Biosensors and Bioelectronics 59 (2014) 307–313
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Graphene functionalized porous Au-paper based electrochemiluminescence device for detection of DNA using luminescent silver nanoparticles coated calcium carbonate/ carboxymethyl chitosan hybrid microspheres as labels Meng Li a, Yanhu Wang a, Yan Zhang a, Jinghua Yu a,n, Shenguang Ge b, Mei Yan a a Key Laboratory of Chemical Sensing & Analysis in Universities of Shandong, School of Chemistry and Chemical Engineering, University of Jinan, Jinan 250022, PR China b Shandong Provincial Key Laboratory of Preparation and Measurement of Building Materials, University of Jinan, Jinan 250022, PR China
art ic l e i nf o
a b s t r a c t
Article history: Received 22 January 2014 Received in revised form 17 March 2014 Accepted 31 March 2014 Available online 8 April 2014
In the paper, a simple and sensitive electrochemiluminescence (ECL) DNA sensor based on graphenemodified porous Au-paper working electrode (GR/Au-PWE) and calcium carbonate/carboxymethyl chitosan (CaCO3/CMC) hybrid microspheres @ luminescent silver nanoparticles (AgNPs) composites was developed. The GR/Au-PWE with excellent conductivity was successfully prepared for the immobilization of capture probe. The CaCO3/CMC hybrid microspheres were prepared by the precipitation of calcium carbonate in an aqueous solution containing CMC. The AgNPs was synthesized by thermal reduction of silver ions in glycine matrix, taking advantage of the solid-state matrix to control the nucleation and migration of reduced silver atoms. The CaCO3/CMC@AgNPs composites exhibited 3.6 times higher ECL intensity than the pure AgNPs-labeled reporter DNA. Taking advantage of dualamplification effects, the paper-based DNA sensor could detect the target DNA quantitatively, in the range of 4.0 10 17–5.0 10 11 M, with a limit of detection as low as 8.5 10 18 M, and perform excellent selectivity. The simple, low-cost, sensitive device could be easily applied for point-of-care testing, public health and environmental monitoring in remote regions, developing or developed countries. & 2014 Elsevier B.V. All rights reserved.
Keywords: Electrochemiluminescence Graphene-modified porous Au-paper electrode Calcium carbonate/carboxymethyl chitosan hybrid microspheres Luminescent silver nanoparticles DNA sensor
1. Introduction Researches on developing precise and convenient detection methodologies that detect DNA and protein samples at extremely low concentration have attract considerable attention due to its potential applications including diagnosis of genetic, environmental monitoring, and food analysis (Lin et al., 2007; Peng et al., 2009). Although large development in the detection of varied types of DNA sensor has been obtained, difficulties still remain to achieve the goal of the point-of-care testing (POCT) applications. Thus, developing simple, fast, inexpensive, highly sensitive, and miniaturized analytical devices was urgent for DNA detection. The last several years witnessed a fast progress in the field of microfluidic paper-based analytical devices (μ-PADs) since the first patterned paper was proposed by Whitesides' group (Martinez
n
Corresponding author. Tel.: þ 86 531 82767161; fax: þ86 531 82765969. E-mail address: [email protected] (J. Yu).
http://dx.doi.org/10.1016/j.bios.2014.03.072 0956-5663/& 2014 Elsevier B.V. All rights reserved.
et al., 2007). Up to now, many detection methods based on μ-PADs, including colorimetric assay (Yildiz et al., 2013), electrochemistry (Nie et al., 2010), chemiluminescence (Yu et al., 2011), thermochromic detection (Siegel et al., 2009), and electrochemiluminescence (ECL) (Shi et al., 2012) have been developed. Thereinto, ECL is a valuable and powerful analytical technique which has attracted a rising attention in development of analytical methods for μ-PADs, owing to its inherent features, such as low cost, rapid determination, wide range of analytes and high sensitivity (Liu and Ju, 2008). Recently, nanoparticles with unique physical and electrical properties have been widely used for the sensitive detection of DNA sequence (Hu et al., 2008). Duo to their long-term stability, superior conductivity, large surface area, and facile biomolecular conjugation, gold nanoparticles (AuNPs) have attracted great attention in different bio-affinity assays (Hu et al., 2008; Yuan et al., 2010). A novel porous Au-paper working electrode (Au-PWE) which combined the porosity of paper and high conductivity of AuNPs was proposed (Yan et al., 2013). The Au-PWE was fabricated
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on a compatibly designed origami electrochemical device through the growth of an interconnected AuNPs layer on the surfaces of cellulose fibers in the paper sample zone to enhance the conductivity of the paper sample zone. To further improve the electrochemical character of Au-PWE, the positive poly(diallyldimethylammonium chloride)-functionalized graphene (PDDA-GR) was used to assemble onto the surface of the Au-coated cellulose fibers in the paper sample zone. In this work, PDDA-GR/Au-PWE was obtained for the immobilization of capture probe DNA, which was of vital importance for the analytical performance, such as sensitivity, selectivity, accuracy, reproducibility and lifetime. Duo to their unique quantum size dependent optical and electrochemical properties, the quantum dots (QDs), such as CdTe (Liu and Ju, 2008), CdSe (Jie et al., 2008), and CdS (Jie et al., 2007), have been extensively used in ECL-based detection. However, the inherent toxicity of the QDs (Jie et al., 2008) compels us to develop novel low-toxicity or nontoxic species. Up to now, some noble metal nanoclusters (NCs), such as AuNCs (Li et al., 2011), AgNCs (Liu et al., 2013) have been developed and applied to the ECLbased detection, but there was no ECL behaviors of metal nanoparticles (NPs) have been studied. Zheng et al. report the observation that polycrystalline silver NPs (AgNPs) with grain sizes down to electron Fermi wavelength exhibit bright luminescence (Zheng et al., 2008). Here, we synthesized the polycrystalline AgNPs and explored the ECL properties of them. Among the inorganic materials, calcium carbonate (CaCO3), a natural mineral with great biocompatibility, has been widely used in industry, technology, medicine, microcapsule fabrication, and many other bio-related fields (Volodkin et al., 2004). To control the particle size, carboxymethyl chitosan were utilized to form hybrid particles, calcium carbonate/carboxymethyl chitosan (CaCO3/CMC) hybrid microspheres (Wang et al., 2010), which have good biocompatibility and biodegradability property, large specific surface area, and fine loading capability. Here, CaCO3/CMC hybrid microspheres were prepared and used as an ECL signal material carrier to obtain a novel biocompatible ECL signal amplifier CaCO3/CMC@AgNPs composites. In the work, a high-performance ECL DNA sensor based on folding μ-PADs was constructed. The PDDA-GR/Au-PWE was successfully prepared for the immobilization of capture probe. In addition, CaCO3/CMC hybrid microspheres with good biocompatibility were synthesized and employed as carriers for immobilization of AgNPs. Complementary ssDNA sequence was covalently bound to AgNPs on the surface of CaCO3/CMC hybrid microspheres. Enhanced sensitivity could be obtained by the increase of AgNPs loading per DNA sensor. The CaCO3/CMC@AgNPs labels were brought to the surface of the PDDA-GR/Au-PWE through subsequent sandwich DNA hybridization. The experimental results indicated that the μ-PADs DNA sensor not only exhibited excellent analytical performance, but also made contributions for simple, high-throughput, low-cost, rapid and portable ECL detection methodologies on μ-PADs.
(diallyldimethylammonium chloride) (PDDA) was purchased from Nektar (Huntsville, AL). Monochloroacetic acid, sodium carbonate (Na2CO3), calcium chloride (CaCl2), and sodium hydroxide were purchased from Shanghai Chemical Reagent Company (Shanghai, China). All chemicals and solvents used were analytical grade available and were used as received. The sequences of oligonucleotides are listed in Table S1. The ultra-pure water was obtained from a Lichun water purification system (Z18 MΩ cm, Jinan, China) and used throughout. The buffers involved in this work are as follows: DNA immobilization buffer, 10 mM Tris–HCl and 0.1 M NaCl (pH 7.4); hybridization buffer, 10 mM phosphate buffered saline (PBS, pH 7.4) with 0.25 mM NaCl; washing buffer, 10 mM PBS, and 0.1 mM NaCl (pH 7.4). Buffer for ECL, 10 mM Tris–HCl buffer (pH 7.4) containing 0.1 M K2S2O8 and 0.1 M KCl as the coreactant. The human serum samples were provided by Shandong Tumor Hospital.
2.2. Apparatus The ECL measurements were conducted on a flow injection luminescence analyzer (IFFM-E, Xi'an Remex Electronic Instrument High-Tech Ltd., Xi'an, China) with the voltage of the photomultiplier tube (PMT) set at 800 V. Cyclic voltammetric measurements (CVs) were performed with a CHI 760D electrochemical workstation (Shanghai CH Instruments, China). Transmission electron microscopy (TEM) images of AgNPs were obtained from a Hitachi H-800 microscope (Japan). Electrochemical impedance spectroscopy (EIS) was carried out on an IM6x electrochemical station (Zahner, Germany). Scanning electron microscope (SEM) images were obtained using a QUANTA FEG 250 thermal field emission SEM (FEI Co., USA). Energy dispersive spectrometer (EDX) was obtained using an Oxford X-MAX50 EDX (Oxford, Britain). Ultraviolet visible (UV–vis) was recorded on a UV-3101 spectrophotometer (Shimadzu, Japan). The photoluminescence characterization was achieved on a LS-55 spectrofluorometer (P.E. USA).
2.3. Preparation of PDDA-GR Graphite oxide (GO) was prepared from natural graphite powder according to a modified Hummer's method (Shao et al., 2010). PDDA-GR was prepared according to the previous literature (Li et al., 2013). First, 0.5 mL of 20% PDDA solution was added to 100 mL of 0.5% GO solution and stirred for 30 min. Then, 0.5 mL of 80% hydrazine hydrate was added and maintained stirring for 24 h at 90 1C. Finally, the black PDDA-GR could be obtained by filtration and washing with water, and then redispersed readily in water upon mild sonication, forming a black suspension.
2.4. Solid-phase synthesis of silver nanoparticles 2. Experiments 2.1. Reagents All oligonucleotides were synthesized and purified from Shanghai Linc-Bio Science Co. Ltd. (Shanghai, China). 6-Mercapto-1hexanol (MCH) and glycine were purchased from Nanoport Co. Ltd. (Shenzhen, China). N, N'-Carbonyldimidazole (CDI), dimethyl sulfoxide, N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC), N-hydroxysuccinimide (NHS, 98%), and gold chloride (HAuCl4) were obtained from Alfa Aesar China Ltd. Silver nitrate, 2-propanol, and chitosan were obtained from Sigma (St. Louis, MO, USA). Poly
The luminescent silver nanoparticles (AgNPs) were prepared according to the reported method (Zheng et al., 2008). Typically, 150 mg of glycine and 15 mg of silver nitrate were first dissolved in distilled water. The solid-phase reaction mix was heated at 172 1C after water evaporation. The reduction reaction of silver nitrate was indicated by a color change from white to dark brown. Then the reaction product was suspended in 10 mL distilled water and sonicated for 24 h. The suspension was centrifuged at 11,000 rpm for 10 min to remove the insoluble aggregates and large nanoparticles. The process was repeated for several times. Finally, the luminescent AgNPs was as-prepared for next use.
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2.5. Preparation of calcium carbonate/carboxymethyl chitosan hybrid microspheres Carboxymethyl chitosan (CMC) was synthesized according to a literature procedure (Liu et al., 2001). Briefly, 6.8 g of sodium hydroxide was dissolved in 10 mL of water, and 40 mL of 2propanol was added to the solution. Then, 5 g of chitosan was dispersed into the solution and stirred at 50 1C for 1 h. After that, 7.5 g of monochloroacetic acid in 10 mL of 2-propanol was added to the mixture dropwise and the reaction was carried out at 50 1C for 4 h. The resulting solution was filtered, and washed with ethanol until the filtrate was neutral. Subsequently, the product was dried in an oven to obtain CMC. The hybrid microspheres were synthesized by the reported method (Wang et al., 2010). In brief, 50 mg of CMC was dissolved in 10 mL of water at room temperature, then 2.5 mL of Na2CO3 (0.5 M) was added and stirred for 0.5 h. After that, 2.5 mL of CaCl2 (0.5 M) was added to the mixtures dropwise and stirred for 12 h. The formed precipitate was collected by centrifugation, and washed several times with water. Finally, the product was dried in an oven to obtain CaCO3/CMC hybrid microspheres.
2.6. Preparation of CaCO3/CMC@AgNPs composites conjugated DNA (S3) According to previous report (Peng et al., 2010), the CaCO3 microparticles prepared in the presence of polysaccharides have a large number of nanopores, which provide a strong capability to load other small particles. Ten milligrams of CaCO3/CMC hybrid microspheres was added into the AgNPs solution under stirring and kept for 12 h. For preparation of CaCO3/CMC@AgNPs composites, the composites were then centrifugated and washed twice by water. Then the prepared CaCO3/CMC@AgNPs composites were dispersed into 2 mL of aqueous solution containing 50 μL of 100 μM S3 and 10 μM EDC–NHS. The mixture was slightly stirred for 8 h. The S3 was immobilized on the CaCO3/CMC@AgNPs composites via the reaction of –NH2 and –COOH of CaCO3/CMC@ AgNPs composites. At last, the S3/CaCO3/CMC@AgNPs conjugation were aged in salts solution (0.1 M NaCl and 10.0 mM acetate buffer) for 24 h, followed by centrifuging at 10,000 rpm for 10 min. The obtained composites were added into immobilization
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buffer to a final concentration of 0.02 μM, and the bioconjugates were stored at 4 1C prior to use.
2.7. Preparation of μ-PAD DNA sensor and the hybridization reaction The preparation of this μ-PAD is described in the Supplemental information (Fig. S1). The fabrication and assay procedure for the folding μ-PAD DNA is represented in Scheme 1. The porous Au-PWE was prepared according to the method described in our previous works (Yan et al., 2013; Ge et al., 2013). The as-prepared Au-PWE was then modified by positively charged PPDDA-GR. Briefly, 20 μL of PDDA-GR solution was dropped into the AuPWE and kept for 30 min, followed by washing with water according to the procedure mentioned in our previous work (Ge et al., 2012). Then, 5 μL of 1% CDI was dropped into the working electrode. Subsequently, the modified working electrode was incubated with 5 μL of 1.0 10 8 M capture probe (S1) for 16 h. The DNA-modified electrode was further treated with 1.0 mM MCH for 2 h to obtain a well-aligned DNA monolayer, followed by washing with the washing buffer and ultrapure water alternately to remove nonspecific adsorbed DNA. For the hybridization reaction, the electrode was incubated with a varying concentration of target DNA (S2) for a desired time at 37 1C. Subsequently, the electrode was hybridized with reporter probe (S3–CaCO3/CMC@ AgNPs bioconjugates) for 2 h at 37 1C. After hybridization, the electrode was extensively rinsed with washing buffer and dried under a stream of nitrogen prior to electrochemical characterization.
2.8. ECL assay procedures of this
μ-PAD
Before the ECL measurement, the sample tab was folded down below the auxiliary tab and clamped into a home-made deviceholder. Thereafter, 40 μL of Tris–HCl buffer (pH 7.4) containing 0.1 M K2S2O8 and 0.1 M KCl was dropped into the paper electrochemical cell and then the device-holder was placed in front of the PMT that was biased at 800 V. The ECL reaction in the PWE was triggered in the scanning range from ( 1.0) to ( 2.5) V with scan rate of 100 mV s 1. The ECL signals related to the target DNA (S2) concentrations could be measured.
Scheme 1. Schematic representation of the fabrication procedures for the μ-PAD DNA sensor.
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3. Results and discussion 3.1. Characterization of the CaCO3/CMC@AgNPs composites As shown in Fig. 1A and B, SEM images of CaCO3/CMC showed that the microspheres exhibit a spherical shape with a diameter about 1 μm. The luminescent AgNPs were synthesized by thermal reduction of silver ions in glycine matrix, taking advantage of the solid-state matrix to control the nucleation and migration of reduced silver atoms. The prepared AgNPs in solution were further characterized by UV–vis absorption spectra and PL (Fig. 1C). The as-obtained nanoparticles showed an absorption feature at 461 nm in the UV–vis absorption spectrum (curve a). The PL spectrum (curve b) showed peak position at 457 nm and 473 nm, respectively. From the photos of the left inset, we can see that the AgNPs aqueous solution exhibited bright blue emission under excitation of 365 nm UV light. The right inset was the TEM image of AgNPs, indicating that particle diameters range from 2 to 20 nm. Fig. 1D and E shows the SEM images of CaCO3/ CMC@AgNPs composites. After coating, the CaCO3/CMC hybrid microspheres were deposited with AgNPs on the surface. To
further investigate the successful preparation of the CaCO3/ CMC@AgNPs composites, an EDX was employed to confirm the presence of elements. Fig. 1F shows the EDX of the CaCO3/ CMC@AgNPs composites, indicating that the composite were composed of C, O, Ag, and Ca elements. 3.2. Characterizations of PDDA-GR/Au-PWE From Fig. 2A, it can be seen that a continuous and dense conducting AuNPs layer with interconnected AuNPs was obtained completely on the cellulose fiber surfaces. The PDDA-GR with positive charges could be absorbed onto the negatively charged AuNPs layer on the surface of interwoven cellulose fibers in AuPWE. As shown in Fig. 2B, the thin PDDA-GR sheets were transparent as a flexible layer attached firmly onto the AuNPs layer. 3.3. EIS In order to verify whether indeed the ECL DNA sensor works as expected, EIS was adopted to monitor the changes in the surface features of the modified electrodes (Hou et al., 2013). The semicircle
Fig. 1. (A) SEM image of the CaCO3/CMC hybrid microspheres; (B) magnification SEM image of the CaCO3/CMC hybrid microspheres; (C) (a) UV–vis absorption and (b) PL spectrum of AgNPs, inset (left: photograph taken under UV light, right: TEM image of the AgNPs); (D) SEM image of CaCO3/CMC@AgNPs composites; (E) magnification SEM image of CaCO3/CMC@AgNPs composites; (F) EDX of CaCO3/CMC@AgNPs composites.
Fig. 2. SEM images of (A) Au-PWE; (B) PDDA-GR/Au-PWE; (C) EIS of the Au-PWE under different surface condition in 10.0 mM [Fe(CN)6]3 /4 solution containing 0.5 M KCl: (a) bare Au-PWE; (b) PDDA-GR/Au-PWE; (c) S1/PDDA-GR/Au-PWE; (d) S2/S1/PDDA-GR/Au-PWE; (e) S3/S2/S1/PDDA-GR/Au-PWE.
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portion at higher frequencies corresponds to the electron-transferlimited process (i.e. electron-transfer resistance, Ret), a change in value of which was associated with the blocking behavior of the modification processes on the surfaces of Au-coated cellulose fibers in Au-PWE. Fig. 2C shows a Nyquist plot of impedance for the stepwise modification process with the Au-PWE. It can be seen that the bare Au-PWE revealed a small semicircle domain (curve a), implying a low Ret value of the redox couple, i.e. [Fe(CN)6]3 /4 . After PDDA-GR was attached on the Au-coated cellulose fiber surface in Au-PWE, a much smaller Ret was observed (curve b), which indicates that GR was an excellent electric conducting material and accelerated the electron transfer. After immobilization of S1, the value of Ret increased (curve c), which was due to the immobilization of negatively charged S1 on the electrode surface resulting in a negatively charged interface that electrostatically repelled the negatively charged redox probe [Fe(CN)6]3 /4 and inhibited interfacial charge transfer (Cho et al., 2006). Subsequently, S2 was hybridized, and the Ret increased again (curve d). Remarkable increase in the Ret was observed after hybridization with S3 loaded on CaCO3/CMC@AgNPs composites (curve e), this was because of the large amount of DNA linked on the CaCO3/CMC@AgNPs composites. 3.4. ECL behavior of CaCO3/CMC@AgNPs composites The success of the biobarcode amplification was closely related to the abundant loading of the AgNPs conjugated with CaCO3/CMC microspheres. Consequently, in order to demonstrate the amplification of CaCO3/CMC@AgNPs composite, a series of experiments was conducted. The Au-PWE was scanned from ( 1.0) V to ( 2.5) V in Tris–HCl buffer (pH 7.4) containing 0.1 M K2S2O8 and 0.1 M KCl. A large increase of ECL intensity was observed from the DNA sensor in which the S3 was labeled by CaCO3/CMC@AgNPs composite (Fig. 3A, curve d), whereas no obvious increase of ECL signal could be observed from PDDA-GR/Au-PWE (Fig. 3A, curve a) and
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S2/S1/PDDA-GR/Au-PWE (Fig. 3A, curve b). These results indicated that enhanced ECL intensity was attributed to attach CaCO3/ CMC@AgNPs/S3, which could react with S2 O8 2 and enhance the ECL signal. Furthermore, from Fig. 3A we can see that the ECL intensity of the DNA sensor using the CaCO3/CMC@AgNPs composite labeled S3 (Fig. 3A, curve d) was 3.6 times higher than the pure AgNPs labeled S3 (Fig. 3A, curve c) at the same S2 concentration, which demonstrated the amplification of ECL signal with CaCO3/ CMC@AgNPs composite as the label. Fig. 3B shows plots of ECL intensity of the DNA sensor vs. the S2 concentration of pure AgNPs (a), and CaCO3/CMC@AgNPs composites (b) labeled S3, respectively. From Fig. 3B, we could see that CaCO3/CMC@AgNPs composites exhibit excellent ECL performance. It was found that the ECL signal was too weak to be distinguished in the absence of S2 O8 2 , indicated that S2 O8 2 was of vital importance in the process of AgNPs as coreactant. In addition, the other coreactant, such as tripropylamine, was considered. The strong ECL signal was only observed in the presence of S2 O8 2 and there was no ECL signal obtained using tripropylamine as coreactant (Fig. 3C, curve a). Moreover, the cathodic ECL of AgNPs was also studied as the potential was cycled between ( 1.0) V and ( 2.5) V. Compared with the ECL between (þ 2.0) V and ( 2.5) V, the cathodic ECL just decreased slightly (Fig. 3D), indicating that the coreactant ECL contributed to the whole ECL dominantly. According to the previous reports concerning the cathodic ECL of QDs (Jie et al., 2009; Wang et al., 2011), it could be confirmed that the cathodic ECL herein was originated from the formation of excited-state AgNPs (Agn) through electron transfer annihilation of negatively charged Agn and the strongly oxidizing n radicals produced by electroreduction of S2 O8 2 . The possible ECL mechanisms were described as follows:
Ag-Agn
(1)
Fig. 3. (A) ECL-potential curves obtained at (a) PDDA-GR/Au-PWE; (b) S2/S1/PDDA-GR/Au-PWE; (c) AgNPs/S3/S2/S1/PDDA-GR/Au-PWE; (d) CaCO3/CMC@AgNPs/S3/S2/S1/ PDDA-GR/Au-PWE in 10 mM Tris–HCl buffer (pH 7.4) containing 0.1 M K2S2O8 and 0.1 M KCl. The S2 concentration was 1.0 10 14 M. (B) Plots of ECL intensity of the DNA sensor vs. S2 concentration of (a) pure AgNPs and (b) CaCO3/CMC@AgNPs labeled S3. (C) ECL-potential curves of AgNPs modified electrode using (a) tripropylamine, and (b) K2S2O8 as coreactant; (D) ECL-potential curves of AgNPs modified electrode with the potential cycled between (a) ( 1.0) V and ( 2.5) V, and (b) ( þ2.0) V and ( 2.5) V. Scan rate: 100 mV s 1. The voltage of the PMT was 800 V.
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Table 1 Detection of Target-ssDNA in spiked samples. Sample
Addeda (fM)
Detectedb (fM)
RSD (%, n¼ 11)c
Recoveryd (%)
1 2 3 4 5 6 7 8
0.04 0.2 1 10 50 500 5000 50,000
0.04104 0.1964 1.035 10.19 48.90 484.50 5135.00 48,800.00
4.1 3.9 4.5 3.2 3.7 4.0 4.6 3.8
102.6 98.2 103.5 101.9 97.8 96.9 102.7 97.6
a
[Added] means the values that we add into human serum sample. [Detected] means the amount of Target-ssDNA obtained according to the standard curve equations from eleven parallel detections. c The RSD of measurements are calculated from eleven independent experiments. d Recovery means the ratio of [Detected]/[Added]. b
Fig. 4. Relationship between ECL intensity and S2 concentration in pH 7.4 Tris–HCl buffer containing 0.1 M K2S2O8 and 0.1 M KCl. Inset (a) ECL profiles of the DNA sensor in the presence of different concentrations of S2. Inset (b) logarithmic calibration curve of the ECL signals (Inset a) for S2.
S2 O8 2 þe -S2 O8 n2
(2)
hybridized DNA was removed through thermal denaturation (Fig. S3C). The results indicated acceptable regeneration.
S2 O8 n2 -SO4 2 þSO4 n
(3)
3.7. Analytical application potential of the
Agn þSO4 n -Agn þ SO4 2
(4)
Agn-Ag þhv
(5)
3.5. Analytical performance The quantitative behavior of the DNA sensor was assessed by monitoring the difference of the ECL intensity upon the concentration of S2 at optical conditions (in Supplemental information Fig. S2). Fig. 4 exhibits the relationship between ECL intensity and S2 concentration. It could be found that the increased ECL intensity was directly related to the concentration of S2 (Inset a). The ECL intensity ascended logarithmically as the S2 concentration increased in the range of 4.0 10 17–5.0 10 11 M. The linear regression equation was IECL ¼872.67 þ354.96 log cDNA (cDNA fM) and the correlation coefficient was 0.9889. The limit of detection (LOD) was 8.5 10 18 M (defined as 3s, where s is the standard deviation of eleven measurements of blank samples), indicative of an acceptable quantitative behavior. Compared with other sensors reported previously, our proposed DNA sensor exhibited a satisfactory detection limit and linear range, and the characteristics of other DNA sensors are summarized in Table S2. 3.6. Specificity, stability, reproducibility, and regeneration of the DNA sensor The specificity of the proposed DNA sensor in discriminating perfect target DNA from two-base mismatched (S4), noncomplementary (S5), and scrambled (S6) DNA sequences was tested via comparing the ECL signal changes under three concentrations (Fig. S3A). The results demonstrated that the proposed DNA sensor exhibited an excellent specificity to the target DNA. Stability of the DNA sensor is a key factor in their application. Fig. S3B shows the ECL signal vs. time under continuous potential scanning for 10 cycles. The results indicated that the μ-PAD DNA sensor had fine storage stability. To investigate the reproducibility of the μ-PAD DNA sensor, intra- and inter-assay were both estimated. The experimental results indicated that the proposed strategy was reliable and can be used for target DNA detection with acceptable reproducibility. In the test, the DNA sensor could be regenerated by incubation of the μ-PAD in hot water (90 1C) for 1 min, by which
μ-PAD DNA sensor
In order to verify the analytical reliability and application potential of the proposed DNA sensor with real samples, eleven replicate determinations of Target-ssDNA in spiked human serum samples were investigated under the optimal conditions. The spiked human serum samples were prepared through adding different amounts of Target-ssDNA (0.04–50,000 fM) into the human serum samples. Table 1 shows the results, that the RSD was less than 5%, and the recoveries were between 96.9% and 103.5%, indicated an acceptable veracity of the proposed method. Thus, it may provide an alternative tool for diagnosis applications.
4. Conclusions In summary, a methodology for low-cost, simple, rapid, portable, disposable, and sensitive DNA detection was developed here by ECL measurement based on GR modified Au-PWE. Taking advantage of dual amplification effects of the GR modified Au-PWE and CaCO3/CMC@AgNPs conposites, the DNA sensor not only performed excellent specificity, but also had a LOD as low as 8.5 10 18 M. The experiments results demonstrated that the μ-PAD DNA sensor exhibited excellent analytical performance and had acceptable application potential in human serum assay. The proposed method provided a promising platform for accurate gene diagnostics at home and in the field. Acknowledgment This work was financially supported by the National Natural Science Foundation of China (21277058 and 21175058); Natural Science Foundation of Shandong Province, China (ZR2012BZ002).
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Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Graphene functionalized porous Au-paper based electrochemiluminescence device for detection of DNA using luminescent silver nanoparticles coated calcium carbonate/ carboxymethyl chitosan hybrid microspheres as labels Meng Li a, Yanhu Wang a, Yan Zhang a, Jinghua Yu a,n, Shenguang Ge b, Mei Yan a a Key Laboratory of Chemical Sensing & Analysis in Universities of Shandong, School of Chemistry and Chemical Engineering, University of Jinan, Jinan 250022, PR China b Shandong Provincial Key Laboratory of Preparation and Measurement of Building Materials, University of Jinan, Jinan 250022, PR China
art ic l e i nf o
a b s t r a c t
Article history: Received 22 January 2014 Received in revised form 17 March 2014 Accepted 31 March 2014 Available online 8 April 2014
In the paper, a simple and sensitive electrochemiluminescence (ECL) DNA sensor based on graphenemodified porous Au-paper working electrode (GR/Au-PWE) and calcium carbonate/carboxymethyl chitosan (CaCO3/CMC) hybrid microspheres @ luminescent silver nanoparticles (AgNPs) composites was developed. The GR/Au-PWE with excellent conductivity was successfully prepared for the immobilization of capture probe. The CaCO3/CMC hybrid microspheres were prepared by the precipitation of calcium carbonate in an aqueous solution containing CMC. The AgNPs was synthesized by thermal reduction of silver ions in glycine matrix, taking advantage of the solid-state matrix to control the nucleation and migration of reduced silver atoms. The CaCO3/CMC@AgNPs composites exhibited 3.6 times higher ECL intensity than the pure AgNPs-labeled reporter DNA. Taking advantage of dualamplification effects, the paper-based DNA sensor could detect the target DNA quantitatively, in the range of 4.0 10 17–5.0 10 11 M, with a limit of detection as low as 8.5 10 18 M, and perform excellent selectivity. The simple, low-cost, sensitive device could be easily applied for point-of-care testing, public health and environmental monitoring in remote regions, developing or developed countries. & 2014 Elsevier B.V. All rights reserved.
Keywords: Electrochemiluminescence Graphene-modified porous Au-paper electrode Calcium carbonate/carboxymethyl chitosan hybrid microspheres Luminescent silver nanoparticles DNA sensor
1. Introduction Researches on developing precise and convenient detection methodologies that detect DNA and protein samples at extremely low concentration have attract considerable attention due to its potential applications including diagnosis of genetic, environmental monitoring, and food analysis (Lin et al., 2007; Peng et al., 2009). Although large development in the detection of varied types of DNA sensor has been obtained, difficulties still remain to achieve the goal of the point-of-care testing (POCT) applications. Thus, developing simple, fast, inexpensive, highly sensitive, and miniaturized analytical devices was urgent for DNA detection. The last several years witnessed a fast progress in the field of microfluidic paper-based analytical devices (μ-PADs) since the first patterned paper was proposed by Whitesides' group (Martinez
n
Corresponding author. Tel.: þ 86 531 82767161; fax: þ86 531 82765969. E-mail address: [email protected] (J. Yu).
http://dx.doi.org/10.1016/j.bios.2014.03.072 0956-5663/& 2014 Elsevier B.V. All rights reserved.
et al., 2007). Up to now, many detection methods based on μ-PADs, including colorimetric assay (Yildiz et al., 2013), electrochemistry (Nie et al., 2010), chemiluminescence (Yu et al., 2011), thermochromic detection (Siegel et al., 2009), and electrochemiluminescence (ECL) (Shi et al., 2012) have been developed. Thereinto, ECL is a valuable and powerful analytical technique which has attracted a rising attention in development of analytical methods for μ-PADs, owing to its inherent features, such as low cost, rapid determination, wide range of analytes and high sensitivity (Liu and Ju, 2008). Recently, nanoparticles with unique physical and electrical properties have been widely used for the sensitive detection of DNA sequence (Hu et al., 2008). Duo to their long-term stability, superior conductivity, large surface area, and facile biomolecular conjugation, gold nanoparticles (AuNPs) have attracted great attention in different bio-affinity assays (Hu et al., 2008; Yuan et al., 2010). A novel porous Au-paper working electrode (Au-PWE) which combined the porosity of paper and high conductivity of AuNPs was proposed (Yan et al., 2013). The Au-PWE was fabricated
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on a compatibly designed origami electrochemical device through the growth of an interconnected AuNPs layer on the surfaces of cellulose fibers in the paper sample zone to enhance the conductivity of the paper sample zone. To further improve the electrochemical character of Au-PWE, the positive poly(diallyldimethylammonium chloride)-functionalized graphene (PDDA-GR) was used to assemble onto the surface of the Au-coated cellulose fibers in the paper sample zone. In this work, PDDA-GR/Au-PWE was obtained for the immobilization of capture probe DNA, which was of vital importance for the analytical performance, such as sensitivity, selectivity, accuracy, reproducibility and lifetime. Duo to their unique quantum size dependent optical and electrochemical properties, the quantum dots (QDs), such as CdTe (Liu and Ju, 2008), CdSe (Jie et al., 2008), and CdS (Jie et al., 2007), have been extensively used in ECL-based detection. However, the inherent toxicity of the QDs (Jie et al., 2008) compels us to develop novel low-toxicity or nontoxic species. Up to now, some noble metal nanoclusters (NCs), such as AuNCs (Li et al., 2011), AgNCs (Liu et al., 2013) have been developed and applied to the ECLbased detection, but there was no ECL behaviors of metal nanoparticles (NPs) have been studied. Zheng et al. report the observation that polycrystalline silver NPs (AgNPs) with grain sizes down to electron Fermi wavelength exhibit bright luminescence (Zheng et al., 2008). Here, we synthesized the polycrystalline AgNPs and explored the ECL properties of them. Among the inorganic materials, calcium carbonate (CaCO3), a natural mineral with great biocompatibility, has been widely used in industry, technology, medicine, microcapsule fabrication, and many other bio-related fields (Volodkin et al., 2004). To control the particle size, carboxymethyl chitosan were utilized to form hybrid particles, calcium carbonate/carboxymethyl chitosan (CaCO3/CMC) hybrid microspheres (Wang et al., 2010), which have good biocompatibility and biodegradability property, large specific surface area, and fine loading capability. Here, CaCO3/CMC hybrid microspheres were prepared and used as an ECL signal material carrier to obtain a novel biocompatible ECL signal amplifier CaCO3/CMC@AgNPs composites. In the work, a high-performance ECL DNA sensor based on folding μ-PADs was constructed. The PDDA-GR/Au-PWE was successfully prepared for the immobilization of capture probe. In addition, CaCO3/CMC hybrid microspheres with good biocompatibility were synthesized and employed as carriers for immobilization of AgNPs. Complementary ssDNA sequence was covalently bound to AgNPs on the surface of CaCO3/CMC hybrid microspheres. Enhanced sensitivity could be obtained by the increase of AgNPs loading per DNA sensor. The CaCO3/CMC@AgNPs labels were brought to the surface of the PDDA-GR/Au-PWE through subsequent sandwich DNA hybridization. The experimental results indicated that the μ-PADs DNA sensor not only exhibited excellent analytical performance, but also made contributions for simple, high-throughput, low-cost, rapid and portable ECL detection methodologies on μ-PADs.
(diallyldimethylammonium chloride) (PDDA) was purchased from Nektar (Huntsville, AL). Monochloroacetic acid, sodium carbonate (Na2CO3), calcium chloride (CaCl2), and sodium hydroxide were purchased from Shanghai Chemical Reagent Company (Shanghai, China). All chemicals and solvents used were analytical grade available and were used as received. The sequences of oligonucleotides are listed in Table S1. The ultra-pure water was obtained from a Lichun water purification system (Z18 MΩ cm, Jinan, China) and used throughout. The buffers involved in this work are as follows: DNA immobilization buffer, 10 mM Tris–HCl and 0.1 M NaCl (pH 7.4); hybridization buffer, 10 mM phosphate buffered saline (PBS, pH 7.4) with 0.25 mM NaCl; washing buffer, 10 mM PBS, and 0.1 mM NaCl (pH 7.4). Buffer for ECL, 10 mM Tris–HCl buffer (pH 7.4) containing 0.1 M K2S2O8 and 0.1 M KCl as the coreactant. The human serum samples were provided by Shandong Tumor Hospital.
2.2. Apparatus The ECL measurements were conducted on a flow injection luminescence analyzer (IFFM-E, Xi'an Remex Electronic Instrument High-Tech Ltd., Xi'an, China) with the voltage of the photomultiplier tube (PMT) set at 800 V. Cyclic voltammetric measurements (CVs) were performed with a CHI 760D electrochemical workstation (Shanghai CH Instruments, China). Transmission electron microscopy (TEM) images of AgNPs were obtained from a Hitachi H-800 microscope (Japan). Electrochemical impedance spectroscopy (EIS) was carried out on an IM6x electrochemical station (Zahner, Germany). Scanning electron microscope (SEM) images were obtained using a QUANTA FEG 250 thermal field emission SEM (FEI Co., USA). Energy dispersive spectrometer (EDX) was obtained using an Oxford X-MAX50 EDX (Oxford, Britain). Ultraviolet visible (UV–vis) was recorded on a UV-3101 spectrophotometer (Shimadzu, Japan). The photoluminescence characterization was achieved on a LS-55 spectrofluorometer (P.E. USA).
2.3. Preparation of PDDA-GR Graphite oxide (GO) was prepared from natural graphite powder according to a modified Hummer's method (Shao et al., 2010). PDDA-GR was prepared according to the previous literature (Li et al., 2013). First, 0.5 mL of 20% PDDA solution was added to 100 mL of 0.5% GO solution and stirred for 30 min. Then, 0.5 mL of 80% hydrazine hydrate was added and maintained stirring for 24 h at 90 1C. Finally, the black PDDA-GR could be obtained by filtration and washing with water, and then redispersed readily in water upon mild sonication, forming a black suspension.
2.4. Solid-phase synthesis of silver nanoparticles 2. Experiments 2.1. Reagents All oligonucleotides were synthesized and purified from Shanghai Linc-Bio Science Co. Ltd. (Shanghai, China). 6-Mercapto-1hexanol (MCH) and glycine were purchased from Nanoport Co. Ltd. (Shenzhen, China). N, N'-Carbonyldimidazole (CDI), dimethyl sulfoxide, N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC), N-hydroxysuccinimide (NHS, 98%), and gold chloride (HAuCl4) were obtained from Alfa Aesar China Ltd. Silver nitrate, 2-propanol, and chitosan were obtained from Sigma (St. Louis, MO, USA). Poly
The luminescent silver nanoparticles (AgNPs) were prepared according to the reported method (Zheng et al., 2008). Typically, 150 mg of glycine and 15 mg of silver nitrate were first dissolved in distilled water. The solid-phase reaction mix was heated at 172 1C after water evaporation. The reduction reaction of silver nitrate was indicated by a color change from white to dark brown. Then the reaction product was suspended in 10 mL distilled water and sonicated for 24 h. The suspension was centrifuged at 11,000 rpm for 10 min to remove the insoluble aggregates and large nanoparticles. The process was repeated for several times. Finally, the luminescent AgNPs was as-prepared for next use.
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2.5. Preparation of calcium carbonate/carboxymethyl chitosan hybrid microspheres Carboxymethyl chitosan (CMC) was synthesized according to a literature procedure (Liu et al., 2001). Briefly, 6.8 g of sodium hydroxide was dissolved in 10 mL of water, and 40 mL of 2propanol was added to the solution. Then, 5 g of chitosan was dispersed into the solution and stirred at 50 1C for 1 h. After that, 7.5 g of monochloroacetic acid in 10 mL of 2-propanol was added to the mixture dropwise and the reaction was carried out at 50 1C for 4 h. The resulting solution was filtered, and washed with ethanol until the filtrate was neutral. Subsequently, the product was dried in an oven to obtain CMC. The hybrid microspheres were synthesized by the reported method (Wang et al., 2010). In brief, 50 mg of CMC was dissolved in 10 mL of water at room temperature, then 2.5 mL of Na2CO3 (0.5 M) was added and stirred for 0.5 h. After that, 2.5 mL of CaCl2 (0.5 M) was added to the mixtures dropwise and stirred for 12 h. The formed precipitate was collected by centrifugation, and washed several times with water. Finally, the product was dried in an oven to obtain CaCO3/CMC hybrid microspheres.
2.6. Preparation of CaCO3/CMC@AgNPs composites conjugated DNA (S3) According to previous report (Peng et al., 2010), the CaCO3 microparticles prepared in the presence of polysaccharides have a large number of nanopores, which provide a strong capability to load other small particles. Ten milligrams of CaCO3/CMC hybrid microspheres was added into the AgNPs solution under stirring and kept for 12 h. For preparation of CaCO3/CMC@AgNPs composites, the composites were then centrifugated and washed twice by water. Then the prepared CaCO3/CMC@AgNPs composites were dispersed into 2 mL of aqueous solution containing 50 μL of 100 μM S3 and 10 μM EDC–NHS. The mixture was slightly stirred for 8 h. The S3 was immobilized on the CaCO3/CMC@AgNPs composites via the reaction of –NH2 and –COOH of CaCO3/CMC@ AgNPs composites. At last, the S3/CaCO3/CMC@AgNPs conjugation were aged in salts solution (0.1 M NaCl and 10.0 mM acetate buffer) for 24 h, followed by centrifuging at 10,000 rpm for 10 min. The obtained composites were added into immobilization
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buffer to a final concentration of 0.02 μM, and the bioconjugates were stored at 4 1C prior to use.
2.7. Preparation of μ-PAD DNA sensor and the hybridization reaction The preparation of this μ-PAD is described in the Supplemental information (Fig. S1). The fabrication and assay procedure for the folding μ-PAD DNA is represented in Scheme 1. The porous Au-PWE was prepared according to the method described in our previous works (Yan et al., 2013; Ge et al., 2013). The as-prepared Au-PWE was then modified by positively charged PPDDA-GR. Briefly, 20 μL of PDDA-GR solution was dropped into the AuPWE and kept for 30 min, followed by washing with water according to the procedure mentioned in our previous work (Ge et al., 2012). Then, 5 μL of 1% CDI was dropped into the working electrode. Subsequently, the modified working electrode was incubated with 5 μL of 1.0 10 8 M capture probe (S1) for 16 h. The DNA-modified electrode was further treated with 1.0 mM MCH for 2 h to obtain a well-aligned DNA monolayer, followed by washing with the washing buffer and ultrapure water alternately to remove nonspecific adsorbed DNA. For the hybridization reaction, the electrode was incubated with a varying concentration of target DNA (S2) for a desired time at 37 1C. Subsequently, the electrode was hybridized with reporter probe (S3–CaCO3/CMC@ AgNPs bioconjugates) for 2 h at 37 1C. After hybridization, the electrode was extensively rinsed with washing buffer and dried under a stream of nitrogen prior to electrochemical characterization.
2.8. ECL assay procedures of this
μ-PAD
Before the ECL measurement, the sample tab was folded down below the auxiliary tab and clamped into a home-made deviceholder. Thereafter, 40 μL of Tris–HCl buffer (pH 7.4) containing 0.1 M K2S2O8 and 0.1 M KCl was dropped into the paper electrochemical cell and then the device-holder was placed in front of the PMT that was biased at 800 V. The ECL reaction in the PWE was triggered in the scanning range from ( 1.0) to ( 2.5) V with scan rate of 100 mV s 1. The ECL signals related to the target DNA (S2) concentrations could be measured.
Scheme 1. Schematic representation of the fabrication procedures for the μ-PAD DNA sensor.
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3. Results and discussion 3.1. Characterization of the CaCO3/CMC@AgNPs composites As shown in Fig. 1A and B, SEM images of CaCO3/CMC showed that the microspheres exhibit a spherical shape with a diameter about 1 μm. The luminescent AgNPs were synthesized by thermal reduction of silver ions in glycine matrix, taking advantage of the solid-state matrix to control the nucleation and migration of reduced silver atoms. The prepared AgNPs in solution were further characterized by UV–vis absorption spectra and PL (Fig. 1C). The as-obtained nanoparticles showed an absorption feature at 461 nm in the UV–vis absorption spectrum (curve a). The PL spectrum (curve b) showed peak position at 457 nm and 473 nm, respectively. From the photos of the left inset, we can see that the AgNPs aqueous solution exhibited bright blue emission under excitation of 365 nm UV light. The right inset was the TEM image of AgNPs, indicating that particle diameters range from 2 to 20 nm. Fig. 1D and E shows the SEM images of CaCO3/ CMC@AgNPs composites. After coating, the CaCO3/CMC hybrid microspheres were deposited with AgNPs on the surface. To
further investigate the successful preparation of the CaCO3/ CMC@AgNPs composites, an EDX was employed to confirm the presence of elements. Fig. 1F shows the EDX of the CaCO3/ CMC@AgNPs composites, indicating that the composite were composed of C, O, Ag, and Ca elements. 3.2. Characterizations of PDDA-GR/Au-PWE From Fig. 2A, it can be seen that a continuous and dense conducting AuNPs layer with interconnected AuNPs was obtained completely on the cellulose fiber surfaces. The PDDA-GR with positive charges could be absorbed onto the negatively charged AuNPs layer on the surface of interwoven cellulose fibers in AuPWE. As shown in Fig. 2B, the thin PDDA-GR sheets were transparent as a flexible layer attached firmly onto the AuNPs layer. 3.3. EIS In order to verify whether indeed the ECL DNA sensor works as expected, EIS was adopted to monitor the changes in the surface features of the modified electrodes (Hou et al., 2013). The semicircle
Fig. 1. (A) SEM image of the CaCO3/CMC hybrid microspheres; (B) magnification SEM image of the CaCO3/CMC hybrid microspheres; (C) (a) UV–vis absorption and (b) PL spectrum of AgNPs, inset (left: photograph taken under UV light, right: TEM image of the AgNPs); (D) SEM image of CaCO3/CMC@AgNPs composites; (E) magnification SEM image of CaCO3/CMC@AgNPs composites; (F) EDX of CaCO3/CMC@AgNPs composites.
Fig. 2. SEM images of (A) Au-PWE; (B) PDDA-GR/Au-PWE; (C) EIS of the Au-PWE under different surface condition in 10.0 mM [Fe(CN)6]3 /4 solution containing 0.5 M KCl: (a) bare Au-PWE; (b) PDDA-GR/Au-PWE; (c) S1/PDDA-GR/Au-PWE; (d) S2/S1/PDDA-GR/Au-PWE; (e) S3/S2/S1/PDDA-GR/Au-PWE.
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portion at higher frequencies corresponds to the electron-transferlimited process (i.e. electron-transfer resistance, Ret), a change in value of which was associated with the blocking behavior of the modification processes on the surfaces of Au-coated cellulose fibers in Au-PWE. Fig. 2C shows a Nyquist plot of impedance for the stepwise modification process with the Au-PWE. It can be seen that the bare Au-PWE revealed a small semicircle domain (curve a), implying a low Ret value of the redox couple, i.e. [Fe(CN)6]3 /4 . After PDDA-GR was attached on the Au-coated cellulose fiber surface in Au-PWE, a much smaller Ret was observed (curve b), which indicates that GR was an excellent electric conducting material and accelerated the electron transfer. After immobilization of S1, the value of Ret increased (curve c), which was due to the immobilization of negatively charged S1 on the electrode surface resulting in a negatively charged interface that electrostatically repelled the negatively charged redox probe [Fe(CN)6]3 /4 and inhibited interfacial charge transfer (Cho et al., 2006). Subsequently, S2 was hybridized, and the Ret increased again (curve d). Remarkable increase in the Ret was observed after hybridization with S3 loaded on CaCO3/CMC@AgNPs composites (curve e), this was because of the large amount of DNA linked on the CaCO3/CMC@AgNPs composites. 3.4. ECL behavior of CaCO3/CMC@AgNPs composites The success of the biobarcode amplification was closely related to the abundant loading of the AgNPs conjugated with CaCO3/CMC microspheres. Consequently, in order to demonstrate the amplification of CaCO3/CMC@AgNPs composite, a series of experiments was conducted. The Au-PWE was scanned from ( 1.0) V to ( 2.5) V in Tris–HCl buffer (pH 7.4) containing 0.1 M K2S2O8 and 0.1 M KCl. A large increase of ECL intensity was observed from the DNA sensor in which the S3 was labeled by CaCO3/CMC@AgNPs composite (Fig. 3A, curve d), whereas no obvious increase of ECL signal could be observed from PDDA-GR/Au-PWE (Fig. 3A, curve a) and
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S2/S1/PDDA-GR/Au-PWE (Fig. 3A, curve b). These results indicated that enhanced ECL intensity was attributed to attach CaCO3/ CMC@AgNPs/S3, which could react with S2 O8 2 and enhance the ECL signal. Furthermore, from Fig. 3A we can see that the ECL intensity of the DNA sensor using the CaCO3/CMC@AgNPs composite labeled S3 (Fig. 3A, curve d) was 3.6 times higher than the pure AgNPs labeled S3 (Fig. 3A, curve c) at the same S2 concentration, which demonstrated the amplification of ECL signal with CaCO3/ CMC@AgNPs composite as the label. Fig. 3B shows plots of ECL intensity of the DNA sensor vs. the S2 concentration of pure AgNPs (a), and CaCO3/CMC@AgNPs composites (b) labeled S3, respectively. From Fig. 3B, we could see that CaCO3/CMC@AgNPs composites exhibit excellent ECL performance. It was found that the ECL signal was too weak to be distinguished in the absence of S2 O8 2 , indicated that S2 O8 2 was of vital importance in the process of AgNPs as coreactant. In addition, the other coreactant, such as tripropylamine, was considered. The strong ECL signal was only observed in the presence of S2 O8 2 and there was no ECL signal obtained using tripropylamine as coreactant (Fig. 3C, curve a). Moreover, the cathodic ECL of AgNPs was also studied as the potential was cycled between ( 1.0) V and ( 2.5) V. Compared with the ECL between (þ 2.0) V and ( 2.5) V, the cathodic ECL just decreased slightly (Fig. 3D), indicating that the coreactant ECL contributed to the whole ECL dominantly. According to the previous reports concerning the cathodic ECL of QDs (Jie et al., 2009; Wang et al., 2011), it could be confirmed that the cathodic ECL herein was originated from the formation of excited-state AgNPs (Agn) through electron transfer annihilation of negatively charged Agn and the strongly oxidizing n radicals produced by electroreduction of S2 O8 2 . The possible ECL mechanisms were described as follows:
Ag-Agn
(1)
Fig. 3. (A) ECL-potential curves obtained at (a) PDDA-GR/Au-PWE; (b) S2/S1/PDDA-GR/Au-PWE; (c) AgNPs/S3/S2/S1/PDDA-GR/Au-PWE; (d) CaCO3/CMC@AgNPs/S3/S2/S1/ PDDA-GR/Au-PWE in 10 mM Tris–HCl buffer (pH 7.4) containing 0.1 M K2S2O8 and 0.1 M KCl. The S2 concentration was 1.0 10 14 M. (B) Plots of ECL intensity of the DNA sensor vs. S2 concentration of (a) pure AgNPs and (b) CaCO3/CMC@AgNPs labeled S3. (C) ECL-potential curves of AgNPs modified electrode using (a) tripropylamine, and (b) K2S2O8 as coreactant; (D) ECL-potential curves of AgNPs modified electrode with the potential cycled between (a) ( 1.0) V and ( 2.5) V, and (b) ( þ2.0) V and ( 2.5) V. Scan rate: 100 mV s 1. The voltage of the PMT was 800 V.
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Table 1 Detection of Target-ssDNA in spiked samples. Sample
Addeda (fM)
Detectedb (fM)
RSD (%, n¼ 11)c
Recoveryd (%)
1 2 3 4 5 6 7 8
0.04 0.2 1 10 50 500 5000 50,000
0.04104 0.1964 1.035 10.19 48.90 484.50 5135.00 48,800.00
4.1 3.9 4.5 3.2 3.7 4.0 4.6 3.8
102.6 98.2 103.5 101.9 97.8 96.9 102.7 97.6
a
[Added] means the values that we add into human serum sample. [Detected] means the amount of Target-ssDNA obtained according to the standard curve equations from eleven parallel detections. c The RSD of measurements are calculated from eleven independent experiments. d Recovery means the ratio of [Detected]/[Added]. b
Fig. 4. Relationship between ECL intensity and S2 concentration in pH 7.4 Tris–HCl buffer containing 0.1 M K2S2O8 and 0.1 M KCl. Inset (a) ECL profiles of the DNA sensor in the presence of different concentrations of S2. Inset (b) logarithmic calibration curve of the ECL signals (Inset a) for S2.
S2 O8 2 þe -S2 O8 n2
(2)
hybridized DNA was removed through thermal denaturation (Fig. S3C). The results indicated acceptable regeneration.
S2 O8 n2 -SO4 2 þSO4 n
(3)
3.7. Analytical application potential of the
Agn þSO4 n -Agn þ SO4 2
(4)
Agn-Ag þhv
(5)
3.5. Analytical performance The quantitative behavior of the DNA sensor was assessed by monitoring the difference of the ECL intensity upon the concentration of S2 at optical conditions (in Supplemental information Fig. S2). Fig. 4 exhibits the relationship between ECL intensity and S2 concentration. It could be found that the increased ECL intensity was directly related to the concentration of S2 (Inset a). The ECL intensity ascended logarithmically as the S2 concentration increased in the range of 4.0 10 17–5.0 10 11 M. The linear regression equation was IECL ¼872.67 þ354.96 log cDNA (cDNA fM) and the correlation coefficient was 0.9889. The limit of detection (LOD) was 8.5 10 18 M (defined as 3s, where s is the standard deviation of eleven measurements of blank samples), indicative of an acceptable quantitative behavior. Compared with other sensors reported previously, our proposed DNA sensor exhibited a satisfactory detection limit and linear range, and the characteristics of other DNA sensors are summarized in Table S2. 3.6. Specificity, stability, reproducibility, and regeneration of the DNA sensor The specificity of the proposed DNA sensor in discriminating perfect target DNA from two-base mismatched (S4), noncomplementary (S5), and scrambled (S6) DNA sequences was tested via comparing the ECL signal changes under three concentrations (Fig. S3A). The results demonstrated that the proposed DNA sensor exhibited an excellent specificity to the target DNA. Stability of the DNA sensor is a key factor in their application. Fig. S3B shows the ECL signal vs. time under continuous potential scanning for 10 cycles. The results indicated that the μ-PAD DNA sensor had fine storage stability. To investigate the reproducibility of the μ-PAD DNA sensor, intra- and inter-assay were both estimated. The experimental results indicated that the proposed strategy was reliable and can be used for target DNA detection with acceptable reproducibility. In the test, the DNA sensor could be regenerated by incubation of the μ-PAD in hot water (90 1C) for 1 min, by which
μ-PAD DNA sensor
In order to verify the analytical reliability and application potential of the proposed DNA sensor with real samples, eleven replicate determinations of Target-ssDNA in spiked human serum samples were investigated under the optimal conditions. The spiked human serum samples were prepared through adding different amounts of Target-ssDNA (0.04–50,000 fM) into the human serum samples. Table 1 shows the results, that the RSD was less than 5%, and the recoveries were between 96.9% and 103.5%, indicated an acceptable veracity of the proposed method. Thus, it may provide an alternative tool for diagnosis applications.
4. Conclusions In summary, a methodology for low-cost, simple, rapid, portable, disposable, and sensitive DNA detection was developed here by ECL measurement based on GR modified Au-PWE. Taking advantage of dual amplification effects of the GR modified Au-PWE and CaCO3/CMC@AgNPs conposites, the DNA sensor not only performed excellent specificity, but also had a LOD as low as 8.5 10 18 M. The experiments results demonstrated that the μ-PAD DNA sensor exhibited excellent analytical performance and had acceptable application potential in human serum assay. The proposed method provided a promising platform for accurate gene diagnostics at home and in the field. Acknowledgment This work was financially supported by the National Natural Science Foundation of China (21277058 and 21175058); Natural Science Foundation of Shandong Province, China (ZR2012BZ002).
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