- Email: [email protected]
A novel PAMAM-Au nanostructure-amplified CdSe quantum dots electrochemiluminescence for ultrasensitive immunoassay
A novel PAMAM-Au nanostructure-amplified CdSe quantum dots electrochemiluminescence for ultrasensitive immunoassay Kai Chen, Zhengkun Lu, Qingmin Meng, Yingqiang Qin, Guifen Jie PII: DOI: Reference:
S1572-6657(15)30016-3 doi: 10.1016/j.jelechem.2015.07.012 JEAC 2180
To appear in: Received date: Revised date: Accepted date:
21 May 2015 28 June 2015 9 July 2015
Please cite this article as: , A novel PAMAM-Au nanostructure-amplified CdSe quantum dots electrochemiluminescence for ultrasensitive immunoassay, (2015), doi: 10.1016/j.jelechem.2015.07.012
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
A
novel
PAMAM-Au
nanostructure-amplified
CdSe
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quantum dots electrochemiluminescence for ultrasensitive
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immunoassay
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Kai Chen, Zhengkun Lu, Qingmin Meng, Yingqiang Qin, Guifen Jie Key Laboratory of Sensor Analysis of Tumor Marker, Ministry of Education, Qingdao University
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of Science and Technology, 266042, P. R. China
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Abstract
A novel PAMAM dendrimers-Au nanostructure was prepared and used to develop an amplified
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CdSe quantum dots (QDs) probe for ultrasensitive electrochemiluminescence (ECL) immunoassay.
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The unique dendrimer-encapsulated Au nanoparticles have numerous functional groups for
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loading enough QDs, and the ECL of QDs was enhanced by the localized surface plasmon resonance (LSPR) of gold nanoparticles (Au NPs). After the PAMAM dendrimer was used to form an effective immobilization matrix for biomolecules, the capture antibody (Ab1) was covalently
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conjugated to the dendrimers. In the presence of the target human IgG (Ag), the secondary antibody (Ab2) modified PAMAM-Au-CdSe QDs signal probe was attached to the electrode. On the basis of the amplified ECL signal of the CdSe QDs, an ultrasensitive signal-on ECL immunosensor was developed. The PAMAM-Au-CdSe QDs nanostructure opens a new promising material direction for ECL biosensing, this method shows considerable promise for diverse clinical applications. Keywords: dendrimers-Au; nanostructure; electrochemiluminescence; immunosensor 1. Introduction Electrogenerated chemiluminescence (ECL) that combines electrochemical and luminescent methods has proved to be a powerful analytical technique for clinical diagnostics
Corresponding author. Tel.: +86-532-84022750; Fax: +86-532-84022750.
E-mail: [email protected] 1
immunoassay[1]
ACCEPTED MANUSCRIPT and DNA analysis,[2] environmental assays,[3] as well as food and water testing.[4] In the past decade, many kinds of semiconductor-based quantum dots (QDs) have been found to have ECL activity,[5] and a series of analytical applications have been developed based on the ECL behaviors of those QDs.[6] However, ECL signal of pure QDs is usually lower than that of luminal or
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Ru(bpy)32+, which is unable to meet the requirements of early diagnosis of low-abundance
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biomarkers. Thus, signal amplification strategies have been developed for ECL trace analysis. The development of novel QDs nanostructure to enhance ECL is of great significance in ECL
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bioassays.
Signal amplification for ECL mainly involves three levels: the matrix, the luminescence quantum efficiency of signal labels, and the number of signal labels. Carbon materials (such as carbon nanotubes,[7] graphene,[8] carbon sphere[9] and carbon nanofiber[10]
and noble metal
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nanostructures are usually used as effective matrix for the indirect signal amplification.[11] In our previous work, an amplified QDs ECL signal probe was developed by using silver-cysteine hybrid
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nanowires.[12]
Gold nanoparticles (GNPs) have attracted much attention in different immunoassay due to their unique physical and chemical properties, such as easily controllable size distribution,
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long-term stability, and friendly biocompatibility with immunospecies. Gold nanoparticles and carbon nanotubes were reported to enhance QDs ECL.[13] Recently, dendrimers polyamidoamine
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(PAMAM), the regular tree-like high branched macromolecules, are receiving considerable attention for applications in chemical and biological areas owing to their numerous functional
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amino groups.[14] It is reported that a unique dendrimers/QDs nanocluster was used to develop an amplified QDs ECL signal probe for cells assays.[6c] A PAMAM-Au/carbon nanotubes nanostructure was used to provide a favorable environment for AChE modification with electrocatalytic characteristics.[15] So far, the PAMAM dendrimers-Au nanostructure has not been
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applied to amplify QDs ECL or develop ECL probe, though they have promising advantages due to numerous functional groups as well as excellent electroconductibility. In this work, we have prepared a new PAMAM-Au nanostructure and used to develop the amplified CdSe QDs ECL probe for ultrasensitive signal-on ECL immunoassay. The dendrimer-encapsulated Au nanoparticles have numerous functional groups for loading enough QDs, and the Au nanoparticles much accelerate the process of ECL reaction due to good conductibility, so ECL signal amplification could be achieved.
On the basis of sandwich
immunoreaction strategy, the amplified Ab2-PAMAM-Au-QDs ECL signal probe was applied to the sensitive ECL immunosensing of human IgG. 2. Experimental 2.1 Syntheses of dual-stabilizers-capped CdSe QDs CdSe QDs were synthesized according to the literature.[16] Briefly, CdCl2 solution (0.20 M, 0.80 mL), HMP (72.5 mg), and MPA (34.6 μL) were dissolved in 50 mL of H2O successively. 2
ACCEPTED MANUSCRIPT Then, pH was adjusted to 9.0, and Na2SeO3 solution (20.0 mM, 0.80 mL) was added to the mixture. After being refluxed at 100 °C for 10 min, the above mixture was added with 3.67 mL of N2H4·H2O and refluxed for another 10 h at 100 °C. The resultant was purified three times by
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isopropyl alcohol with centrifugation at 10000 rpm and stored in the dark at 4 °C. The
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concentration of dual-stabilizers-capped CdSe QDs stock solution was estimated to be 7.10 μM
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with an empirical equation.[17] 2.2 Synthesis of PAMAM-Au nanocomposite
Our approach for the preparation of dendrimer-encapsulated Au metal particles is similar to the
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previously proposed solutions for Pt.[18] PAMAM-Au nanocomposites were prepared as follows: 2.5 ml HAuCl4 solution (0.3 mM) was added to 2.5 mL PAMAM (0.1 mM) with vigorous stirring
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for 20 min. Then, 2.5 mL of formic acid (0.1 mM) was incrementally added (with at least 15 min between additions) into the previous solution. When zerovalent Au complex was formed, the color
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changed from yellow to fuchsia. This reaction took over 4 h. To obtain the different sizes of
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PAMAM-Au particles, various volumes of HAuCl4 were added to the same volume of PAMAM together with an excess of reducing agent to make sure that most of the HAuCl4 was reduced to
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Au. The nanocomposites growth kinetics was followed by UV–Vis spectroscopy. And the morphology and particle sizes of the nanocomposites were characterized using a transmission electron microscopy (TEM) image.
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2.3 Preparation of the PAMAM-Au-CdSe QDs ECL probe The CdSe QDs solution (250 μL) was activated with EDC (0.1 M, 10 μL) and NHS (0.025 M, 10 μL) for 30 min, then 50 μL of PAMAM-Au and 25 μL of Ab2 (2.5×10-3 mg/mL) were added and reacted at 37 ◦C for 12 h with gentle shaking. After centrifugation at 5000 rpm for 15 min to remove excess reagents. The obtained PAMAM-Au-CdSe QDs-Ab2 were washed with PBS containing 0.05% Tween-20 three times and resuspended in 250 μL of buffer. Then 100 μL of 10-5 M DNA was added and reacted at 37 ◦C for 12 h with gentle shaking. The resulting PAMAM-Au-CdSe QDs-Ab2 was then centrifugated at 10000 rpm for 15 min, and resuspended in 500 μL of buffer. 2.4 Amplified ECL immunoassay based on PAMAM-Au nanostructure 7 μL of 2% PAMAM solution was dropped on the cleaned electrodes and dried. After 100 μL
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ACCEPTED MANUSCRIPT of 0.28 mg/mL Ab1 was activated with EDC (10 μL of 0.1 M) and NHS (10 μL of 0.025 M) for 30 min, 280 μL of PBS（0.01 mol/L, pH=7.4）was added and shaked homogeneously. Then the electrodes were immersed in the activated Ab1 solution and incubated at 25 °C in a moisture
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atmosphere for at least 12 h. After incubating in 1% BSA at 37 ◦C for 50 min, the Ab1-modified
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electrodes were incubated in human IgG (Ag) of different concentrations at 37 °C for 50 min, then
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in PAMAM-Au-QDs ECL probe for 50 min, and washed with pH 7.4 PBS for ECL measurements. The modified electrodes above were in contact with 0.1 mol·L-1 PBS (pH 7.4) containing 0.1 mol·L-1 K2S2O8 and 0.1 mol·L-1 KCl and scanned from 0 to –1.5 V. ECL signals related to the IgG
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concentrations were measured. 3. Results and discussion
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3.1. Preparation and characterization of the PAMAM-Au-CdSe QDs probe Figure 1A showed the transmission electron microscopy (TEM) image of the CdSe QDs, the
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average diameter was about 10 nm. Figure 1B presented the photoluminescence (PL) spectra of
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the CdSe QDs, the PL emission peak was at 583 nm, and the intensity was high, indicating the
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CdSe QDs possess good luminescent property.
Figure 1. Transmission electron microscopy (TEM) image (A), and photoluminescence (PL) spectra (B) of the CdSe QDs Figure S1 dispalys the absorption spectra of the PAMAM-HAuCl4 solution (curve a) and dendrimer-encapsulated AuNPs (curve b), respectively. PAMAM-HAuCl4 has a ligand–metal charge-transfer band at 226 nm. After reduction of the composite, the spectrum changes significantly, there is absorption peak at 520 nm (curve b), which results from AuNPs. The fabrication procedure of the PAMAM-Au-CdSe QDs probe is shown in Scheme 1. First,
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ACCEPTED MANUSCRIPT as the PAMAM are high branched macromolecules with numerous amino groups, large number of CdSe QDs were assembled on the PAMAM-Au nanostructure by covalent bond, so the CdSe QDs ECL was amplified, which could improved the detection sensitivity. Then, the antibody (Ab2) and
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bio-bar-code (bbc) DNA with amino-groups were covalently conjugated to the nanostructure. Finally, BSA was used to block nonspecific binding sites of the nanostructure. The resulting
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PAMAM-Au-CdSe QDs probe was characterized by TEM image. As shown in Figure 2A, the pure PAMAM-Au nanostructure has an average diameter of 30-40 nm, the particle was very smooth and clear. By comparison, after the CdSe QDs, Ab2 and bbc DNA were linked to the nanostructure
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to fabricate the QDs probe (Figure 2B), the distinctive difference in the topography can be observed, the particle surface became more vague and rough, and the diameter was obviously
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larger (Figure 2B). The spherical structure was attributed to the core of gold nanoparticles. In addition, the structure of the TEM image was amplified (Figure 2C), the black particles on the
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surface were the CdSe QDs. These results indicate that the PAMAM-Au-CdSe QDs probe was
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successfully fabricated.
Scheme 1. The principle for amplified ECL immunoassay based on PAMAM-Au-CdSe QDs probe
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Figure 2. TEM image of the PAMAM-Au nanostructure (A), and the PAMAM-Au-CdSe QDs
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probe (B). Inset C: The amplified TEM image of (B).
3.2. Amplified ECL immunoassay based on PAMAM-Au-CdSe QDs probe
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The principle for amplified ECL immunoassay based on the PAMAM-Au-CdSe QDs probe is
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depicted in Scheme 1. The dendrimers PAMAM with high stability and bioactivity were firstly assembled on the Au electrode, then the capture antibody (Ab1) was covalently linked to the
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PAMAM. After BSA was used to block nonspecific binding sites, Ab1 specifically bind to its target-antigen IgG, followed by immunoreaction with PAMAM-Au-CdSe QDs signal probe. On the basis of sandwich-type immunoassay format, the signal-on ECL immunosensor was
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developed.
The fabrication process of the ECL immunosensor was characterized by electrochemical impedance spectroscopy (EIS). As shown in Figure S2, when PAMAM was firstly immobilized on the electrode, the electron transfer resistance obviously increased (curve b) compared with that at the bare Au electrode (curve a). Subsequently, with the step-by-step immobilization of Ab1, BSA and Ag on the electrode, the electron transfer resistance gradually increased (curve c, d) because of the insulating protein layer. Finally, the PAMAM-Au-CdSe QDs signal probe was linked to the electrode, the EIS became much larger (curve e). Therefore, EIS results confirm that the ECL immunosensor was successfully fabricated. In addition, Figure S3 shows the field-emission scanning electron microscopy (FE-SEM) images of the modified electrode. When the pure PAMAM dendrimer was firstly immobilized on
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ACCEPTED MANUSCRIPT the Au electrode, it was observed that many nanospheres with an average diameter of 30 nm are uniform in size and morphology (Figure S3A). Subsequently, after Ab1 was covalently conjugated to the dendrimers, the Ab2 modified PAMAM-Au-CdSe QDs signal probe was attached to the
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electrode via sandwich immunoreaction. It was found that a thick film with many nanoparticles was homogeneously formed on the electrode (Figure S3B), indicating the successful fabrication of
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the ECL immunosensor.
The feasibility of the sandwich ECL immunoassay of IgG was further examined. As shown in inset of Figure 3A, in the absence of target IgG (Ag), the ECL signal was very low, indicating that
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little unspecific binding occurs. In the presence of target IgG, after the sandwich immunoreaction of Ab2-PAMAM-Au-CdSe QDs signal probe with IgG, the electrode displayed obvious higher
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ECL signal (curves a~g), suggesting the ECL immunosensor was feasible for the IgG detection. Figure 3A (curves a~g) showed the typical ECL signal responses for different concentrations of
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IgG. With the increase of IgG concentrations, the ECL peak signal gradually increased. As the
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specific immunoreaction of IgG with more QDs signal probe enable more QDs to assemble on the electrode, thus the ECL signal gradually increased, indicating that the ECL immunosensor could
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be applied to signal-on ECL detection of IgG concentration.
Figure 3. (A) ECL signal responses upon different concentrations of IgG (a~g), the concentration of IgG was (a) 0.1, (b) 1, (c) 10, (d) 50, (e) 100, (f) 500, (g) 1000 pg mL−1 ; Inset: ECL signal response of the electrode in the absence of IgG; (B) Relationship between ECL signals and IgG concentrations In addition, the control experiment was conducted by using the Au-DNA-CdSe QDs as ECL signal probe, the ECL response was shown in the Figure S4. By comparison, ECL signal was obviously lower than that of Ab2-PAMAM-Au-CdSe QDs signal probe. The reason may be that
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ACCEPTED MANUSCRIPT limited QDs were linked to the gold nanoparticles by bbc DNA, while large number of QDs were assembled on the PAMAM-Au nanostructure and the LSPR also enhanced the ECL of QDs. Figure 3B displays the ECL peak intensity increased with IgG concentrations in the range of
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0.1−1000 pg mL−1, and the standard calibration curve for IgG detection was shown in Figure 4A. The ECL signal was logarithmically related to the IgG concentrations in the range of 0.1−500 pg
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mL−1 (Figure 4A) with a detection limit of 0.05 pg mL−1 (R = 0.994) at 3σ, which is comparable to other nanomaterials-based ECL immunosensor.[12-13] According to the linear equation, the IgG concentration was quantitatively measured. A series of five duplicate measurements of 50 pg mL−1
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were used for estimating the precision, and the relative standard deviation (RSD) was 5.3%,
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showing good reproducibility of the ECL method.
Figure 4. (A) Standard calibration curve for IgG detection; (B) Specificity for the determination
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of human IgG using the ECL immunosensor: (a) IgG; (b) BSA, (c) thrombin, (d) lysozyme The specificity of the ECL immunosensor for IgG detection was studied, the influences of some other proteins such as BSA, thrombin, and lysozyme were examined by analyzing the IgG solutions containing interfering substances. Figure 4B shows that none of these proteins caused obvious ECL change even at 100 pg mL−1, while only 10 pg mL−1 of IgG resulted in significant ECL enhancement, indicating those proteins did not interfere with the ECL assay of IgG. The ECL immunosensor exhibited good specificity for IgG. After the immunosensor was stored in pH 7.4 PBS at 4 ◦C over 2 weeks, the analytical performances did not show an obvious decline, demonstrating that the immunosensor had good stability. The reproducibility of the proposed method was estimated by determining 100 pg mL−1 IgG with four immunosensors made at the same electrode. Four measurements from the batch resulted in a relative standard deviation of 6.3%, indicating good reproducibility of the immunosensor. 8
ACCEPTED MANUSCRIPT 3.3. Application of the ECL immunosensor in human IgG levels The feasibility of the ECL immunosensor for clinical applications was investigated by analyzing IgG in human serum. Table 1 shows the comparison between the assay results obtained
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by the immunosensor and the reference values by the ELISA method. There is no obvious difference between the results and ELISA method. Thus, the developed immunosensor could be
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satisfactorily applied to the clinical determination of IgG levels in human serum. Table 1. Comparison of Human Serum IgG by the ECL Immunosensor and ELISA 1
ECL immunosensor
5.0
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serum samples
[pg mL−1]a ELISA [pg mL−1]
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4.75
relative deviation [%]
3
20.0
100.0
19.2
103.6
4.2
-3.5
value from five successive measurements.
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a Average
5.3
2
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4. Conclusions
In summary, we have prepared a novel PAMAM dendrimers-Au nanostructure with good
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bioactivity and electroconductibility, and used it to develop an amplified CdSe QDs ECL probe for ultrasensitive immunoassay. The unique nanostructure was used not only to assemble a large
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number of QDs signal labels, but also to accelerate the ECL reaction efficiency, which thus greatly amplify the QDs ECL signal. The PAMAM dendrimer was applied to construct an amplified immobilization platform for antibody molecules. Based on the specific sandwich immunoreactions, an ultrasensitive signal-on ECL immunosensor using the amplified QDs ECL signal probe was developed for IgG assay. The PAMAM dendrimers-Au-QDs nanostructure opened a new kind of QDs nanocomposites in ECL bioassay. This method provided a promising alternative tool for detection of protein in clinical laboratory. Acknowledgments This work was supported by the National Natural Science Foundation of China (No. 21175078). References [1] C. Dodeigne, L.Thunus, R. Lejeune, Talanta 51 (2000) 415−439. [2] (a) Y. Chen, J. Xu, J. Su, Y. Xiang, R.Yuan, Y. Chai, Anal. Chem. 84 (2012) 7750−7755; (b) G. 9
ACCEPTED MANUSCRIPT F. Jie, Y. Q. Qin, Q. M. Meng, J. L.Wang, Analyst 140 (2015) 79 – 82. 3 L.
mi - racia, A. M.
arc a-Campa a, . . Huertas-P rez, F. J. Lara, Anal. Chim. Acta.
640 (2009) 7−28.
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[4] M. M. Richter, Chem. Rev. 104 (2004) 3003−3036.
[5] (a) Z. Ding, B. M. Quinn, S. K. Haram, L. E. Pell, B. A. Korgel, A. J. Bard, Science 296
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(2002) 1293−1297; (b) Y. Bae, N. Myung, A. J. Bard, Nano Lett. 4 (2004) 1153−1161. [6] (a) G. F. Jie, J. X. Yuan, Anal. Chem. 84 (2012) 2811−2817; (b) H. Jiang, H. Ju, Anal. Chem. 79 (2007) 6690−6696; (c) G. F. Jie, L. Wang, J. X. Yuan, S. S. Zhang, Anal. Chem. 83 (2011)
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3873–3880; (d) Y. Shan, J. Xu, H. Chen, Chem. Commun. 8 (2009) 905−907. [7] J. Li, L. R. Guo, W. Gao, X. H. Xia, L. M. Zheng, Chem. Commun. 48 (2009) 7545−7547.
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[8] (a) X. Zhao, S. Zhou, L. Jiang, W. Hou, Q. Shen, J. Zhu, Chem. Eur. J. 18 (2012) 4974−4981; (b) S. Zhang, Y. Shao, H.-g. Liao, J. Liu, I. A. Aksay, G. Yin, Y. Lin, Chem. Mater. 23 (2011)
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1079−1081; (c) S. Wang, X.Wang, S. P. Jiang, Phys. Chem. Chem. Phys. 13 (2011)
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6883−6891.
[9] S. B. Yoon, K. Sohn, J. Y. Kim, C. H. Shin, J. S. Yu, T. Hyeon, Adv. Mater. 14 (2002) 19−21.
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[10] Q. Zhu, M. Han, H. Wang, L. Liu, J. Bao, Z. Dai, J. Shen, Analyst 135 (2010) 2579−2584. [11] J. Wang, H. Han, X. Jiang, L. Huang, L. Chen, N. Li, Anal. Chem. 84 (2012) 4893−4899. [12] T. Y. Huang, Q. M. Meng, G. F. Jie, Biosens. Bioelectron. 66 (2015) 84 – 88
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[13] (a) G. F. Jie, L. L. Li, C. Chen, J. Xuan, J. J. Zhu, Biosens. Bioelectron. 24 (2009) 3352–3358; (b) G. F. Jie, J. J. Zhang, D. C. Wang, C. Cheng, H. Y. Chen, J. J. Zhu, Anal. Chem. 80 (2008) 4033–4039.
[14] (a) P. Antoni, Y. Hed, A. Hordberg, D. Nyström, H. Holst, A. Hult, M. Malkoch, Angew. Chem. 121 (2009) 2160–2164; (b) X. Peng, Q. Pan, G. L. Rempel, Chem. Soc. Rev. 37 (2008) 1619–1628. [15] Y. Qu, Q. Sun, F. Xiao, G. Shi, L. Jin, Bioelectrochemistry 77 (2010) 139–144. [16] S. Liu, X. Zhang, Y. Yu, G. Zou, Biosens. Bioelectron. 55 (2014) 203−208. [17] W. W. Yu, L. H. Qu, W. Z. Guo, X. G. Peng, Chem. Mater. 15 (2003) 2854−2860. [18] M. Q. Zhao, M. C. Richard, Angew. Chem. Int. Ed. 38 (1999) 364–366.
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Figure captions
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Figure 1. Transmission electron microscopy (TEM) image (A), and photoluminescence (PL)
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spectra (B) of the CdSe QDs
Scheme 1. The principle for amplified ECL immunoassay based on PAMAM-Au-CdSe QDs
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probe
Figure 2. TEM image of the PAMAM-Au nanostructure, (A) and the PAMAM-Au-CdSe QDs probe (B). Inset C: The amplified TEM image of (B).
Figure 3. (A) ECL signal responses upon different concentrations of IgG (a~g), Inset: ECL signal
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response of the electrode in the absence of IgG; (B) Relationship between ECL signals and IgG concentrations
Figure 4. (A) Standard calibration curve for IgG detection; (B) Specificity for the determination
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of human IgG using the ECL immunosensor: (a) IgG; (b) BSA, (c) thrombin, (d) lysozyme
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Graphical abstract
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ACCEPTED MANUSCRIPT Highlights
A novel amplified PAMAM-Au-CdSe QDs ECL probe was fabricated.
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An ultrasensitive signal-on ECL immunosensor was developed using the ECL probe.
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The QDs nanostructure opens a new promising material for ECL biosensing.
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1.
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S1572-6657(15)30016-3 doi: 10.1016/j.jelechem.2015.07.012 JEAC 2180
To appear in: Received date: Revised date: Accepted date:
21 May 2015 28 June 2015 9 July 2015
Please cite this article as: , A novel PAMAM-Au nanostructure-amplified CdSe quantum dots electrochemiluminescence for ultrasensitive immunoassay, (2015), doi: 10.1016/j.jelechem.2015.07.012
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
A
novel
PAMAM-Au
nanostructure-amplified
CdSe
T
quantum dots electrochemiluminescence for ultrasensitive
IP
immunoassay
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Kai Chen, Zhengkun Lu, Qingmin Meng, Yingqiang Qin, Guifen Jie Key Laboratory of Sensor Analysis of Tumor Marker, Ministry of Education, Qingdao University
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of Science and Technology, 266042, P. R. China
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Abstract
A novel PAMAM dendrimers-Au nanostructure was prepared and used to develop an amplified
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CdSe quantum dots (QDs) probe for ultrasensitive electrochemiluminescence (ECL) immunoassay.
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The unique dendrimer-encapsulated Au nanoparticles have numerous functional groups for
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loading enough QDs, and the ECL of QDs was enhanced by the localized surface plasmon resonance (LSPR) of gold nanoparticles (Au NPs). After the PAMAM dendrimer was used to form an effective immobilization matrix for biomolecules, the capture antibody (Ab1) was covalently
AC
conjugated to the dendrimers. In the presence of the target human IgG (Ag), the secondary antibody (Ab2) modified PAMAM-Au-CdSe QDs signal probe was attached to the electrode. On the basis of the amplified ECL signal of the CdSe QDs, an ultrasensitive signal-on ECL immunosensor was developed. The PAMAM-Au-CdSe QDs nanostructure opens a new promising material direction for ECL biosensing, this method shows considerable promise for diverse clinical applications. Keywords: dendrimers-Au; nanostructure; electrochemiluminescence; immunosensor 1. Introduction Electrogenerated chemiluminescence (ECL) that combines electrochemical and luminescent methods has proved to be a powerful analytical technique for clinical diagnostics
Corresponding author. Tel.: +86-532-84022750; Fax: +86-532-84022750.
E-mail: [email protected] 1
immunoassay[1]
ACCEPTED MANUSCRIPT and DNA analysis,[2] environmental assays,[3] as well as food and water testing.[4] In the past decade, many kinds of semiconductor-based quantum dots (QDs) have been found to have ECL activity,[5] and a series of analytical applications have been developed based on the ECL behaviors of those QDs.[6] However, ECL signal of pure QDs is usually lower than that of luminal or
T
Ru(bpy)32+, which is unable to meet the requirements of early diagnosis of low-abundance
IP
biomarkers. Thus, signal amplification strategies have been developed for ECL trace analysis. The development of novel QDs nanostructure to enhance ECL is of great significance in ECL
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bioassays.
Signal amplification for ECL mainly involves three levels: the matrix, the luminescence quantum efficiency of signal labels, and the number of signal labels. Carbon materials (such as carbon nanotubes,[7] graphene,[8] carbon sphere[9] and carbon nanofiber[10]
and noble metal
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nanostructures are usually used as effective matrix for the indirect signal amplification.[11] In our previous work, an amplified QDs ECL signal probe was developed by using silver-cysteine hybrid
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nanowires.[12]
Gold nanoparticles (GNPs) have attracted much attention in different immunoassay due to their unique physical and chemical properties, such as easily controllable size distribution,
D
long-term stability, and friendly biocompatibility with immunospecies. Gold nanoparticles and carbon nanotubes were reported to enhance QDs ECL.[13] Recently, dendrimers polyamidoamine
TE
(PAMAM), the regular tree-like high branched macromolecules, are receiving considerable attention for applications in chemical and biological areas owing to their numerous functional
CE P
amino groups.[14] It is reported that a unique dendrimers/QDs nanocluster was used to develop an amplified QDs ECL signal probe for cells assays.[6c] A PAMAM-Au/carbon nanotubes nanostructure was used to provide a favorable environment for AChE modification with electrocatalytic characteristics.[15] So far, the PAMAM dendrimers-Au nanostructure has not been
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applied to amplify QDs ECL or develop ECL probe, though they have promising advantages due to numerous functional groups as well as excellent electroconductibility. In this work, we have prepared a new PAMAM-Au nanostructure and used to develop the amplified CdSe QDs ECL probe for ultrasensitive signal-on ECL immunoassay. The dendrimer-encapsulated Au nanoparticles have numerous functional groups for loading enough QDs, and the Au nanoparticles much accelerate the process of ECL reaction due to good conductibility, so ECL signal amplification could be achieved.
On the basis of sandwich
immunoreaction strategy, the amplified Ab2-PAMAM-Au-QDs ECL signal probe was applied to the sensitive ECL immunosensing of human IgG. 2. Experimental 2.1 Syntheses of dual-stabilizers-capped CdSe QDs CdSe QDs were synthesized according to the literature.[16] Briefly, CdCl2 solution (0.20 M, 0.80 mL), HMP (72.5 mg), and MPA (34.6 μL) were dissolved in 50 mL of H2O successively. 2
ACCEPTED MANUSCRIPT Then, pH was adjusted to 9.0, and Na2SeO3 solution (20.0 mM, 0.80 mL) was added to the mixture. After being refluxed at 100 °C for 10 min, the above mixture was added with 3.67 mL of N2H4·H2O and refluxed for another 10 h at 100 °C. The resultant was purified three times by
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isopropyl alcohol with centrifugation at 10000 rpm and stored in the dark at 4 °C. The
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with an empirical equation.[17] 2.2 Synthesis of PAMAM-Au nanocomposite
Our approach for the preparation of dendrimer-encapsulated Au metal particles is similar to the
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previously proposed solutions for Pt.[18] PAMAM-Au nanocomposites were prepared as follows: 2.5 ml HAuCl4 solution (0.3 mM) was added to 2.5 mL PAMAM (0.1 mM) with vigorous stirring
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for 20 min. Then, 2.5 mL of formic acid (0.1 mM) was incrementally added (with at least 15 min between additions) into the previous solution. When zerovalent Au complex was formed, the color
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changed from yellow to fuchsia. This reaction took over 4 h. To obtain the different sizes of
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PAMAM-Au particles, various volumes of HAuCl4 were added to the same volume of PAMAM together with an excess of reducing agent to make sure that most of the HAuCl4 was reduced to
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Au. The nanocomposites growth kinetics was followed by UV–Vis spectroscopy. And the morphology and particle sizes of the nanocomposites were characterized using a transmission electron microscopy (TEM) image.
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2.3 Preparation of the PAMAM-Au-CdSe QDs ECL probe The CdSe QDs solution (250 μL) was activated with EDC (0.1 M, 10 μL) and NHS (0.025 M, 10 μL) for 30 min, then 50 μL of PAMAM-Au and 25 μL of Ab2 (2.5×10-3 mg/mL) were added and reacted at 37 ◦C for 12 h with gentle shaking. After centrifugation at 5000 rpm for 15 min to remove excess reagents. The obtained PAMAM-Au-CdSe QDs-Ab2 were washed with PBS containing 0.05% Tween-20 three times and resuspended in 250 μL of buffer. Then 100 μL of 10-5 M DNA was added and reacted at 37 ◦C for 12 h with gentle shaking. The resulting PAMAM-Au-CdSe QDs-Ab2 was then centrifugated at 10000 rpm for 15 min, and resuspended in 500 μL of buffer. 2.4 Amplified ECL immunoassay based on PAMAM-Au nanostructure 7 μL of 2% PAMAM solution was dropped on the cleaned electrodes and dried. After 100 μL
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ACCEPTED MANUSCRIPT of 0.28 mg/mL Ab1 was activated with EDC (10 μL of 0.1 M) and NHS (10 μL of 0.025 M) for 30 min, 280 μL of PBS（0.01 mol/L, pH=7.4）was added and shaked homogeneously. Then the electrodes were immersed in the activated Ab1 solution and incubated at 25 °C in a moisture
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atmosphere for at least 12 h. After incubating in 1% BSA at 37 ◦C for 50 min, the Ab1-modified
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electrodes were incubated in human IgG (Ag) of different concentrations at 37 °C for 50 min, then
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in PAMAM-Au-QDs ECL probe for 50 min, and washed with pH 7.4 PBS for ECL measurements. The modified electrodes above were in contact with 0.1 mol·L-1 PBS (pH 7.4) containing 0.1 mol·L-1 K2S2O8 and 0.1 mol·L-1 KCl and scanned from 0 to –1.5 V. ECL signals related to the IgG
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concentrations were measured. 3. Results and discussion
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3.1. Preparation and characterization of the PAMAM-Au-CdSe QDs probe Figure 1A showed the transmission electron microscopy (TEM) image of the CdSe QDs, the
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the CdSe QDs, the PL emission peak was at 583 nm, and the intensity was high, indicating the
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Figure 1. Transmission electron microscopy (TEM) image (A), and photoluminescence (PL) spectra (B) of the CdSe QDs Figure S1 dispalys the absorption spectra of the PAMAM-HAuCl4 solution (curve a) and dendrimer-encapsulated AuNPs (curve b), respectively. PAMAM-HAuCl4 has a ligand–metal charge-transfer band at 226 nm. After reduction of the composite, the spectrum changes significantly, there is absorption peak at 520 nm (curve b), which results from AuNPs. The fabrication procedure of the PAMAM-Au-CdSe QDs probe is shown in Scheme 1. First,
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ACCEPTED MANUSCRIPT as the PAMAM are high branched macromolecules with numerous amino groups, large number of CdSe QDs were assembled on the PAMAM-Au nanostructure by covalent bond, so the CdSe QDs ECL was amplified, which could improved the detection sensitivity. Then, the antibody (Ab2) and
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bio-bar-code (bbc) DNA with amino-groups were covalently conjugated to the nanostructure. Finally, BSA was used to block nonspecific binding sites of the nanostructure. The resulting
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PAMAM-Au-CdSe QDs probe was characterized by TEM image. As shown in Figure 2A, the pure PAMAM-Au nanostructure has an average diameter of 30-40 nm, the particle was very smooth and clear. By comparison, after the CdSe QDs, Ab2 and bbc DNA were linked to the nanostructure
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to fabricate the QDs probe (Figure 2B), the distinctive difference in the topography can be observed, the particle surface became more vague and rough, and the diameter was obviously
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larger (Figure 2B). The spherical structure was attributed to the core of gold nanoparticles. In addition, the structure of the TEM image was amplified (Figure 2C), the black particles on the
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Scheme 1. The principle for amplified ECL immunoassay based on PAMAM-Au-CdSe QDs probe
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Figure 2. TEM image of the PAMAM-Au nanostructure (A), and the PAMAM-Au-CdSe QDs
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probe (B). Inset C: The amplified TEM image of (B).
3.2. Amplified ECL immunoassay based on PAMAM-Au-CdSe QDs probe
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The principle for amplified ECL immunoassay based on the PAMAM-Au-CdSe QDs probe is
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depicted in Scheme 1. The dendrimers PAMAM with high stability and bioactivity were firstly assembled on the Au electrode, then the capture antibody (Ab1) was covalently linked to the
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PAMAM. After BSA was used to block nonspecific binding sites, Ab1 specifically bind to its target-antigen IgG, followed by immunoreaction with PAMAM-Au-CdSe QDs signal probe. On the basis of sandwich-type immunoassay format, the signal-on ECL immunosensor was
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The fabrication process of the ECL immunosensor was characterized by electrochemical impedance spectroscopy (EIS). As shown in Figure S2, when PAMAM was firstly immobilized on the electrode, the electron transfer resistance obviously increased (curve b) compared with that at the bare Au electrode (curve a). Subsequently, with the step-by-step immobilization of Ab1, BSA and Ag on the electrode, the electron transfer resistance gradually increased (curve c, d) because of the insulating protein layer. Finally, the PAMAM-Au-CdSe QDs signal probe was linked to the electrode, the EIS became much larger (curve e). Therefore, EIS results confirm that the ECL immunosensor was successfully fabricated. In addition, Figure S3 shows the field-emission scanning electron microscopy (FE-SEM) images of the modified electrode. When the pure PAMAM dendrimer was firstly immobilized on
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electrode via sandwich immunoreaction. It was found that a thick film with many nanoparticles was homogeneously formed on the electrode (Figure S3B), indicating the successful fabrication of
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the ECL immunosensor.
The feasibility of the sandwich ECL immunoassay of IgG was further examined. As shown in inset of Figure 3A, in the absence of target IgG (Ag), the ECL signal was very low, indicating that
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little unspecific binding occurs. In the presence of target IgG, after the sandwich immunoreaction of Ab2-PAMAM-Au-CdSe QDs signal probe with IgG, the electrode displayed obvious higher
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ECL signal (curves a~g), suggesting the ECL immunosensor was feasible for the IgG detection. Figure 3A (curves a~g) showed the typical ECL signal responses for different concentrations of
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IgG. With the increase of IgG concentrations, the ECL peak signal gradually increased. As the
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specific immunoreaction of IgG with more QDs signal probe enable more QDs to assemble on the electrode, thus the ECL signal gradually increased, indicating that the ECL immunosensor could
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Figure 3. (A) ECL signal responses upon different concentrations of IgG (a~g), the concentration of IgG was (a) 0.1, (b) 1, (c) 10, (d) 50, (e) 100, (f) 500, (g) 1000 pg mL−1 ; Inset: ECL signal response of the electrode in the absence of IgG; (B) Relationship between ECL signals and IgG concentrations In addition, the control experiment was conducted by using the Au-DNA-CdSe QDs as ECL signal probe, the ECL response was shown in the Figure S4. By comparison, ECL signal was obviously lower than that of Ab2-PAMAM-Au-CdSe QDs signal probe. The reason may be that
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0.1−1000 pg mL−1, and the standard calibration curve for IgG detection was shown in Figure 4A. The ECL signal was logarithmically related to the IgG concentrations in the range of 0.1−500 pg
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mL−1 (Figure 4A) with a detection limit of 0.05 pg mL−1 (R = 0.994) at 3σ, which is comparable to other nanomaterials-based ECL immunosensor.[12-13] According to the linear equation, the IgG concentration was quantitatively measured. A series of five duplicate measurements of 50 pg mL−1
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were used for estimating the precision, and the relative standard deviation (RSD) was 5.3%,
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showing good reproducibility of the ECL method.
Figure 4. (A) Standard calibration curve for IgG detection; (B) Specificity for the determination
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of human IgG using the ECL immunosensor: (a) IgG; (b) BSA, (c) thrombin, (d) lysozyme The specificity of the ECL immunosensor for IgG detection was studied, the influences of some other proteins such as BSA, thrombin, and lysozyme were examined by analyzing the IgG solutions containing interfering substances. Figure 4B shows that none of these proteins caused obvious ECL change even at 100 pg mL−1, while only 10 pg mL−1 of IgG resulted in significant ECL enhancement, indicating those proteins did not interfere with the ECL assay of IgG. The ECL immunosensor exhibited good specificity for IgG. After the immunosensor was stored in pH 7.4 PBS at 4 ◦C over 2 weeks, the analytical performances did not show an obvious decline, demonstrating that the immunosensor had good stability. The reproducibility of the proposed method was estimated by determining 100 pg mL−1 IgG with four immunosensors made at the same electrode. Four measurements from the batch resulted in a relative standard deviation of 6.3%, indicating good reproducibility of the immunosensor. 8
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satisfactorily applied to the clinical determination of IgG levels in human serum. Table 1. Comparison of Human Serum IgG by the ECL Immunosensor and ELISA 1
ECL immunosensor
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[pg mL−1]a ELISA [pg mL−1]
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4. Conclusions
In summary, we have prepared a novel PAMAM dendrimers-Au nanostructure with good
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bioactivity and electroconductibility, and used it to develop an amplified CdSe QDs ECL probe for ultrasensitive immunoassay. The unique nanostructure was used not only to assemble a large
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number of QDs signal labels, but also to accelerate the ECL reaction efficiency, which thus greatly amplify the QDs ECL signal. The PAMAM dendrimer was applied to construct an amplified immobilization platform for antibody molecules. Based on the specific sandwich immunoreactions, an ultrasensitive signal-on ECL immunosensor using the amplified QDs ECL signal probe was developed for IgG assay. The PAMAM dendrimers-Au-QDs nanostructure opened a new kind of QDs nanocomposites in ECL bioassay. This method provided a promising alternative tool for detection of protein in clinical laboratory. Acknowledgments This work was supported by the National Natural Science Foundation of China (No. 21175078). References [1] C. Dodeigne, L.Thunus, R. Lejeune, Talanta 51 (2000) 415−439. [2] (a) Y. Chen, J. Xu, J. Su, Y. Xiang, R.Yuan, Y. Chai, Anal. Chem. 84 (2012) 7750−7755; (b) G. 9
ACCEPTED MANUSCRIPT F. Jie, Y. Q. Qin, Q. M. Meng, J. L.Wang, Analyst 140 (2015) 79 – 82. 3 L.
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arc a-Campa a, . . Huertas-P rez, F. J. Lara, Anal. Chim. Acta.
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[5] (a) Z. Ding, B. M. Quinn, S. K. Haram, L. E. Pell, B. A. Korgel, A. J. Bard, Science 296
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(2002) 1293−1297; (b) Y. Bae, N. Myung, A. J. Bard, Nano Lett. 4 (2004) 1153−1161. [6] (a) G. F. Jie, J. X. Yuan, Anal. Chem. 84 (2012) 2811−2817; (b) H. Jiang, H. Ju, Anal. Chem. 79 (2007) 6690−6696; (c) G. F. Jie, L. Wang, J. X. Yuan, S. S. Zhang, Anal. Chem. 83 (2011)
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3873–3880; (d) Y. Shan, J. Xu, H. Chen, Chem. Commun. 8 (2009) 905−907. [7] J. Li, L. R. Guo, W. Gao, X. H. Xia, L. M. Zheng, Chem. Commun. 48 (2009) 7545−7547.
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[8] (a) X. Zhao, S. Zhou, L. Jiang, W. Hou, Q. Shen, J. Zhu, Chem. Eur. J. 18 (2012) 4974−4981; (b) S. Zhang, Y. Shao, H.-g. Liao, J. Liu, I. A. Aksay, G. Yin, Y. Lin, Chem. Mater. 23 (2011)
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1079−1081; (c) S. Wang, X.Wang, S. P. Jiang, Phys. Chem. Chem. Phys. 13 (2011)
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[9] S. B. Yoon, K. Sohn, J. Y. Kim, C. H. Shin, J. S. Yu, T. Hyeon, Adv. Mater. 14 (2002) 19−21.
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[10] Q. Zhu, M. Han, H. Wang, L. Liu, J. Bao, Z. Dai, J. Shen, Analyst 135 (2010) 2579−2584. [11] J. Wang, H. Han, X. Jiang, L. Huang, L. Chen, N. Li, Anal. Chem. 84 (2012) 4893−4899. [12] T. Y. Huang, Q. M. Meng, G. F. Jie, Biosens. Bioelectron. 66 (2015) 84 – 88
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[13] (a) G. F. Jie, L. L. Li, C. Chen, J. Xuan, J. J. Zhu, Biosens. Bioelectron. 24 (2009) 3352–3358; (b) G. F. Jie, J. J. Zhang, D. C. Wang, C. Cheng, H. Y. Chen, J. J. Zhu, Anal. Chem. 80 (2008) 4033–4039.
[14] (a) P. Antoni, Y. Hed, A. Hordberg, D. Nyström, H. Holst, A. Hult, M. Malkoch, Angew. Chem. 121 (2009) 2160–2164; (b) X. Peng, Q. Pan, G. L. Rempel, Chem. Soc. Rev. 37 (2008) 1619–1628. [15] Y. Qu, Q. Sun, F. Xiao, G. Shi, L. Jin, Bioelectrochemistry 77 (2010) 139–144. [16] S. Liu, X. Zhang, Y. Yu, G. Zou, Biosens. Bioelectron. 55 (2014) 203−208. [17] W. W. Yu, L. H. Qu, W. Z. Guo, X. G. Peng, Chem. Mater. 15 (2003) 2854−2860. [18] M. Q. Zhao, M. C. Richard, Angew. Chem. Int. Ed. 38 (1999) 364–366.
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Figure captions
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Figure 1. Transmission electron microscopy (TEM) image (A), and photoluminescence (PL)
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spectra (B) of the CdSe QDs
Scheme 1. The principle for amplified ECL immunoassay based on PAMAM-Au-CdSe QDs
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probe
Figure 2. TEM image of the PAMAM-Au nanostructure, (A) and the PAMAM-Au-CdSe QDs probe (B). Inset C: The amplified TEM image of (B).
Figure 3. (A) ECL signal responses upon different concentrations of IgG (a~g), Inset: ECL signal
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response of the electrode in the absence of IgG; (B) Relationship between ECL signals and IgG concentrations
Figure 4. (A) Standard calibration curve for IgG detection; (B) Specificity for the determination
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of human IgG using the ECL immunosensor: (a) IgG; (b) BSA, (c) thrombin, (d) lysozyme
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Graphical abstract
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ACCEPTED MANUSCRIPT Highlights
A novel amplified PAMAM-Au-CdSe QDs ECL probe was fabricated.
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An ultrasensitive signal-on ECL immunosensor was developed using the ECL probe.
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The QDs nanostructure opens a new promising material for ECL biosensing.
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