DOI: 10.1002/asia.201500105
Full Paper
Immunoassays
Biometallization-Based Electrochemical Magnetoimmunosensing Strategy for Avian Influenza A (H7N9) Virus Particle Detection Chuan-Hua Zhou,[a, b] Zhen Wu,[a] Jian-Jun Chen,*[c] Chaochao Xiong,[c] Ze Chen,[d] DaiWen Pang,[a] and Zhi-Ling Zhang*[a] Abstract: A highly sensitive electrochemical immunosensor for avian influenza A (H7N9) virus (H7N9 AIV) detection was proposed by using electrochemical magnetoimmunoassay coupled with biometallization and anodic stripping voltammetry. This strategy could accumulate the enzyme-generated product on the surface of the magneto electrode by means of silver deposition, which amplified the detection signal about 80 times. The use of magnetic beads (MBs) and the magneto electrode could also amplify the detection
Introduction A novel avian origin influenza A (H7N9) virus (H7N9 AIV) has caused over three hundred human infections since its emergence in China in February 2013.[1] Although the virus is not capable of efficient human-to-human transmission, its biological features and pandemic potential have caused global concern.[2] Meanwhile, fast and sensitive virus detection techniques are urgently needed to provide immediate and appropriate [a] Dr. C.-H. Zhou,+ Z. Wu,+ Prof. D.-W. Pang, Prof. Z.-L. Zhang Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education) College of Chemistry and Molecular Sciences State Key Laboratory of Virology Wuhan University Wuhan, 430072 (P. R. China) E-mail: [email protected] [b] Dr. C.-H. Zhou+ Key Laboratory of Medicinal Chemistry for Natural Resource (Ministry of Education) School of Chemical Science and Technology Yunnan University Kunming, 650091 (P. R. China) [c] Prof. J.-J. Chen, C. Xiong CAS Key Laboratory of Special Pathogens and Biosafety Wuhan Institute of Virology Chinese Academy of Sciences Wuhan, 430071 (P. R. China) E-mail: [email protected] [d] Prof. Z. Chen Shanghai Institute of Biological Products (P. R. China) [+] These authors contributed equally to this work. Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/asia.201500105. Chem. Asian J. 2015, 10, 1387 – 1393
signal. Furthermore, a bi-electrode signal transduction system was introduced into this immunosensor, which is also beneficial to the immunoassay. A concentration as low as 0.011 ng mL¢1 of H7N9 AIV could be detected in about 1.5 h with good specificity. This study not only provides a simple and sensitive approach for virus detection but also offers an effective signal enhancement strategy for the development of highly sensitive MB-based electrochemical immunoassays.
clinical strategies to control the spread of the virus. The traditional virus detection methods including virus culture, serological tests, enzyme-linked immunoassays (ELISA), and polymerase chain reaction (PCR) are widely used in virus detection. However, these methods suffer from different drawbacks, such as long analysis times, low sensitivity or require expensive reagents, facilities operators, and sophisticated instruments.[3] As a consequence, there is extensive interest to develop simple and sensitive virus detection methods. Electrochemical immunoassays have attracted considerable interest due to their intrinsic advantages of low cost, high sensitivity, portability, and low power requirement.[4] However, the practical application of electrochemical immunosensors is restricted by the pollution of the working electrode. The magnetic bead (MB)-based immunosensing strategy combines the advantages of electrochemical detection and immunomagnetic separation, which could overcome this shortcoming very well.[5] The sensitivity could also be increased due to the excellent properties of MBs.[6] A series of MB-based electrochemical biosensors have been fabricated and applied to the detection of virus particles,[5, 7] pathogenic bacteria,[8] antibodies,[9] pesticide residues,[10] cancer biomarkers,[11] nucleic acids[12] and so on. In order to achieve higher sensitive detection in biomedical analysis, different kinds of signal amplification strategies were proposed to improve the sensitivity of bioassays, such as biobarcode amplification,[13] silver enhancement,[14] the use of multiple labels on a nanomaterial,[15] and biotin–streptavidin amplification[16] . The silver enhancement strategy could enhance the sensitivity greatly, but the main drawback of this method is the high background signal.[17] Biometallization, which enables highly specific metal deposition only in the presence of
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Full Paper used as the reaction carrier based on a sandwich-typed immunoassay (Scheme 1 A). The biotin–streptavidin system was introduced into this immunosensor by using a biotinylated antibody and alkaline phosphatase-labelled streptavidin (SA-ALP) as signal tags. The target virus could be captured by its antibody on the surface of IMBs. Due to the fast reaction kinetics and effective immunomagnetic separation of IMBs, the target virus could be separated from the complex matrix rapidly without any pretreatment. After the immunoreaction process, the immunocomplex-coated MBs were transferred to the surface of the magneto-controlled home-made gold nanoparticles (AuNPs)-modified electrode (M-AuE). Then ALP on the surface of MB catalyzed p-aminophenyl phosphate monohydrate (pAPP) into the reducing agent p-aminophenol (p-AP), which reduced Ag + to Ag0 spontaneously, leading to the deposition of Ag0 on the surface of the M-AuE. As a consequence, the concentration of the target virus could be detected by the anodic stripping current of the deposited silver through this biometallization-based electrochemical magnetoimmunosensing strategy, realizing the detection of virus particles with high sensitivity. To investigate the feasibility of this strategy, ALP was covalently linked to MBs directly, and the conjugates (ALP-MBs) were accumulated on the M-AuE with a magnet underneath Results and Discussion followed by a biometallization reaction and anodic stripping Biometallization-based electrochemical magnetoimmunodetection. As shown in Figure S1 in the Supporting Informasensing tion, when bare MBs were trapped on the M-AuE, no detectaThe principle of the biometallization-based electrochemical ble signal was obtained, which indicated that the unreacted pmagnetoimmunosensor is illustrated in Scheme 1. The detecAPP could not reduce Ag + . By contrast, the presence of MBtion method could be divided into three steps: immunoreacALP on the surface of the magnet electrode could produce tions in a centrifuge tube (Scheme 1 A), enzyme-induced silver a large stripping current due to the enzyme-induced silver deposition on the magneto-controlled electrode (Scheme 1 B), deposition reaction on the surface of M-AuE. As a consequence, and electrochemical detection of the deposited silver the presence of ALP was crucial for the biometallization reac(Scheme 1 C). The immunomagnetic beads (IMBs), which were tion and this strategy was available for the detection of ALP modified with the monoclonal antibody of H7N9 virus, were on the surface of MBs. Since the half-wave potentials of p-AP and Ag + versus NHE are 0.097 V and 0.799 V, respectively, p-AP can reduce Ag + to Ag0 spontaneously. But this reduction process is too slow without the AuNP nucleation sites.[19] As a result, the presence of AuNPs on the electrode could greatly enhance the stripping signal, owing to the pronounced effect of AuNPs on the silver deposition process coupled with the increased number of nucleation sites on the electrode. As a consequence, the AuNPs-modified electrode was preferable for the deposition of silver. The biometallization reaction parameters such as the concenScheme 1. Illustration of the protocol for the biometallization-based electrochemical magnetoimmunoassay for trations of Ag + and enzyme subH7N9 AIV. (A) Immunoreactions in a centrifuge tube, (B) enzyme-induced metallization reaction on a magnetostrate p-APP, the pH of the solucontrolled home-made electrode and (C) the electrochemical detection procedure. enzyme, could overcome this drawback, and this strategy could be used to amplify the detection signals tremendously.[18] Herein, we exploited a highly sensitive electrochemical magnetoimmunosensor for H7N9 AIV detection based on biometallization. The target virus could be captured and separated from complex samples without any pretreatment. A magnetocontrolled home-made electrode was used to capture the immunocomplex-coated MBs for subsequent biometallization reaction. The deposition of silver took place on the surface of the magneto electrode for a relatively long period followed by highly sensitive anodic stripping analysis, which amplified the detection signal dramatically. In addition, the bi-electrode signal transduction system was introduced into this biometallization-based electrochemical magnetoimmunosensor by separating the biometallization reaction on two different electrodes. This strategy could avoid the possible influence of silver ions and silver deposition on the enzyme activity, and the stripping analysis of the deposited silver could be realized more easily. The proposed method shows a broad prospect for on-site diagnostic applications.
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Figure 1. The effects of (A) p-APP concentration, (B) Ag + concentration, (C) pH of the detection solution, and (D) the reaction time on the detection signal.
ure 2 A, the linear sweep voltammetry (LSV) signals of deposited silver increased steeply with increasing H7N9 AIV concentrations. The presence of the target virus resulted in the binding of SA-ALP on the IMBs which could induce silver deposition on the M-AuE, producing relatively large stripping currents. As a result, this method could be used for H7N9 AIV detection with high sensitivity. The integration of the biometallization with the electrochemical magnetoimmunosensing could greatly enhance the sensitivity of the immunoassay. When AgNO3 was absent in the biometallization reaction solution, the
tion, and the reaction time were evaluated and optimized. The amount of deposited Ag on the M-AuE depended on the amount of p-AP, which is the product of the enzymatic hydrolysis reaction. As a result, the anodic stripping current increased with the concentration of p-APP up to 2 mm (Figure 1 A). A sufficient amount of Figure 2. (A) Typical LSV signals of the immunosensor in the presence of different concentrations of H7N9 AIV: Ag + was also needed in the (a) 0, (b) 0.5, (c) 1, (d) 5, (e) 50 ng mL¢1. (B) Comparison of the electrochemical signals using different signal amplifienzyme reaction solution to oxi- cation strategies. dize the p-AP intermediate as soon as it was produced on the presence of ALP could catalyze the conversion of p-APP into psurface of the M-AuE. As shown in Figure 1 B, the anodic stripAP, which could be oxidized directly on the surface of the Mping signal increased remarkably with an increase in the conAuE. Thus, the oxidation signal of p-AP was obtained, which centrations of AgNO3 of up to 2 mm and then reached a plawas much smaller than the LSV signal of deposited Ag0 obteau. The dependence of the anodic stripping signal on the pH tained via the biometallization strategy (Figure 2 B). The inteof the enzyme reaction solution is shown in Figure 1 C, and the gration of the biometallization strategy could enhance the optimum pH was found to be 9.8. Thus, a 1 m diethanolamine electrochemical signals about 80 times when the deposition solution of pH 9.8 containing 2 mm p-APP and 2 mm AgNO3 time was 30 min (Figure 2 B). The reasons are as follows: The was selected as the biometallization reaction solution. enzymatic product of ALP can reduce Ag + to Ag0 on the surThe deposition time also affected the electrochemical signal greatly, which would affect the sensitivity of the immunoassay. face of the magnet electrode by means of biometallization, As shown in Figure 1 D, the stripping current increased signifiwhich made it possible to accumulate the electrochemical cantly with the biometallization time, which indicated that active product for a relatively long period via metal deposition more silver had been deposited on the surface of the M-AuE. and the diffusion of the enzymatic product could also be The slope decreased significantly with time, especially after avoided. Meanwhile, this strategy could be combined with the a deposition time over 30 min, which may be attributed to the highly sensitive stripping analysis, thus offering a substantial partial deactivation of ALP caused by silver deposition. signal amplification of the electrochemical signal. To verify the capability of this method on virus detection, H7N9 AIV was selected as the model analyte. The target virus was captured by the IMBs based on a sandwich-typed immunoassay and then detected on the M-AuE. As shown in FigChem. Asian J. 2015, 10, 1387 – 1393
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Full Paper the deposition of Ag0 on the detection electrode (the reaction mechanism is presented in the Supporting Information). Then the target virus could be detected by the anodic stripping analysis of the deposited silver on the detection electrode with In the biometallization-based electrochemical magnetoimmuhigh sensitivity (Scheme 2 C). The deposition of silver on the nosensing strategy, the immunocomplex-coated MBs were acsurface of the IMBs could be avoided via the bi-electrode cumulated on the surface of the magneto electrode directly signal transduction system, which was beneficial to the immuand then the electrochemical signal was obtained by means of noassay. enzyme-induced silver deposition. This strategy combines the The detection conditions of the bi-electrode signal transduchighly sensitive anodic stripping analysis and biometallization, tion system-based biometallization magnetoimmunosensor thus providing an immunoassay with high sensitivity. However, were also optimized to enhance the detection sensitivity (see the Ag + ions coexisting in the substrate solution might inhibit the Supporting Information for details). Diethanolamine buffer the ALP activity, which could influence the electrochemical (1 m, pH 9.8) containing 2 mm p-APP and 0.05 % Tween 20 was signal. The silver deposition on IMBs, on which the enzyme is chosen as the enzyme reaction solution, which was added in present, could also affect the biometallization detection. Morethe poly(dimethylsiloxane) (PDMS) cell of the M-InkE and used over, the relatively high concentration of enzymatic product as the anolyte. Tris-base solution (1 m) containing 2 mm AgNO3 could lead to the nucleation of silver atoms, which could result in the generation of silver nanoparticles in solution and, as was used as the silver deposition solution which was used as a result, the electrochemical signal obtained from the anodic the catholyte. stripping analysis was diminished. The amplification efficiency of the magneto electrode was The bi-electrode signal transduction system was introduced investigated under the optimal conditions. The LSV signal obinto this electrochemical magnetoimmunosensor, and the biotained from the bi-electrode signal transduction system with metallization process could be separated to take place on two or without magnet underneath the home-made ink electrode independent electrodes, and thus the above-mentioned probwas compared (Figure 3 A). When the external permanent lems could be solved. The principle of the strategy is illustrated magnet was set underneath the home-made ink electrode, all in Scheme 2. The IMBs were also used as the reaction carrier to of the IMBs in the electrolytic cell could be accumulated on the surface of the working electrode which was beneficial to the oxidation of the enzymatic product p-AP. As a result, the electrochemical signal could be greatly amplified. The accumulation of the immunocomplexcoated MBs on the magneto electrode could enhance the detection signal about 6 times (Figure 3 A). The bi-electrode signal transduction system was beneficial to the immunoassay as well. This biometallization-based electrochemical magnetoimmunoassay combines the highly sensitive anodic stripping analysis and biScheme 2. Illustration of the protocol for the bi-electrode signal transduction system-based biometallization magnetoimmunosensor. (A) Immunoreactions in a centrifuge tube, (B) biometallization reaction utilizing the bi-elecometallization, providing the imtrode strategy and (C) the electrochemical detection procedure. munoassay with high sensitivity. However, the Ag + coexisting in the substrate solution might incapture the target virus in a complex matrix, and the immunohibit the ALP activity, which would influence the electrochemireaction took place in a centrifuge tube (Scheme 2 A). Then the cal signal. The silver deposition on IMBs, on which the enzyme bi-electrode device, which was constructed by the magnetois present, would also affect the biometallization detection.[20] controlled home-made ink electrode (M-InkE) as the anode The application of the bi-electrode signal transduction system and the AuNPs-modified gold detection electrode as the cathcould solve these problems very well. The biometallization reode of the galvanic cell, was used for the MB-based biometalliaction could be separated into two independent processes zation reaction (Scheme 2 B). Alkaline phosphatase (ALP) through this bi-electrode strategy which could effectively tagged on the surface of MB could catalyze p-APP into the reavoid the influence of the Ag + and silver deposition on the acducing agent p-AP, which could be oxidized on the surface of tivity of ALP. The deposition of silver on the IMBs could also be the M-InkE while Ag + was reduced at the cathode, leading to avoided. In comparison with the single-electrode strategy, the The bi-electrode signal transduction system-based biometallization signal amplification strategy
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Full Paper was from 0.02 to 50 ng mL¢1 with a correlation coefficient of 0.996 (Figure 4 B). The detection limit was 0.011 ng mL¢1 at a signal-to-noise ratio of 3, which is lower than that of other viral detection methods.[21] Five independent measurements were performed by using 10 and 50 ng mL¢1 H7N9 AIV samples to estimate the precision of this method. The relative standard deviations were 3.8 and 4.6 % with mean LSV signals of 87.8 and 344.6 mA, respectively, which proved the good precision of this method. The specificity of this method was evaluated by using other viruses such as inactivated avian influenza A (H5N1) virus (H5N1 AIV), porcine pseudorabies virus (PRV) and newcastle disease virus (NDV) as negative controls (Figure 5 A; the con-
Figure 3. (A) Typical LSV signals for H7N9 AIV detection using the bi-electrode signal transduction system (a) with or (b) without magnet underneath the home-made ink electrode. (B) Typical LSV signals for 50 ng mL¢1 H7N9 AIV using different strategies for biometallization.
LSV signal obtained from the bi-electrode strategy was larger and sharper (Figure 3 B), and thus was more suitable for the quantitative determination of the target virus. The sensitivity and detection range of this immunosensor were then evaluated. As shown in Figure 4 A, the LSV signal of deposited silver increased steeply with increasing H7N9 AIV concentration. The linear range of this method for H7N9 AIV Figure 5. (A) Histogram for the specificity of this method by using H7N9 AIV samples and other viruses, such as inactivated H5N1 AIV, PRV, and NDV as negative controls (50 ng mL¢1). (B) LSV responses of the immunosensor in the presence (black) and absence (hatched) of H7N9 AIV (50 ng mL¢1) in different media.
Figure 4. (A) Typical LSV signals of the immunosensor in the presence of different concentrations of H7N9 AIV: (a) 0, (b) 0.02, (c) 0.5, (d) 5, (e) 10, (f) 20, (g) 30, (h) 50 ng mL¢1. (B) Electrochemical current response versus the concentration of H7N9 AIV. Chem. Asian J. 2015, 10, 1387 – 1393
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centration of these viruses was 50 ng mL¢1). The LSV signals of negative samples were negligible; only in the presence of the target virus a large electrochemical signal was observed, which demonstrated that this method has sufficient specificity for the determination of H7N9 AIV. The highly specific interaction between H7N9 AIV and its antibody ensured the specificity of the analysis. The anti-interference ability of this immunosensor was also investigated. As shown in Figure 5 B, all the positive samples showed obvious LSV signals, and H7N9 AIV could be detected with a high signal-to-noise ratio in complex matrices such as chicken serum and chicken liver. Accordingly, the proposed method could be directly applied to complex samples. The use of MBs as the immunoreaction carrier not only improved the detection sensitivity due to the magnetic enrichment but also enhanced the anti-interference due to an effective immunomagnetic separation.[10]
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Full Paper Conclusions In conclusion, a simple and highly sensitive biometallizationbased electrochemical magnetoimmunosensing strategy is proposed based on the integration of electrochemical magnetoimmunosensing and biometallization reaction. This strategy could accumulate the enzymatic product for a relatively long period by means of silver deposition, which could amplify the detection signal about 80 times. The use of MBs as immunoreaction carrier and the magneto-controlled electrode as the detection electrode could also amplify the detection signal due to the magnetic enrichment. Furthermore, the bi-electrode signal transduction system was applied in this biometallization-based electrochemical magnetoimmunosensor, which could avoid the possible influence of silver deposition and heavy metal ions on the enzyme activity, and the deposition of silver on IMBs could also be avoided. This strategy could be applied to H7N9 AIV detection with good specificity and strong anti-interference ability, and a concentration as low as 0.011 ng mL¢1 H7N9 AIV could be detected in 1.5 h. The proposed immunosensor not only provides a rapid, simple and sensitive approach for virus detection with high sensitivity and selectivity but also presents a new signal amplification approach for MB-based electrochemical immunoassays.
Experimental Section Materials and Reagents Inactivated H7N9 AIV, murine origin H7N9 hemagglutinin (HA) specific monoclonal antibody (mAb) and rabbit derived polyclonal antibody (pAb), as well as other viruses such as inactivated H5N1 AIV, NDV and PRV were obtained from the Wuhan Institute of Virology, Chinese Academy of Sciences. N-(3-dimethylamino-propyl)-N’-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were purchased from Sigma–Aldrich (St. Louis, MO). p-Aminophenyl phosphate monohydrate (p-APP) was purchased from Santa Cruz Biotechnology, Inc. EZ-link sulfo-NHS-LC-biotinylation kit was purchased from Pierce Biotechnology (Rockford, IL). Superparamagnetic magnetic beads (500 nm in diameter) were purchased from Ademtech SA (Pessac, France). Alkaline phosphatase labelled streptavidin (SA-ALP) was purchased from Vector (Burlingame, CA). Skim milk was received from Becton, Dickinson and Company. All other chemical reagents were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).The immunomagnetic beads (MB-mAb conjugates, IMBs) were prepared following a two-step EDC/NHS chemistry, and the biotinylated-pAb (B-pAb) was prepared using the EZ-link sulfo-NHS-LC-biotinylation kit according to our previous report.[7]
Preparation of the Magneto-controlled Home-made Electrodes The magneto-controlled electrodes were prepared using traditional photoresist lithography according to our previous report with some modification.[5] (1) The M-AuE was fabricated using indium tin oxide (ITO) glass as the substrate, and the protocol is illustrated in Figure S3 in the Supporting Information. Briefly, the ITO glass slides (Sigma) were cut into pieces of approximately 25 Õ 25 mm2, then cleaned via ulChem. Asian J. 2015, 10, 1387 – 1393
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trasonic cleaning in ethanol and ultrapure water. The positive resist (AZ 4620) was spin-coated on the surface of the slides and then soft-baked on a hot-plate at 75 8C for 3 min followed by 5 min at 105 8C. The substrates were placed in contact with a photomask and exposed to an UV light source for an appropriate time, and the photoresist was then developed in an AZ developer (1:3 v/v AZ400K/H2O) for 2 min. Concentrated hydrochloric acid was used to etch the unprotected indium tin oxide and then the poly(dimethylsiloxane) (PDMS) cell was irreversibly linked to the slides by O2 plasma treatment. Finally, a copper wire was welded to the ITO glass using conductive adhesive to form the home-made ITO electrode (Figure S3C in the Supporting Information). The photoresist was washed away from the electrode via ultrasonic rinsing in ethanol and then the electrode was soaked in piranha solution (7:3 v/v concentrated H2SO4/30 % H2O2 ; Caution! The piranha solution should be handled with extreme care) for 10 min. Then Au nanoparticles were deposited on the surface of the working electrode using electrochemical deposition (a photograph of the homemade AuNPs-modified electrode is shown in Figure S3D in the Supporting Information). An external permanent magnet was placed under the working electrode to form the M-AuE. (2) The M-InkE was fabricated using glass slide as the substrate. As shown in Figure S4 in the Supporting Information, glass slides (Sigma) were cut into pieces of approximately 25 Õ 25 mm2, then cleaned via ultrasonic rising in ethanol and ultrapure water, and immersed in piranha solution at 90 8C for 0.5 h. After rinsing with ultrapure water and drying under a stream of nitrogen, the slides were patterned with a positive resist (AZ 4620) using the traditional photoresist lithography. The conductive ink was brushed on the surface of the slides. The photoresist was then washed away from the electrode via ultrasonic rinsing in ethanol, and the PDMS cell was irreversibly linked to the slides by O2 plasma treatment. Finally, a copper wire was welded to the glass slide using conductive adhesive to form the home-made ink electrode (Figure S4C in the Supporting Information). An external permanent magnet was placed under the working electrode to form the M-InkE.
Immunoreaction Procedure The immunoreaction was performed in a 1.5 mL centrifuge tube by three steps and the IMBs were used as the reaction carrier based on a sandwich immunoassay, as shown in Scheme 1 A. (1) The IMBs were added to 1 mL virus samples and then incubated for 20 min at 37 8C with gentle shaking (200 rpm) to capture the target virus. (2) After separation from the suspension and washing with 400 mL rising buffer (RB; consisting of 0.1 % skim milk, 0.05 % Tween 20 in 0.1 m phosphate buffer, pH 7.2), the bead-virus composites were incubated with B-pAb (5 mg mL¢1) in 100 mL binding buffer (BB; consisting of 0.3 % skim milk, 0.05 % Tween 20 in 0.1 m phosphate buffer, pH 7.2) for 20 min at 37 8C. Then the composites of IMB/virus/B-pAb were washed twice with 400 mL RB. (3) SA-ALP (10 mg mL¢1 in 100 mL BB) was applied to react with the composites, and the mixture was incubated for 10 min at 37 8C with gentle shaking. Finally, the composites (IMB/virus/B-pAb/SA-ALP) were washed thrice with RB. The time consumed for the immunoassay was about 1 h.
Electrochemical Detection Electrochemical measurements were carried out with a CHI660a electrochemical workstation (CH Instruments, Inc. Shanghai, China). To detect the target virus with high sensitivity, two detection strategies based on the magneto-controlled electrode and enzyme-induced metallization were proposed.
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Full Paper (1) The M-AuE was used to capture the immunocomplex-coated MBs by placing an external BaFe12O19 magnet underneath. Then a biometallization reaction solution containing 2 mm p-APP, 2 mm AgNO3 and 0.05 % Tween 20 in 1 m diethanolamine solution (1 m, pH 9.8) was added to the PDMS cell. The electrode was incubated at 37 8C for 30 min. Then the M-AuE was washed with water, and 1 m KCl was added to the PDMS cell. A Pt wire counter electrode and an Ag/AgCl reference electrode were immersed into the supporting electrolyte. LSV measurements from ¢0.1 to 0.4 V (vs. Ag/ AgCl) with a 100 mV s¢1 scanning rate was performed for the electrochemical detection. For comparison, the immunocomplex-coated MBs were accumulated on the M-AuE with an external magnet underneath, and subsequently 200 mL detection solution of 1 m pH 9.8 diethanolamine containing 2 mm p-APP and 0.05 % Tween 20 was added to the PDMS cell. After incubation at 37 8C for 30 min, the LSV signal of pAP oxidation was monitored and this enzymatic signal was compared with the signal obtained from the biometallization strategy. (2) The bi-electrode signal transduction system could also be used for the biometallization-based electrochemical detection. As shown in Scheme 2, after the immunoreaction procedure, the composites of IMB/virus/B-pAb/SA-ALP were accumulated on the M-InkE with an external magnet underneath, and then 100 mL enzyme reaction solution (consisting of 2 mm p-APP and 0.05 % Tween 20 in 1 m diethanolamine buffer, pH 9.8) was added to the PDMS cell of the MInkE. The M-InkE was wired to an AuNPs-modified gold detection electrode (6 mm in diameter), which was immerged in 1 mL silver deposition solution composed of 2 mm AgNO3 and 1 m Tris-base. These two solutions were connected with a salt bridge. Thus, a bielectrode signal transduction device was constructed with the MInkE as the anode and the detection electrode as the cathode. The bi-electrode device was maintained to work at 37 8C for 30 min. Then, the detection electrode was taken out of the cell, rinsed with water, and then immersed in 1 mol L¢1 KCl for electrochemical detection. LSV measurements were performed at a potential range from ¢0.1 to 0.3 V (vs. Ag/AgCl) with a 100 mV s¢1 scanning rate using the three-electrode system.
Detection of H7N9 AIV in Complex Matrices Fresh liver from healthy chicken was crushed, and the supernatant was separated by centrifugation. Then 50 ng mL¢1 H7N9 AIV was added to the supernatant or chicken serum to form the synthetic complex samples. These samples were then analyzed using the bielectrode signal transduction system-based biometallization magnetoimmunosensor. The control experiments were performed with the same supernatant or chicken serum in the absence of H7N9 AIV.
Acknowledgements This work was supported by the National Basic Research Program of China (2011CB933600), the 863 Program (2013AA032204), the National Natural Science Foundation of China (21175100, 21475099), the Natural Science Foundation of Hubei (2014CFA003), the Fundamental Research Funds for the Central Universities (2042014kf0196). Keywords: avian influenza A (H7N9) virus · bi-electrode signal transduction · biometallization · electrochemical immunosensors · immunoassays · magnetic beads Chem. Asian J. 2015, 10, 1387 – 1393
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Received: January 30, 2015 Published online on April 17, 2015
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Immunoassays
Biometallization-Based Electrochemical Magnetoimmunosensing Strategy for Avian Influenza A (H7N9) Virus Particle Detection Chuan-Hua Zhou,[a, b] Zhen Wu,[a] Jian-Jun Chen,*[c] Chaochao Xiong,[c] Ze Chen,[d] DaiWen Pang,[a] and Zhi-Ling Zhang*[a] Abstract: A highly sensitive electrochemical immunosensor for avian influenza A (H7N9) virus (H7N9 AIV) detection was proposed by using electrochemical magnetoimmunoassay coupled with biometallization and anodic stripping voltammetry. This strategy could accumulate the enzyme-generated product on the surface of the magneto electrode by means of silver deposition, which amplified the detection signal about 80 times. The use of magnetic beads (MBs) and the magneto electrode could also amplify the detection
Introduction A novel avian origin influenza A (H7N9) virus (H7N9 AIV) has caused over three hundred human infections since its emergence in China in February 2013.[1] Although the virus is not capable of efficient human-to-human transmission, its biological features and pandemic potential have caused global concern.[2] Meanwhile, fast and sensitive virus detection techniques are urgently needed to provide immediate and appropriate [a] Dr. C.-H. Zhou,+ Z. Wu,+ Prof. D.-W. Pang, Prof. Z.-L. Zhang Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education) College of Chemistry and Molecular Sciences State Key Laboratory of Virology Wuhan University Wuhan, 430072 (P. R. China) E-mail: [email protected] [b] Dr. C.-H. Zhou+ Key Laboratory of Medicinal Chemistry for Natural Resource (Ministry of Education) School of Chemical Science and Technology Yunnan University Kunming, 650091 (P. R. China) [c] Prof. J.-J. Chen, C. Xiong CAS Key Laboratory of Special Pathogens and Biosafety Wuhan Institute of Virology Chinese Academy of Sciences Wuhan, 430071 (P. R. China) E-mail: [email protected] [d] Prof. Z. Chen Shanghai Institute of Biological Products (P. R. China) [+] These authors contributed equally to this work. Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/asia.201500105. Chem. Asian J. 2015, 10, 1387 – 1393
signal. Furthermore, a bi-electrode signal transduction system was introduced into this immunosensor, which is also beneficial to the immunoassay. A concentration as low as 0.011 ng mL¢1 of H7N9 AIV could be detected in about 1.5 h with good specificity. This study not only provides a simple and sensitive approach for virus detection but also offers an effective signal enhancement strategy for the development of highly sensitive MB-based electrochemical immunoassays.
clinical strategies to control the spread of the virus. The traditional virus detection methods including virus culture, serological tests, enzyme-linked immunoassays (ELISA), and polymerase chain reaction (PCR) are widely used in virus detection. However, these methods suffer from different drawbacks, such as long analysis times, low sensitivity or require expensive reagents, facilities operators, and sophisticated instruments.[3] As a consequence, there is extensive interest to develop simple and sensitive virus detection methods. Electrochemical immunoassays have attracted considerable interest due to their intrinsic advantages of low cost, high sensitivity, portability, and low power requirement.[4] However, the practical application of electrochemical immunosensors is restricted by the pollution of the working electrode. The magnetic bead (MB)-based immunosensing strategy combines the advantages of electrochemical detection and immunomagnetic separation, which could overcome this shortcoming very well.[5] The sensitivity could also be increased due to the excellent properties of MBs.[6] A series of MB-based electrochemical biosensors have been fabricated and applied to the detection of virus particles,[5, 7] pathogenic bacteria,[8] antibodies,[9] pesticide residues,[10] cancer biomarkers,[11] nucleic acids[12] and so on. In order to achieve higher sensitive detection in biomedical analysis, different kinds of signal amplification strategies were proposed to improve the sensitivity of bioassays, such as biobarcode amplification,[13] silver enhancement,[14] the use of multiple labels on a nanomaterial,[15] and biotin–streptavidin amplification[16] . The silver enhancement strategy could enhance the sensitivity greatly, but the main drawback of this method is the high background signal.[17] Biometallization, which enables highly specific metal deposition only in the presence of
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Full Paper used as the reaction carrier based on a sandwich-typed immunoassay (Scheme 1 A). The biotin–streptavidin system was introduced into this immunosensor by using a biotinylated antibody and alkaline phosphatase-labelled streptavidin (SA-ALP) as signal tags. The target virus could be captured by its antibody on the surface of IMBs. Due to the fast reaction kinetics and effective immunomagnetic separation of IMBs, the target virus could be separated from the complex matrix rapidly without any pretreatment. After the immunoreaction process, the immunocomplex-coated MBs were transferred to the surface of the magneto-controlled home-made gold nanoparticles (AuNPs)-modified electrode (M-AuE). Then ALP on the surface of MB catalyzed p-aminophenyl phosphate monohydrate (pAPP) into the reducing agent p-aminophenol (p-AP), which reduced Ag + to Ag0 spontaneously, leading to the deposition of Ag0 on the surface of the M-AuE. As a consequence, the concentration of the target virus could be detected by the anodic stripping current of the deposited silver through this biometallization-based electrochemical magnetoimmunosensing strategy, realizing the detection of virus particles with high sensitivity. To investigate the feasibility of this strategy, ALP was covalently linked to MBs directly, and the conjugates (ALP-MBs) were accumulated on the M-AuE with a magnet underneath Results and Discussion followed by a biometallization reaction and anodic stripping Biometallization-based electrochemical magnetoimmunodetection. As shown in Figure S1 in the Supporting Informasensing tion, when bare MBs were trapped on the M-AuE, no detectaThe principle of the biometallization-based electrochemical ble signal was obtained, which indicated that the unreacted pmagnetoimmunosensor is illustrated in Scheme 1. The detecAPP could not reduce Ag + . By contrast, the presence of MBtion method could be divided into three steps: immunoreacALP on the surface of the magnet electrode could produce tions in a centrifuge tube (Scheme 1 A), enzyme-induced silver a large stripping current due to the enzyme-induced silver deposition on the magneto-controlled electrode (Scheme 1 B), deposition reaction on the surface of M-AuE. As a consequence, and electrochemical detection of the deposited silver the presence of ALP was crucial for the biometallization reac(Scheme 1 C). The immunomagnetic beads (IMBs), which were tion and this strategy was available for the detection of ALP modified with the monoclonal antibody of H7N9 virus, were on the surface of MBs. Since the half-wave potentials of p-AP and Ag + versus NHE are 0.097 V and 0.799 V, respectively, p-AP can reduce Ag + to Ag0 spontaneously. But this reduction process is too slow without the AuNP nucleation sites.[19] As a result, the presence of AuNPs on the electrode could greatly enhance the stripping signal, owing to the pronounced effect of AuNPs on the silver deposition process coupled with the increased number of nucleation sites on the electrode. As a consequence, the AuNPs-modified electrode was preferable for the deposition of silver. The biometallization reaction parameters such as the concenScheme 1. Illustration of the protocol for the biometallization-based electrochemical magnetoimmunoassay for trations of Ag + and enzyme subH7N9 AIV. (A) Immunoreactions in a centrifuge tube, (B) enzyme-induced metallization reaction on a magnetostrate p-APP, the pH of the solucontrolled home-made electrode and (C) the electrochemical detection procedure. enzyme, could overcome this drawback, and this strategy could be used to amplify the detection signals tremendously.[18] Herein, we exploited a highly sensitive electrochemical magnetoimmunosensor for H7N9 AIV detection based on biometallization. The target virus could be captured and separated from complex samples without any pretreatment. A magnetocontrolled home-made electrode was used to capture the immunocomplex-coated MBs for subsequent biometallization reaction. The deposition of silver took place on the surface of the magneto electrode for a relatively long period followed by highly sensitive anodic stripping analysis, which amplified the detection signal dramatically. In addition, the bi-electrode signal transduction system was introduced into this biometallization-based electrochemical magnetoimmunosensor by separating the biometallization reaction on two different electrodes. This strategy could avoid the possible influence of silver ions and silver deposition on the enzyme activity, and the stripping analysis of the deposited silver could be realized more easily. The proposed method shows a broad prospect for on-site diagnostic applications.
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Figure 1. The effects of (A) p-APP concentration, (B) Ag + concentration, (C) pH of the detection solution, and (D) the reaction time on the detection signal.
ure 2 A, the linear sweep voltammetry (LSV) signals of deposited silver increased steeply with increasing H7N9 AIV concentrations. The presence of the target virus resulted in the binding of SA-ALP on the IMBs which could induce silver deposition on the M-AuE, producing relatively large stripping currents. As a result, this method could be used for H7N9 AIV detection with high sensitivity. The integration of the biometallization with the electrochemical magnetoimmunosensing could greatly enhance the sensitivity of the immunoassay. When AgNO3 was absent in the biometallization reaction solution, the
tion, and the reaction time were evaluated and optimized. The amount of deposited Ag on the M-AuE depended on the amount of p-AP, which is the product of the enzymatic hydrolysis reaction. As a result, the anodic stripping current increased with the concentration of p-APP up to 2 mm (Figure 1 A). A sufficient amount of Figure 2. (A) Typical LSV signals of the immunosensor in the presence of different concentrations of H7N9 AIV: Ag + was also needed in the (a) 0, (b) 0.5, (c) 1, (d) 5, (e) 50 ng mL¢1. (B) Comparison of the electrochemical signals using different signal amplifienzyme reaction solution to oxi- cation strategies. dize the p-AP intermediate as soon as it was produced on the presence of ALP could catalyze the conversion of p-APP into psurface of the M-AuE. As shown in Figure 1 B, the anodic stripAP, which could be oxidized directly on the surface of the Mping signal increased remarkably with an increase in the conAuE. Thus, the oxidation signal of p-AP was obtained, which centrations of AgNO3 of up to 2 mm and then reached a plawas much smaller than the LSV signal of deposited Ag0 obteau. The dependence of the anodic stripping signal on the pH tained via the biometallization strategy (Figure 2 B). The inteof the enzyme reaction solution is shown in Figure 1 C, and the gration of the biometallization strategy could enhance the optimum pH was found to be 9.8. Thus, a 1 m diethanolamine electrochemical signals about 80 times when the deposition solution of pH 9.8 containing 2 mm p-APP and 2 mm AgNO3 time was 30 min (Figure 2 B). The reasons are as follows: The was selected as the biometallization reaction solution. enzymatic product of ALP can reduce Ag + to Ag0 on the surThe deposition time also affected the electrochemical signal greatly, which would affect the sensitivity of the immunoassay. face of the magnet electrode by means of biometallization, As shown in Figure 1 D, the stripping current increased signifiwhich made it possible to accumulate the electrochemical cantly with the biometallization time, which indicated that active product for a relatively long period via metal deposition more silver had been deposited on the surface of the M-AuE. and the diffusion of the enzymatic product could also be The slope decreased significantly with time, especially after avoided. Meanwhile, this strategy could be combined with the a deposition time over 30 min, which may be attributed to the highly sensitive stripping analysis, thus offering a substantial partial deactivation of ALP caused by silver deposition. signal amplification of the electrochemical signal. To verify the capability of this method on virus detection, H7N9 AIV was selected as the model analyte. The target virus was captured by the IMBs based on a sandwich-typed immunoassay and then detected on the M-AuE. As shown in FigChem. Asian J. 2015, 10, 1387 – 1393
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Full Paper the deposition of Ag0 on the detection electrode (the reaction mechanism is presented in the Supporting Information). Then the target virus could be detected by the anodic stripping analysis of the deposited silver on the detection electrode with In the biometallization-based electrochemical magnetoimmuhigh sensitivity (Scheme 2 C). The deposition of silver on the nosensing strategy, the immunocomplex-coated MBs were acsurface of the IMBs could be avoided via the bi-electrode cumulated on the surface of the magneto electrode directly signal transduction system, which was beneficial to the immuand then the electrochemical signal was obtained by means of noassay. enzyme-induced silver deposition. This strategy combines the The detection conditions of the bi-electrode signal transduchighly sensitive anodic stripping analysis and biometallization, tion system-based biometallization magnetoimmunosensor thus providing an immunoassay with high sensitivity. However, were also optimized to enhance the detection sensitivity (see the Ag + ions coexisting in the substrate solution might inhibit the Supporting Information for details). Diethanolamine buffer the ALP activity, which could influence the electrochemical (1 m, pH 9.8) containing 2 mm p-APP and 0.05 % Tween 20 was signal. The silver deposition on IMBs, on which the enzyme is chosen as the enzyme reaction solution, which was added in present, could also affect the biometallization detection. Morethe poly(dimethylsiloxane) (PDMS) cell of the M-InkE and used over, the relatively high concentration of enzymatic product as the anolyte. Tris-base solution (1 m) containing 2 mm AgNO3 could lead to the nucleation of silver atoms, which could result in the generation of silver nanoparticles in solution and, as was used as the silver deposition solution which was used as a result, the electrochemical signal obtained from the anodic the catholyte. stripping analysis was diminished. The amplification efficiency of the magneto electrode was The bi-electrode signal transduction system was introduced investigated under the optimal conditions. The LSV signal obinto this electrochemical magnetoimmunosensor, and the biotained from the bi-electrode signal transduction system with metallization process could be separated to take place on two or without magnet underneath the home-made ink electrode independent electrodes, and thus the above-mentioned probwas compared (Figure 3 A). When the external permanent lems could be solved. The principle of the strategy is illustrated magnet was set underneath the home-made ink electrode, all in Scheme 2. The IMBs were also used as the reaction carrier to of the IMBs in the electrolytic cell could be accumulated on the surface of the working electrode which was beneficial to the oxidation of the enzymatic product p-AP. As a result, the electrochemical signal could be greatly amplified. The accumulation of the immunocomplexcoated MBs on the magneto electrode could enhance the detection signal about 6 times (Figure 3 A). The bi-electrode signal transduction system was beneficial to the immunoassay as well. This biometallization-based electrochemical magnetoimmunoassay combines the highly sensitive anodic stripping analysis and biScheme 2. Illustration of the protocol for the bi-electrode signal transduction system-based biometallization magnetoimmunosensor. (A) Immunoreactions in a centrifuge tube, (B) biometallization reaction utilizing the bi-elecometallization, providing the imtrode strategy and (C) the electrochemical detection procedure. munoassay with high sensitivity. However, the Ag + coexisting in the substrate solution might incapture the target virus in a complex matrix, and the immunohibit the ALP activity, which would influence the electrochemireaction took place in a centrifuge tube (Scheme 2 A). Then the cal signal. The silver deposition on IMBs, on which the enzyme bi-electrode device, which was constructed by the magnetois present, would also affect the biometallization detection.[20] controlled home-made ink electrode (M-InkE) as the anode The application of the bi-electrode signal transduction system and the AuNPs-modified gold detection electrode as the cathcould solve these problems very well. The biometallization reode of the galvanic cell, was used for the MB-based biometalliaction could be separated into two independent processes zation reaction (Scheme 2 B). Alkaline phosphatase (ALP) through this bi-electrode strategy which could effectively tagged on the surface of MB could catalyze p-APP into the reavoid the influence of the Ag + and silver deposition on the acducing agent p-AP, which could be oxidized on the surface of tivity of ALP. The deposition of silver on the IMBs could also be the M-InkE while Ag + was reduced at the cathode, leading to avoided. In comparison with the single-electrode strategy, the The bi-electrode signal transduction system-based biometallization signal amplification strategy
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Full Paper was from 0.02 to 50 ng mL¢1 with a correlation coefficient of 0.996 (Figure 4 B). The detection limit was 0.011 ng mL¢1 at a signal-to-noise ratio of 3, which is lower than that of other viral detection methods.[21] Five independent measurements were performed by using 10 and 50 ng mL¢1 H7N9 AIV samples to estimate the precision of this method. The relative standard deviations were 3.8 and 4.6 % with mean LSV signals of 87.8 and 344.6 mA, respectively, which proved the good precision of this method. The specificity of this method was evaluated by using other viruses such as inactivated avian influenza A (H5N1) virus (H5N1 AIV), porcine pseudorabies virus (PRV) and newcastle disease virus (NDV) as negative controls (Figure 5 A; the con-
Figure 3. (A) Typical LSV signals for H7N9 AIV detection using the bi-electrode signal transduction system (a) with or (b) without magnet underneath the home-made ink electrode. (B) Typical LSV signals for 50 ng mL¢1 H7N9 AIV using different strategies for biometallization.
LSV signal obtained from the bi-electrode strategy was larger and sharper (Figure 3 B), and thus was more suitable for the quantitative determination of the target virus. The sensitivity and detection range of this immunosensor were then evaluated. As shown in Figure 4 A, the LSV signal of deposited silver increased steeply with increasing H7N9 AIV concentration. The linear range of this method for H7N9 AIV Figure 5. (A) Histogram for the specificity of this method by using H7N9 AIV samples and other viruses, such as inactivated H5N1 AIV, PRV, and NDV as negative controls (50 ng mL¢1). (B) LSV responses of the immunosensor in the presence (black) and absence (hatched) of H7N9 AIV (50 ng mL¢1) in different media.
Figure 4. (A) Typical LSV signals of the immunosensor in the presence of different concentrations of H7N9 AIV: (a) 0, (b) 0.02, (c) 0.5, (d) 5, (e) 10, (f) 20, (g) 30, (h) 50 ng mL¢1. (B) Electrochemical current response versus the concentration of H7N9 AIV. Chem. Asian J. 2015, 10, 1387 – 1393
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centration of these viruses was 50 ng mL¢1). The LSV signals of negative samples were negligible; only in the presence of the target virus a large electrochemical signal was observed, which demonstrated that this method has sufficient specificity for the determination of H7N9 AIV. The highly specific interaction between H7N9 AIV and its antibody ensured the specificity of the analysis. The anti-interference ability of this immunosensor was also investigated. As shown in Figure 5 B, all the positive samples showed obvious LSV signals, and H7N9 AIV could be detected with a high signal-to-noise ratio in complex matrices such as chicken serum and chicken liver. Accordingly, the proposed method could be directly applied to complex samples. The use of MBs as the immunoreaction carrier not only improved the detection sensitivity due to the magnetic enrichment but also enhanced the anti-interference due to an effective immunomagnetic separation.[10]
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Full Paper Conclusions In conclusion, a simple and highly sensitive biometallizationbased electrochemical magnetoimmunosensing strategy is proposed based on the integration of electrochemical magnetoimmunosensing and biometallization reaction. This strategy could accumulate the enzymatic product for a relatively long period by means of silver deposition, which could amplify the detection signal about 80 times. The use of MBs as immunoreaction carrier and the magneto-controlled electrode as the detection electrode could also amplify the detection signal due to the magnetic enrichment. Furthermore, the bi-electrode signal transduction system was applied in this biometallization-based electrochemical magnetoimmunosensor, which could avoid the possible influence of silver deposition and heavy metal ions on the enzyme activity, and the deposition of silver on IMBs could also be avoided. This strategy could be applied to H7N9 AIV detection with good specificity and strong anti-interference ability, and a concentration as low as 0.011 ng mL¢1 H7N9 AIV could be detected in 1.5 h. The proposed immunosensor not only provides a rapid, simple and sensitive approach for virus detection with high sensitivity and selectivity but also presents a new signal amplification approach for MB-based electrochemical immunoassays.
Experimental Section Materials and Reagents Inactivated H7N9 AIV, murine origin H7N9 hemagglutinin (HA) specific monoclonal antibody (mAb) and rabbit derived polyclonal antibody (pAb), as well as other viruses such as inactivated H5N1 AIV, NDV and PRV were obtained from the Wuhan Institute of Virology, Chinese Academy of Sciences. N-(3-dimethylamino-propyl)-N’-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were purchased from Sigma–Aldrich (St. Louis, MO). p-Aminophenyl phosphate monohydrate (p-APP) was purchased from Santa Cruz Biotechnology, Inc. EZ-link sulfo-NHS-LC-biotinylation kit was purchased from Pierce Biotechnology (Rockford, IL). Superparamagnetic magnetic beads (500 nm in diameter) were purchased from Ademtech SA (Pessac, France). Alkaline phosphatase labelled streptavidin (SA-ALP) was purchased from Vector (Burlingame, CA). Skim milk was received from Becton, Dickinson and Company. All other chemical reagents were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).The immunomagnetic beads (MB-mAb conjugates, IMBs) were prepared following a two-step EDC/NHS chemistry, and the biotinylated-pAb (B-pAb) was prepared using the EZ-link sulfo-NHS-LC-biotinylation kit according to our previous report.[7]
Preparation of the Magneto-controlled Home-made Electrodes The magneto-controlled electrodes were prepared using traditional photoresist lithography according to our previous report with some modification.[5] (1) The M-AuE was fabricated using indium tin oxide (ITO) glass as the substrate, and the protocol is illustrated in Figure S3 in the Supporting Information. Briefly, the ITO glass slides (Sigma) were cut into pieces of approximately 25 Õ 25 mm2, then cleaned via ulChem. Asian J. 2015, 10, 1387 – 1393
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trasonic cleaning in ethanol and ultrapure water. The positive resist (AZ 4620) was spin-coated on the surface of the slides and then soft-baked on a hot-plate at 75 8C for 3 min followed by 5 min at 105 8C. The substrates were placed in contact with a photomask and exposed to an UV light source for an appropriate time, and the photoresist was then developed in an AZ developer (1:3 v/v AZ400K/H2O) for 2 min. Concentrated hydrochloric acid was used to etch the unprotected indium tin oxide and then the poly(dimethylsiloxane) (PDMS) cell was irreversibly linked to the slides by O2 plasma treatment. Finally, a copper wire was welded to the ITO glass using conductive adhesive to form the home-made ITO electrode (Figure S3C in the Supporting Information). The photoresist was washed away from the electrode via ultrasonic rinsing in ethanol and then the electrode was soaked in piranha solution (7:3 v/v concentrated H2SO4/30 % H2O2 ; Caution! The piranha solution should be handled with extreme care) for 10 min. Then Au nanoparticles were deposited on the surface of the working electrode using electrochemical deposition (a photograph of the homemade AuNPs-modified electrode is shown in Figure S3D in the Supporting Information). An external permanent magnet was placed under the working electrode to form the M-AuE. (2) The M-InkE was fabricated using glass slide as the substrate. As shown in Figure S4 in the Supporting Information, glass slides (Sigma) were cut into pieces of approximately 25 Õ 25 mm2, then cleaned via ultrasonic rising in ethanol and ultrapure water, and immersed in piranha solution at 90 8C for 0.5 h. After rinsing with ultrapure water and drying under a stream of nitrogen, the slides were patterned with a positive resist (AZ 4620) using the traditional photoresist lithography. The conductive ink was brushed on the surface of the slides. The photoresist was then washed away from the electrode via ultrasonic rinsing in ethanol, and the PDMS cell was irreversibly linked to the slides by O2 plasma treatment. Finally, a copper wire was welded to the glass slide using conductive adhesive to form the home-made ink electrode (Figure S4C in the Supporting Information). An external permanent magnet was placed under the working electrode to form the M-InkE.
Immunoreaction Procedure The immunoreaction was performed in a 1.5 mL centrifuge tube by three steps and the IMBs were used as the reaction carrier based on a sandwich immunoassay, as shown in Scheme 1 A. (1) The IMBs were added to 1 mL virus samples and then incubated for 20 min at 37 8C with gentle shaking (200 rpm) to capture the target virus. (2) After separation from the suspension and washing with 400 mL rising buffer (RB; consisting of 0.1 % skim milk, 0.05 % Tween 20 in 0.1 m phosphate buffer, pH 7.2), the bead-virus composites were incubated with B-pAb (5 mg mL¢1) in 100 mL binding buffer (BB; consisting of 0.3 % skim milk, 0.05 % Tween 20 in 0.1 m phosphate buffer, pH 7.2) for 20 min at 37 8C. Then the composites of IMB/virus/B-pAb were washed twice with 400 mL RB. (3) SA-ALP (10 mg mL¢1 in 100 mL BB) was applied to react with the composites, and the mixture was incubated for 10 min at 37 8C with gentle shaking. Finally, the composites (IMB/virus/B-pAb/SA-ALP) were washed thrice with RB. The time consumed for the immunoassay was about 1 h.
Electrochemical Detection Electrochemical measurements were carried out with a CHI660a electrochemical workstation (CH Instruments, Inc. Shanghai, China). To detect the target virus with high sensitivity, two detection strategies based on the magneto-controlled electrode and enzyme-induced metallization were proposed.
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Full Paper (1) The M-AuE was used to capture the immunocomplex-coated MBs by placing an external BaFe12O19 magnet underneath. Then a biometallization reaction solution containing 2 mm p-APP, 2 mm AgNO3 and 0.05 % Tween 20 in 1 m diethanolamine solution (1 m, pH 9.8) was added to the PDMS cell. The electrode was incubated at 37 8C for 30 min. Then the M-AuE was washed with water, and 1 m KCl was added to the PDMS cell. A Pt wire counter electrode and an Ag/AgCl reference electrode were immersed into the supporting electrolyte. LSV measurements from ¢0.1 to 0.4 V (vs. Ag/ AgCl) with a 100 mV s¢1 scanning rate was performed for the electrochemical detection. For comparison, the immunocomplex-coated MBs were accumulated on the M-AuE with an external magnet underneath, and subsequently 200 mL detection solution of 1 m pH 9.8 diethanolamine containing 2 mm p-APP and 0.05 % Tween 20 was added to the PDMS cell. After incubation at 37 8C for 30 min, the LSV signal of pAP oxidation was monitored and this enzymatic signal was compared with the signal obtained from the biometallization strategy. (2) The bi-electrode signal transduction system could also be used for the biometallization-based electrochemical detection. As shown in Scheme 2, after the immunoreaction procedure, the composites of IMB/virus/B-pAb/SA-ALP were accumulated on the M-InkE with an external magnet underneath, and then 100 mL enzyme reaction solution (consisting of 2 mm p-APP and 0.05 % Tween 20 in 1 m diethanolamine buffer, pH 9.8) was added to the PDMS cell of the MInkE. The M-InkE was wired to an AuNPs-modified gold detection electrode (6 mm in diameter), which was immerged in 1 mL silver deposition solution composed of 2 mm AgNO3 and 1 m Tris-base. These two solutions were connected with a salt bridge. Thus, a bielectrode signal transduction device was constructed with the MInkE as the anode and the detection electrode as the cathode. The bi-electrode device was maintained to work at 37 8C for 30 min. Then, the detection electrode was taken out of the cell, rinsed with water, and then immersed in 1 mol L¢1 KCl for electrochemical detection. LSV measurements were performed at a potential range from ¢0.1 to 0.3 V (vs. Ag/AgCl) with a 100 mV s¢1 scanning rate using the three-electrode system.
Detection of H7N9 AIV in Complex Matrices Fresh liver from healthy chicken was crushed, and the supernatant was separated by centrifugation. Then 50 ng mL¢1 H7N9 AIV was added to the supernatant or chicken serum to form the synthetic complex samples. These samples were then analyzed using the bielectrode signal transduction system-based biometallization magnetoimmunosensor. The control experiments were performed with the same supernatant or chicken serum in the absence of H7N9 AIV.
Acknowledgements This work was supported by the National Basic Research Program of China (2011CB933600), the 863 Program (2013AA032204), the National Natural Science Foundation of China (21175100, 21475099), the Natural Science Foundation of Hubei (2014CFA003), the Fundamental Research Funds for the Central Universities (2042014kf0196). Keywords: avian influenza A (H7N9) virus · bi-electrode signal transduction · biometallization · electrochemical immunosensors · immunoassays · magnetic beads Chem. Asian J. 2015, 10, 1387 – 1393
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Received: January 30, 2015 Published online on April 17, 2015
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