that the two ECL signals showed no overlap at the peak potential of each other (Figure 1); thus, the cross talk could be well avoided, showing similar value during the individual (Figure 6A, b) and simultaneous (Figure 6C, a) detection. In the presence of substrate HQ at 1 mM, the self-produced coreactant H2O2 was consumed via eq E


Article

Analytical Chemistry 13.33 Therefore, the ECL intensity (I) of the immunosensor decreased with the increasing immobilized quantity of HRP, which positively correlated to concentration of analytes. Under the optimal conditions, the linear ranges of AFP-L3 (Figure 6D) and AFP (Figure 6E) detection were 3.24 pg mL−1−32.4 ng mL−1 and 1.0 pg mL−1−20 ng mL−1, respectively, with limits of detection (LODs) of 3.24 pg mL−1 and 1.0 pg mL−1 (take the ECL intensity changes ΔI > 10%). HRP


HQ + H 2O2 ⎯⎯⎯⎯→ quinone + H 2O

serum samples were appropriately diluted with the standard dilution of ELISA kits prior to the assay. In addition, the SDs between the R-value and D-value (SD1), and reference ratio (R-ratio) and detected ratio (D-ratio, SD2) were calculated and listed in Table 1. For all of the 12 D-values, RSDs of 3 replicates of detection were between 1.25% and 10.60% (Table S1 in Supporting Information). These experimental data indicated an acceptable accuracy of the proposed method for further application-oriented research. Considering that our detection platform is easily prepared and disposable, it exhibits great promising applications for future point-of-care testing.

(13)

■

Interference, Stability, and Repeatability Investigation of the Immunosensor. The common protein such as BSA at 5 μg mL−1, compounds such as ascorbic acid, Zn(NO3)2, and FeCl3 at 10 μM showed a negligible effect on the ECL signals from DMSA-CdTe QDs and TiO2-GSH-CdTe QDs composites (Figure S4 in Supporting Information). To evaluate the repeatability, intra- and inter-relative standard deviation (RSD) for ECL signals were calculated, by collecting ECL intensities at −0.89 V (for DMSA-CdTe QDs) and −1.25 V (for GSH-CdTe QDs). The RSDs for five parallel measurements with one immunosensor (intra-assay) was 6.86% at −0.89 V and 9.84% at −1.25 V, indicating a good precision. The detection of five immunosensors fabricated independently (interassay) showed RSDs of 8.69% at −0.89 V and 3.43% at −1.25 V, giving an acceptable fabrication reproducibility of the immunosensors. In addition, the immunosensor stored at 4 °C showed almost consistent intensity when detected in the 1st, 2nd, 5th, and 30th days, with RSD of 5.67%. Analysis of Human Serum Samples. To evaluate the analytical reliability and application potential of the proposed detection method, the dual targets immunosensor was employed to test AFP-L3% in clinical human serum samples by simultaneous detection of AFP and AFP-L3 contents. In Table 1, the assay results of human serum samples using the proposed method (D-value, ng mL−1) were compared with reference values (R-value, ng mL−1) obtained by commercial chemiluminescent immunoassays (Abbott Laboratories). If the levels of AFP-L3 and AFP were out of the calibration range, the

CONCLUSION A dual targets nano-ECL immunosensor was designed, based on potential-resolution by DMSA-CdTe QDs/TiO2-GSHCdTe QDs composites and spatial discrimination of ITO electrodes. By immobilizing the DMSA-CdTe QDs-anti-AFP and TiO2-GSH-CdTe QDs-anti-AFP-L3 on ITO electrodes surface, combined with the enzymatic amplification strategy, on-step test of AFP-L3% could be realized by simultaneous detection of AFP and AFP-L3. The novel methodology showed acceptable sensitivity, precision, and accuracy. The ECL emission of CdTe QDs could be generated in the presence of the self-produced coreactant H2O2. In the presence of enzymatic substrate in the detection solution, the enzymatic amplification greatly increased the sensitivity and extended the detectable concentration range. The immunosensor with simple manipulations showed a wide linear range and good reproducibility. This work provided a new immunoassay strategy for multiple-components detection based on the nano-ECL technique with high sensitivity. One-step detection methodology for the subtype of tumor biomarker AFP, AFPL3%, makes for great convenience for HCC early screening. It would extend the application of QDs in clinical applications.

■

* Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.5b02660. TEM images, FT-IR spectrum, ECL curves, and table of D-values and RSDs (PDF)

Table 1. Comparison of R-value/ratio and D-value/ratio by As-Constructed Dual Targets Immunosensor in Clinical Serum Sample Assays sample 1

2

3

AFP AFPL3 AFP AFPL3 AFP AFPL3 AFP AFPL3 AFP AFPL3 AFP AFPL3

R-ratio (%)

D-ratio (%)

SD2

0.013 0.002

11.6

11.1

0.354

5.75 0.52

0.403 0.056

11.6

9.0

1.838

12.29 2.600

11.38 3.0

0.643 0.283

21.2

26.4

3.677

1.229 0.260

1.46 0.25

0.163 0.007

21.2

17.1

2.900

13.20 0.540

12.91 0.552

0.205 0.008

40.9

42.7

1.273

6.600 0.270

6.847 0.271

0.175 0.001

40.9

39.6

0.919

R-value

D-value

SD1

1.036 0.120

1.054 0.117

5.180 0.600

ASSOCIATED CONTENT

S

■

AUTHOR INFORMATION

Corresponding Authors

*(X.L.) Phone/fax: +86-25-83626060. E-mail: silentsign@163. com. *(H.J.) E-mail: [email protected]. Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS We gratefully acknowledge the support of National Natural Science Foundation of China (Grant 21205012), Natural Science Foundation of Jiangsu Province (Grant BK2012077), and Medical Scientific Research Projects from Jiangsu Ministry of Health (Grant H201236). F


Article



■

(35) Doever, E. H.; Zammit, E.; Barbante, G. J.; Francis, N. W. Chem. Sci. 2013, 4, 977−982. (36) Han, F. F.; Jiang, H.; Fang, D. J.; Jiang, D. C. Anal. Chem. 2014, 86, 6896−6902. (37) Wang, S. W.; Ge, L.; Zhang, Y.; Song, X. R.; Li, N. Q.; Ge, S. G.; Yu, J. H. Lab Chip 2012, 12, 4489−4498. (38) Liu, X.; Zhang, Y. Y.; Lei, J. P.; Xue, Y. D.; Cheng, L. X.; Ju, H. X. Anal. Chem. 2010, 82, 7351−7356. (39) Ge, C. W.; Xu, M.; Liu, J.; Lei, J. P.; Ju, H. X. Chem. Commun. 2008, 450−452.

REFERENCES

(1) El-Serag, H. B.; Rudolph, K. L. Gastroenterology 2007, 132, 2557− 2576. (2) Bruix, J.; Sherman, M. Hepatology 2005, 42, 1208−1236. (3) Ikai, I.; Arii, S.; Okazaki, M.; Okita, K.; Omata, M.; Kojiro, M.; Takayasu, K.; Nakanuma, Y.; Makuuchi, M.; Matsuyama, Y.; Monden, M.; Kudo, M. Hepatol. Res. 2007, 37, 676−691. (4) Stravitz, R. T.; Heuman, D. M.; Chand, N. Am. J. Med. 2008, 121, 119−126. (5) Yu, E. W.; Chie, W. C.; Chen, T. H. Cancer J. 2004, 10, 317−325. (6) Malaguarnera, G.; Giordano, M.; Paladina, I.; Berretta, M.; Cappellani, A.; Malaguarnera, M. Dig. Dis. Sci. 2010, 55, 2744−2755. (7) Xu, W. J.; Guo, B. L.; Han, Y. G.; Shi, L.; Ma, W. S. Tumor Biol. 2014, 35, 12069−12074. (8) Yamamoto, K.; Imamura, H.; Matsuyama, Y.; Kume, Y.; Ikeda, H.; Norman, G. L.; Shums, Z.; Aoki, T.; Hasegawa, K.; Beck, Y.; Sugawara, Y.; Kokudo, N. J. Gastroenterol. 2010, 45, 1272−1282. (9) Kagebayashi, C.; Yamaguchi, I.; Akinaga, A.; Kitano, H.; Yokoyama, K.; Satomura, M.; Kurosawa, T.; Watanabe, M.; Kawabata, T.; Chang, W. Anal. Biochem. 2009, 388, 306−311. (10) Huang, C.; Sheng, S. L.; Sun, X. G.; Liu, J. J.; Huang, G. Clin. Chim. Acta 2014, 431, 232−238. (11) Morimoto, M.; Numata, K.; Nozaki, A.; Kondo, M.; Moriya, S.; Taguri, M.; Morita, S.; Konno, M.; Sugo, A.; Miyajima, E.; Maeda, S.; Tanaka, K. Int. J. Clin. Oncol. 2012, 17, 373−379. (12) Zou, G. Z.; Ju, H. X. Anal. Chem. 2004, 76, 6871−6876. (13) Liu, X.; Cheng, L. X.; Lei, J. P.; Liu, H.; Ju, H. X. Chem. - Eur. J. 2010, 16, 10764−10770. (14) He, L. J.; Wu, M. S.; Xu, J. J.; Chen, H. Y. Chem. Commun. 2013, 49, 1539−1541. (15) Jiang, H.; Wang, X. M. Anal. Chem. 2014, 86, 6872−6878. (16) Ding, S. N.; Gao, B. H.; Shan, D.; Sun, Y. M.; Cosnier, S. Chem. - Eur. J. 2012, 18, 1595−1598. (17) Liu, Y. T.; Lei, J. P.; Huang, Y.; Ju, H. X. Anal. Chem. 2014, 86, 8735−8741. (18) Cheng, Y.; Huang, Y.; Lei, J. P.; Zhang, L.; Ju, H. X. Anal. Chem. 2014, 86, 5158−5163. (19) Yang, M. L.; Chen, Y.; Xiang, Y.; Yuan, R.; Chai, Y. Q. Biosens. Bioelectron. 2013, 50, 393−398. (20) Zhao, P.; Zhou, L. F.; Nie, Z.; Xu, X. H.; Li, W.; Huang, Y.; He, K. Y.; Yao, S. Z. Anal. Chem. 2013, 85, 6279−6286. (21) Jie, G. F.; Zhang, J.; Jie, G. X.; Wang, L. Biosens. Bioelectron. 2014, 52, 69−75. (22) Zhang, L.; Jiang, J. H.; Luo, J. J.; Zhang, L.; Cai, J. Y.; Teng, J. W.; Yang, P. H. Biosens. Bioelectron. 2013, 49, 46−52. (23) Song, Y. Y.; Zhuang, Q. L.; Li, C. Y.; Liu, H. F.; Cao, J.; Gao, Z. D. Electrochem. Commun. 2012, 16, 44−48. (24) Lu, Q.; Wei, W.; Zhou, Z. X.; Zhou, Z. X.; Zhang, Y. J.; Liu, S. Q. Analyst 2014, 139, 2404−2410. (25) Dong, Y. P.; Gao, T. T.; Zhou, Y.; Zhu, J. J. Anal. Chem. 2014, 86, 11373−11379. (26) Shi, G. F.; Cao, J. T.; Zhang, J. J.; Huang, K. J.; Liu, Y. M.; Chen, Y. H.; Ren, S. W. Analyst 2014, 139, 5827−5834. (27) Lu, J. J.; Yan, M.; Ge, L.; Ge, S. G.; Wang, S. W.; Yan, J. X.; Yu, J. H. Biosens. Bioelectron. 2013, 47, 271−277. (28) Perez-Larraya, J. G.; Paris, S.; Idbaih, A.; Dehais, C.; LaigleDonadey, F.; Navarro, S.; Capelle, L.; Mokhtari, K.; Marie, Y.; Sanson, M. Cancer 2014, 120, 3972−3980. (29) Schwab, B.; Rudolph, A.; Jost, E.; Klinge, U. Anticancer Res. 2014, 34, 2745−2754. (30) Noor, M. O.; Krull, U. J. Anal. Chem. 2014, 86, 10331−10339. (31) Qian, Z. S.; Shan, X. Y.; Chai, L. J.; Chen, J. R.; Feng, H. Chem. Eur. J. 2014, 20, 16065−16069. (32) Freeman, R.; Liu, X.; Willner, I. Nano Lett. 2011, 11, 4456− 4461. (33) Liu, G. D.; Wang, J.; Kim, J.; Jan, M. R. Anal. Chem. 2004, 76, 7126−7130. (34) Doeven, E. H.; Barbante, G. J.; Kerr, E.; Hogan, C. F.; Endler, J. A.; Francis, P. S. Anal. Chem. 2014, 86, 2727−2732. G


Quantum Dots Based Potential-Resolution Dual-Targets

Recommend Documents