Digital Single Virus Electrochemical Enzyme-Linked Immunoassay for

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Digital Single Virus Electrochemical Enzyme-Linked Immunoassay for Ultrasensitive H7N9 Avian Influenza Virus Counting Zhen Wu, Wen-Jing Guo, Yi-Yan Bai, Li Zhang, Jiao Hu, Dai-Wen Pang, and Zhi-Ling Zhang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b03281 • Publication Date (Web): 20 Dec 2017 Downloaded from http://pubs.acs.org on December 20, 2017

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Analytical Chemistry

Digital Single Virus Electrochemical Enzyme-Linked Immunoassay for Ultrasensitive H7N9 Avian Influenza Virus Counting Zhen Wu, Wen-Jing Guo, Yi-Yan Bai, Li Zhang, Jiao Hu, Dai-Wen Pang, Zhi-Ling 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. *Corresponding author: Zhi-Ling Zhang, Email: [email protected].

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ABSTRACT: Electrochemistry has been widely used to explore fundamental properties of single molecules due to its fast response and high specificity. However, the lack of efficient signal amplification strategies and quantitative method limit its clinical application. Here, we proposed a digital single virus electrochemical enzyme-linked immunoassay (digital ELISA) for H7N9 avian influenza virus (H7N9 AIV) counting by integration of digital analysis, bifunctional fluorescence magnetic nanospheres (bi-FMNs) with monolayer gold nanoparticles (Au NPs) modified microelectrode array (MA). Bi-FMNs are fabricated by co-immobilizing polyclonal antibody (pAb) and alkaline phosphatase (ALP). At most one target will be captured per bi-FMNs by controlling the proportion of bi-FMNs to target concentrations (≥ 5:1). The introduction of digital analysis can solve signal fluctuation and the reliability of single virus detection, enabling the digital ELISA to be sensitively and accurately applied for H7N9 AIV detection with a low detection limit of 7.8 fg/mL, which is greatly promising in single biomolecular detection, early diagnosis of disease and practical application.

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The ability to detect a single biomolecule is of great interest for food safety and clinical diagnosis at the early stage of infection.1,2 Although powerful and universal, the conventional enzyme-linked immunoassay (ELISA) suffers from several limitations. The concentration of many biomarkers in blood ranges from 10-16 to 10-12 M, such as the blood samples from cancer patients and the early stage of virus infection. Current immunoassays typically detect biomarkers at concentrations above 10-12 M, suffering from insufficient sensitivity.3 Furthermore, microplate ELISA usually requires large volumes of reagent and long reaction time. From January to February of 2017, a total of 304 cases of human infection with H7N9 AIV (including 36 deaths) according to the World Health Organization (WHO).4 Sensitive detection methods can realize early diagnosis and treatment. The development of digital single molecule electrochemical enzyme-linked immunoassay (digital ELISA) based on droplets or microwells can effectively save reagent and time,5-8 with high spatial and temporal resolution and sensitivity. To detect low concentration proteins in blood, Walt et al. captured them on microbeads with an enzyme reporter. After isolating the beads in 50-fl reaction chambers, they used fluorescence imaging to detect single protein molecules.3,9 Subsequently, they achieved four kinds of cytokines detection by using bead encoding techniques and digital ELISA.10 After that, Zhang et al. proposed a digital ELISA based on the Förster resonance energy transfer (FRET) between single quantum dots (QDs) and fluorescent dyes to detect DNA, MicroRNA, renin, protein kinase, and telomerase.11-13 However, FRET requires accurate regulation of the distance and angle between the donor and acceptor. In addition to fluorescence 3



immunoassay, the use of advanced tools and strategies in other fields may bring new breakthroughs in digital ELISA. Electrochemistry has many wonderful characteristics, including direct signal transduction, high sensitivity, fast response and low cost, which is enormously promising in digital ELISA. But the electrochemical signals of single molecules are too small to be detected, effective signal amplification methods are urgently needed to improve the sensitivity. Fan and Bard firstly employed redox cycling to trap single ferrocene molecules between a Pt-Ir tip and ITO ultramicroelectrode, demonstrating the feasibility of single molecule electrochemistry (SME).14 They also proposed an approach for observing electrocatalytic currents based on the collisions of single nanoparticles at a microelectrode, such as Pt, TiO2 and ZnO nanoparticles.15-17 Moreover, the use of enzymes in electrochemistry brings in many signal amplification methods, including substrate circle amplification, enzyme-induced metallization (EIM) and multiplenzyme labeling.18-20 For example, an electrochemical DNA biosensor was reported with a detection limit of 0.065 fM, providing the possibility of SME.20 However, the nondeterminism of Brownian motion and the lack of accurate quantitative method limit the practical application of SME. The introduction of digital analysis can realize low concentration of targets detection as signals are denoted as “0” or “1”, no longer considering the concrete strength of signals.21-24 The quantitative theoretical basis of digital analysis is Poisson distribution that stands for a probability distribution, requiring a lot of parallel data. Therefore, the preparation of uniform and conductive microelectrode array (MA) is necessary. 4


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Currently, Hutchison et al. proposed a covalently bound molecular linker method to modify small Au NPs on boron doped diamond electrodes.25 The modified electrode exhibited nearly ideal, reproducible electrochemical behavior with narrow redox peaks and small peak separations, which was beneficial to prepare MA. Here, based on the integration of SME, bifunctional fluorescence magnetic nanospheres (bi-FMNs) with digital analysis, we propose a digital ELISA for ultrasensitive H7N9 AIV counting. As shown in Scheme 1, bi-FMNs are fabricated by co-immobilizing pAb and ALP to separate low concentrations of H7N9 AIV from complex samples. At most one virus will be captured per bi-FMNs by controlling the proportion of bi-FMNs to virus concentration (≥ 5:1). Then target/bi-FMNs complexes are individually separated into antibody modified MA. After sandwich immunoreaction, alkaline phosphatase (ALP) on bi-FMNs surface can catalyze the dephosphorylation of p-aminophenyl phosphate monohydrate (p-APP) to produce p-aminophenol (p-AP). The strong reduced p-AP instantly reduces Ag+ to Ag0 since the half-wave potentials of p-AP and Ag+ (vs NHE) are 0.097 and 0.799 V, respectively. After 30 min enzyme-induced metallization (EIM) reaction, much Ag0 deposit on microelectrode array (MA) surface. Then the MAs are rinsed with ultrapure water and 1 µL KCl is added into each microelectrode to perform linear sweep voltammetry (LSV) from -0.1 to 0.3 V with an Ag/AgCl wire as reference and a Pt wire as counter electrode respectively. Finally, signals are counted as “0” or “1” based on digital analysis, and then H7N9 AIV concentrations can be calculated through the probability of “0”. 5



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Experimental Section Reagents and Instruments. Calf intestine alkaline phosphatase (ALP) was purchased from Sigma-Aldrich (St. Louis, MO). The p-aminophenyl phosphate monohydrate (p-APP) and p-nitrophenol sodium phosphate hexahydrate (p-NPP) were purchased from Santa Cruz Biotechnology, Inc. Dylight 488-labeled goat anti-rabbit IgG was purchased from Abbkine. N-hydroxysuccinimide (NHS) was purchased

from

thermo.

Bovine

serum

N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide

albumin

hydrochloride

(BSA)

and

(EDC)

were

purchased from Sigma-Aldrich. Poly(dimethylsiloxane) (PDMS) was purchased from GE (GE Toshiba Silicones. Co., Ltd., Japan). Inactivated H7N9 AIV, H5N1 AIV, Pseudo rabies virus (PRV), and Newcastle disease virus (NDV) were obtained from Wuhan Institute of Virology, Chinese Academy of Sciences. Anti-H7N9 hemagglutinin (HA) rabbit monoclonal antibody (mAb) and pAb were purchased from Sino Biological Inc. (Beijing, China). All other chemical reagents were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). All electrochemical assays were performed on a CHI660a electrochemical workstation (CH Instruments, Inc. Shanghai, China). Dynamic light scattering (DLS) measurements were performed on a Zetasizer Nano ZS instrument (Malvern). TEM images were obtained by a FEI Tecnai G2 20 TWIN electron microscope. SEM (ZEISS SIGMA FESEM) and AFM (Veeco Instruments Inc.) were used to measure the surface structure of MA. Preparation of bi-FMNs. Uniform and dispersive FMNs were prepared.26,27 6


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Owing to the coordination effect between the amino of polyethyleneimine (PEI) and metal elements on the surface of nanoparticles, four layers of Fe3O4 nanoparticles and three

layers

of

CdSe/ZnS

QDs

were

assembled

on

the

surface

of

poly(styrene/acrylamide) copolymer nanospheres. To prepare bi-FMNs, 5 µL FMNs (about 16 mg/mL) were reacted with 200 µL 0.01 M pH 6.1 phosphate buffer solution (PBS) containing 5 mM EDC/NHS. After reacting at 37 °C for 30 min and washing with pH 7.2 PBS 3 times, 200 µL PBS containing 0.063 mg/mL mAb/ALP was added and incubated for 2 h to obtain bi-FMNs. Finally, the bi-FMNs were washed with PBS 3 times and stored at 4 °C for further use. Fabrication of Monolayer Au NPs/mAb Modified MA. Firstly, Au NPs were prepared by sodium citrate reduction method. About 500 µL 25 mM HAuCl4 was added to 50 mL ultrapure water and heated to boiling under stirring. After boiling for 5 min, 2 mL 10% sodium citrate was added and continued reaction for 15 min. The solution color changed from pale yellow to deep red. Au NPs solution was transferred into a 50 K ultrafiltration tube followed by centrifuging for 3 min at 4000 rpm and stored at 4 °C for further use. Different diameters of Au NPs could be prepared by simply changing the volume of sodium citrate. Meanwhile, ITO glasses were cut into 3×2 cm2 rectangles and soaked in piranha solution (7:3 concentrated H2SO4/H2O2) for 5 min. After washing with ultrapure water, the slides were reacted with 3-aminopropyltriethoxysilane (APTES) solution (40 µL APTES, 186 µL methanol, 2 µL acetate, and 980 µL ultrapure water) for 1 h with 7



gentle shaking. After washing, 400 µL 0.01 M pH 7.2 PBS containing 10 µM EDC and 50 µM Au NPs was added followed by incubating for 15 min at 25 °C with gentle shaking (120 rpm) to prepare monolayer Au NPs modified slides. Subsequently, the slides were further reacted with 200 µL PBS containing 50 µg/mL mAb after activating with EDC to prepare mAb functioned slides. To fabricate MA, 5 g PDMS (10:1 w/w RTV615A/RTV615B) was added onto silicon wafer and put on the spin coater with 600 rpm for 15 s to obtain 200 µm thin membranes. After heating at 75 °C for 30 min, the PDMS was peeled off, cut into 2 × 2 cm2 squares and punched with an 800 µm diameter needle to get 100 holes. The PDMS cell was irreversibly bonded to the above slides to fabricate 10×10 individual monolayer Au NPs/mAb MA. The MA was stored at 4 °C for further use. Ultrasensitive H7N9 AIV Detection by Digital ELISA. Every 10 µL bi-FMNs was reacted with different concentrations of H7N9 AIV. After incubating at 25 °C for 30 min with gentle shaking, the target/bi-FMNs complexes were washed with PBS for 3 times and re-dispersed into 100 µL PBS. By controlling the proportion of bi-FMNs to H7N9 AIV (≥ 5:1), at most one virus would be captured per bi-FMNs. Then every 0.5 µL target/bi-FMNs solution was added into Au NPs/mAb modified MA to guarantee that each microelectrode contained one compound. The fluorescence imaging of bi-FMNs was used to monitor and demonstrate the feasibility of individually separating target/bi-FMNs complexes into microelectrodes to perform single virus immunoassay. After reacting at 25 °C for 30 min with gentle shaking, the MAs were rinsed with ultrapure water and then 1 µL 0.5 M pH 9.8 diethanolamine 8


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solution containing 2 µM AgNO3 and 0.5 mM p-APP were added to perform EIM. After 30 min reaction and rinsed with ultrapure water, 1 µL 0.1 M KCl was added to perform LSV from -0.1 to 0.3 V with an Ag/AgCl wire as reference and a Pt wire as counter electrode respectively. The sweep rate was 0.1 V/s and 500 microelectrodes could be detected in about one hour. The electrochemical signals were denoted as “0” and “1” to obtain the average bi-FMNs number per microelectrode (ANE). Then the target concentrations could be calculated based on digital analysis and Poisson distribution.

Results and Discussion Bi-FMNs Enabled Single Virus Immunoassay. To effectively capture low concentrations of targets from complex samples, uniform FMNs was prepared based on the layer-by-layer (LBL) assembly method previously reported by our group,26,27 as shown in Figure 1A. The average diameter was 380±10 nm by randomly counting 200 FMNs (Figure 1B). According to the magnetic response curve in Figure 1C, almost 100% FMNs could be captured by a magnetic scaffold in 150 s, indicating a rapid magnetic response. The use of FMNs as capture carriers can effectively simplify operation and save time. In view of its large specific area, an appropriate concentration of pAb and ALP was simultaneously reacted with FMNs to prepare bi-FMNs. As shown in Figure S1 in the Supporting Information, the hydrated size and surface charge of FMNs changed from 396.7 nm and -25.8 mV to 413.6 nm and -16.6 mV respectively after pAb and ALP modification, demonstrating the successful construction of bi-FMNs. Dylight Fluor 9



488-labeled goat anti-rabbit IgG were used to react with bi-FMNs and observed under a fluorescence inverse microscope. The red fluorescence of FMNs changed to yellow-green fluorescence after reacting with Dylight, indicating that pAb was successfully conjugated to FMNs (Figure S2, Supporting Information). Furthermore, the input concentration ratio of pAb to ALP was optimized (100:1, 10:1, 1:1, 1:10, 1:100), as shown in Figure S3 in the Supporting Information. The capture efficiency of bi-FMNs to H7N9 AIV increased with pAb and the current enhanced with ALP concentrations. In order to guarantee both highly capture efficiency and electrochemical signals, the ratio of 1:1 was finally chose for the subsequent experiment. Based on the linear curves in Figure S4 in the Supporting Information, the number of pAb and ALP per FMNs was calculated to be approximately 63 and 111 respectively. The optimal bi-FMNs could not only capture low concentrations of targets with rich binding sites, but also provide an efficient signal amplification strategy by increasing ALP load.19 By controlling the proportion of bi-FMNs to target concentrations (≥ 5:1), the transmission electron microscope (TEM) image of target/bi-FMNs complexes in Figure 1D demonstrated that at most one virus was captured per bi-FMNs as well as the possibility of single virus immunoassay.3,10 Otherwise, more than one virus would be captured per bi-FMNs (Figure S5A, Supporting Information). In order to investigate the storage stability of bi-FMNs, the catalytic activity of ALP and biological activity of pAb were continuously recorded for 5 weeks, as shown in Figure S5B-C in the Supporting Information. The stable currents and fluorescence resulted from the good storage 10


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stability of bi-FMNs. In general, a new kind of bi-FMNs was prepared by co-immobilizing pAb and ALP, which served as capture carriers and signal carriers simultaneously. The bi-FMNs could save time, simplify operation and improve the load of pAb/ALP, providing the possibility for single virus immunoassay. Monolayer Au NPs/mAb Modified MA Facilitated High-throughput Parallel Tests. Monolayer Au NPs was formed based on a covalent coupling between the carboxyl of Au NPs and the amino of ITO under the activation of EDC, as shown in Figure 2A. Firstly, Au NPs was prepared through a sodium citrate reduction method, which was simple, fast and high yield.28 The Au NPs were uniform and well-dispersed with an average diameter of 18.5±2 nm (Figure 2C). Moreover, the hydrated size and surface potential of Au NPs were 29.2 nm and -27.7 mV respectively, demonstrating its good dispersity and rich carboxyl group capable of covalent coupling. Different methods were tried to modify Au NPs on the surface of ITO glasses and displayed by scanning electron microscopy (SEM). As shown in Figure 2B, some Au NPs could be modified on ITO surface by drop-casting, but the distribution was not uniform and easy to fall off during operation. Only in the presence of EDC, uniform monolayer Au NPs could be achieved resulted from a covalent coupling. Atomic force microscope (AFM) was further applied to characterize the height distribution of Au NPs on ITO surface. As shown in Figure 2D-E, the Au NPs were uniform with a height of approximately 18 nm, confirming the successful modification of monolayer 11



Au NPs. Based on SEM and AFM images, the coverage of Au NPs was about 5×109 per cm2. The size and concentration of Au NPs were optimized. The SEM images (Figure S6, Supporting Information) displayed that the coverage increased with Au NPs concentrations and reached saturation at 50 µM due to the limited binding sites of the ITO surface. Different sizes of Au NPs were prepared by simply changing the volume of sodium citrate (11.1, 13.4, 18.5, 25.2, 33.4, 41 nm), as shown in Figure S7 in the Supporting Information. The currents increased with the size of Au NPs and tended to a platform until 18.5 nm. The reasons might be that the greater the size, the bigger of the specific surface area of modified electrodes under the same coverage. But the stability of Au NPs would be decreased if the size was too big, resulting in uneven distribution and fluctuant currents. The surface area of each microelectrode was calculated to be about 0.013 mm2 based on the cyclic voltammogram (CV) in 0.5 M H2SO4. The mAb was further modified on the surface of MA by using the remaining carboxyl of Au NPs and the detail was shown in Figure S8 in the Supporting Information. A PDMS membrane with 100 micropores was bonded to the surface of Au NPs/mAb modified ITO slides to fabricate 10×10 individual MA. The diameter, depth and volume of each reactor were 800 µm, 200 µm and 0.5 µL respectively. The CVs of ten parallel microelectrodes in [Fe(CN)6]3-/4- with a sweep rate of 0.1 V/s indicated that the homemade MA were uniform enough to perform high-throughput parallel experiments (Figure S9). Water contact angles were further measured to characterize 12


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the change of ITO glasses after modification, as shown in Figure S10 in the Supporting Information. The contact angle of ITO decreased from 80º to 53º after monolayer Au NPs modification and further decreased to 38º after mAb modification, indicating its good hydrophilic. After covering a hydrophobic PDMS membrane (103º) on the surface of Au NPs/mAb modified ITO glasses, a hydrophilic hole with hydrophobic around could be formed. Therefore, the reaction solution could be well confined into the little volume of microelectrodes cell to perform EIM. In summary, monolayer Au NPs/mAb modified MA by covalent coupling had the advantages of uniformity and fast electron transfer rate, which satisfied the requirement of high-throughput parallel experiment for digital ELISA. Feasibility of Single Virus Electrochemical ELISA. Efficient signal amplification methods are urgently needed since the charge of single molecules is too small to detect.29,30 Compared with direct oxidation currents of p-AP, EIM can amplify signals by about 100 times, providing the possibility for single molecule detection.24,31 Firstly, the sensitivity of EIM was investigated by comparing the LSV currents in the presence of ALP+AgNO3, ALP+p-APP, AgNO3+ALP, or ALP+p-APP+AgNO3 respectively. As shown in Figure 3A, a strong current appeared only when ALP, p-APP, and AgNO3 existed simultaneously, demonstrating good selectively of EIM. Moreover, the concentrations of AgNO3 and p-APP were optimized (Figure S11, Supporting Information). The highest signal-to-background ratio of 15 could be achieved when the final concentrations of AgNO3 and p-APP were 2 µM and 0.5 mM respectively. Under optimal conditions, the current of 13



different ALP concentrations were recorded, as shown in Figure 3B. Signals decreased with ALP concentrations and appeared fluctuation when the concentration was low to 1 aM.32 Therefore, conventional quantitative method based on a calibration curve was not appropriate. In order to investigate the effect of FMNs on electrochemical signals, the current with or without FMNs under different ALP concentrations were recorded (Figure 3C). The current decreased to 3/5 of original signals, which might be that FMNs occupied some active sites of microelectrodes. However, 111 ALP molecules could be loaded per FMNs and the influence of FMNs on signals could be ignored. To effectively capture H7N9 AIV, uniform bi-FMNs were fabricated. By controlling the proportion of bi-FMNs to target concentrations (≥ 5:1), at most one virus would be captured per bi-FMNs. Then the target/bi-FMNs complexes were individually separated into MA to perform single virus immunoassay, which was monitored by the red fluorescence imaging of FMNs (Figure 3D). The SEM images of 20 microelectrodes after immunoreaction further indicated the successful single virus ELISA, as shown in Figure 3E. To verify the feasibility of H7N9 AIV detection, four control experiments were designed by changing immunoreaction conditions. As shown in Figure 3F, the currents were negligible in the absence of mAb, H7N9 AIV, or bi-FMNs. However, an obvious signal appeared in the presence of 1 pg/mL H7N9 AIV, demonstrating that the LSV signals only came from ALP catalysis based on specific immunoreaction and sensitive EIM. The detection process was that ALP catalyzed its substrates to reduced 14


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p-AP and Ag+ was instantly reduced to Ag0. After 30 min deposition time, a stripping signal could be obtained by LSV. According to the Faraday’s law: Q = n

(1),

where F is the Faraday’s constant (96500 C/mol), m is the Ag0 deposition amount and n is the electron transfer number. Then the relationship between Ag0 amount and time under different deposition time could be obtained (Figure S12A, Supporting Information). Therefore, the deposition rate of Ag+ can be calculated by the equation: v=

[ ]

(2) based on a differential operation (Figure S12B, Supporting

Information). The deposition rate was calculated to be about 0.95 pg/min. And the theoretical stripping signal per bi-FMNs after 30 min deposition time was calculated to be approximately 25.4 nC. When bi-FMNs were diluted to single particles level, every 0.5 µL solution was added into MA to perform EIM. By random counting 50 microelectrode signals, obvious current fluctuation appeared. The possible reasons might be that one more or less a molecule had great influence on signal amplification efficiency and the number heterogeneity of ALP molecules per FMNs. Overall, based on the efficient amplification of EIM and bi-FMNs together with monolayer Au NPs modified MA, a single virus electrochemical ELISA for H7N9 AIV detection could be realized. Reliable Digital ELISA for Ultrasensitive H7N9 AIV Counting. The introduction of digital analysis can effectively solve the problem of signal fluctuation and the heterogeneity of bi-FMNs, because the signal is denoted as “0” or “1” without considering the concrete intensity of signals, providing a reliable and sensitive quantitative method for low concentration targets detection. Here, a digital 15



ELISA was constructed for ultrasensitive H7N9 AIV counting based on the integration of bi-FMNs, monolayer Au NPs modified MA with digital analysis. When the target concentration is low, each microelectrode will be randomly distributed by 0, 1, 2, or more bi-FMNs according to Poisson distribution, whose probability can be calculated by the Poisson law: = =

!

(3), where x is the particles

number and λ is the average bi-FMNs number per microelectrode. When x = 0, the deformation of equation 3 is: λ = −ln"# (4). Then the whole bi-FMNs concentration could be calculated by: $ =

%& '

(5), where V is the reaction volume

(0.5 µL). Then the λ0 and C0 could be obtained by simply counting the probability of “0” among 500 microelectrodes. Different concentrations of bi-FMNs were prepared and each 0.5 µL was added into MA to perform EIM. Their electrochemical signals distribution were shown in Figure 4A, big current fluctuation appeared at each concentration and the 500 microelectrodes could be divided into four groups containing 0, 1, 2, and more bi-FMNs with the current of 2±1.0, 10±3.0, 16±3.0 and 21±3.0 nA respectively. Among them, 10±3.0 nA could represent the signal from single bi-FMNs and had a good agreement with the theoretical result. The experimental results matched well with the theoretical results of Poisson distribution (Figure 4B). Furthermore, with the decreasing of λ, P(x>1) tended to zero, only P(x=0) and P(x=1) existed, which demonstrated the feasibility of digital analysis by counting signals as “0” and “1”. In order to simplify statistical process, 500 electrochemical signals were directly output into a colorful imaging through a MATLAB program (Figure 4C). The probabilities of “0” or “1” could be easily obtained by counting dark 16


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and bright balls. Based on the integration of bi-FMNs, monolayer Au NPs/mAb modified MA with digital analysis, a digital ELISA was proposed for H7N9 AIV detection (Figure 5A). The relationship between H7N9 AIV concentrations and the average bi-FMNs number per microelectrode (ANE) was shown in Figure 5B. A good linear curve appeared from 0.01 to 1.5 pg/mL with a low detection limit of 7.8 fg/mL, which was 1-3 orders of magnitude more sensitive than other H7N9 AIV detection.19,33-35 The possible reasons might be as follows: (1) Compared with direct currents of enzyme products, EIM could amplify signals by about 100 times,22 providing an efficient amplification method for single virus detection; (2) A single bi-FMNs could load many pAb and ALP to further enhance signal-to-background ratio, showing the possibility of single virus immunoassay; (3) Monolayer Au NPs modified MA with excellent homogeneity was fabricated by a covalent coupling capable of high-throughput parallel experiments; (4) The combination of digital analysis effectively avoided current fluctuation, offering a reliable and accurate quantitative method. In practical application, the samples are complicated and the specificity of detection methods is necessary to guarantee the accuracy of the results. Other control virus, such as H9N2 AIV (5.2 pg/mL), H5N1 AIV (12 pg/mL), PRV (23.3 pg/mL), and NDV (0.78 ng/mL) were used to perform the same digital ELISA. As shown in Figure 5C, only 1 pg/mL H7N9 AIV achieved higher ANE than other control virus, even when their concentration were 1-3 orders of magnitude more concentrated than H7N9 AIV. The ANE of H7N9 AIV was approximately 29-fold stronger than control 17



samples, indicating high specificity of the digital ELISA as well as its potential application in complex systems. The sensitivity and reliability of the digital ELISA for H7N9 AIV detection were further investigated in complex samples. Here, chicken serum and liver were used as blood and tissue samples respectively to demonstrate the anti-interference ability of the method. An amount of H7N9 AIV was added to ground chicken liver and serum followed by centrifuging to remove solid insolubles. The control samples were also prepared by the same method without H7N9 AIV. As shown in Figure 5D, the ANE in experimental samples were significantly higher than those in control groups, either in PBS buffer or complex media, which demonstrated the good anti-interference ability of the digital ELISA. In general, the proposed digital ELISA for H7N9 AIV counting has the advantages of high sensitivity, specificity, reliability, and anti-interference ability.

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Conclusions In summary, a novel digital ELISA was fabricated for ultrasensitive H7N9 AIV counting by integration of bi-FMNs, monolayer Au NPs modified MA with digital analysis. By controlling the proportion of bi-FMNs to target concentrations (≥ 5:1), at most one target will be captured per bi-FMNs, providing the possibility of single virus immunoassay. Using covalent coupling, monolayer Au NPs modified MA with good homogeneity was fabricated to satisfy the high-throughput parallel experiments requirement. The introduction of digital analysis can solve the problem of signal fluctuation and reliability of single virus detection by counting the electrochemical signals as “0” or “1”. Taking advantages of the bi-FMNs, MA and digital analysis, a digital ELISA was successfully performed for H7N9 AIV counting with a low detection limit of 7.8 fg/mL without complicated sample pretreatment or sophisticated instruments, showing broad potential in early diagnosis of disease and single cell detection.

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FIGURES Scheme 1. Illustration of the Digital ELISA for H7N9 AIV Counting: (A) Capture of Single Virus per bi-FMNs and Individual Separation of Target/bi-FMNs

Complexes

into

Microelectrode

Enzyme-induced Metallization (EIM) and Digital Analysis

20


Array

(MA);

(B)

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Figure 1. Characterization of bi-FMNs. (A) TEM image of FMNs. (B) Histogram for the size distribution of FMNs (n=200). (C) Capture efficiencies of FMNs at different attraction time with a commercial magnetic scaffold. (D) TEM image of target/bi-FMNs complexes.

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Figure 2. Characterization of MA. (A) Fabrication diagram of monolayer Au NPs modified ITO microelectrodes. (B) SEM images of bare ITO (a) and Au NPs modified ITO by drop-casting (b) or covalent coupling (c). (C) TEM image of Au NPs. (D) AFM image of monolayer Au NPs modified ITO surface. (E) Height distribution of Au NPs along the white line in Figure D.

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Figure 3. (A) Stripping curves of different solution compositions: (a) ALP + p-APP; (b) AgNO3 + ALP; (c) AgNO3 + p-APP; (d) AgNO3 + p-APP + ALP. (B) Current distribution of 50 parallel tests under different ALP concentrations. (C) Current intensity in the absence and presence of FMNs. Fluorescence (D) and SEM (E) images of 20 microelectrodes after single virus ELISA. (F) Stripping curves under different reaction conditions: (a) without virus, (b) without mAb, (c) without bi-FMNs, and (d) 1 pg/mL H7N9 AIV.

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Figure 4. (A) Current distribution of 500 microelectrodes under different bi-FMNs concentrations. (B) Comparison of the probability distribution between experimental results and Poisson distribution. (C) Transformation of 500 electrochemical signals to colorful balls through a MATLAB program.

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Figure 5. (A) Diagram of the sandwich immunoreaction in each microelectrode. (B) Linear curve between ANE and original input H7N9 AIV concentrations (0.01, 0.05, 0.1, 0.2, 0.4, 0.8, 1.1, 1.5 pg/mL). (C) Histogram for the specificity of digital ELISA for H7N9 AIV detection. (D) Histogram of the ANE in the presence (black) and absence (white) of H7N9 AIV in different complex media.

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AUTHOR INFORMATION Corresponding Author *Zhi-Ling Zhang, Email: [email protected]; Phone: 0086-27-68756759, Fax: 0086-27-68754067. Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (21475099, 21535005, 21775111). Inactivated H7N9 AIV was obtained from professor Jian-Jun Chen of Wuhan Institute of Virology, Chinese Academy of Sciences.

SUPPORTING INFORMATION Figures S1−S12. Figure S1, characterization of bi-FMNs. Figure S2, demonstration of pAb modified FMNs. Figure S3, optimization of the number of pAb and ALP per FMNs. Figure S4, characterization of the number of pAb and ALP. Figure S5, demonstration of single virus immunoassay. Figure S6, optimization of Au NPs concentrations. Figure S7, optimization of Au NPs sizes. Figure S8, characterization of mAb modified MA. Figure S9, uniformity of microelectrodes. Figure S10, characterization of the change of contact angles. Figure S11, optimizing conditions of EIM. Figure S12, theoretical calculation of EIM.

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REFERENCES (1) Dunevall, J.; Fathali, H.; Najafinobar, N.; Lovric, J.; Wigström, J.; Cans, A. S.; Ewing, A. G. J.

Am. Chem. Soc. 2015, 137, 4344-4346. (2) Liu, Z.; Ding, S. Y.; Chen, Z. B.; Wang, X.; Tian, J. H.; Anema, J. R.; Zhou, X. S.; Wu, D. Y.; Mao, B. W.; Xu, X.; Ren, B.; Tian, Z. Q. Nat. Commun. 2011, 2, 305-310. (3) Rissin, D. M.; Kan, C. W.; Campbell, T. G.; Howes, S. C.; Fournier, D. R.; Song, L.; Piech, T.; Patel, P. P.; Chang, L.; Rivnak, A. J.; Ferrell, E. P.; Randall, J. D.; Provuncher, G. K.; Walt, D. R.; Duffy, D. Nat. Biotechnol. 2010, 28, 595–599. (4) W.H.O. Website. Human infection with avian influenza A (H7N9) virus. Update 20 February 2017. ‹http://www.who.int/csr/don/20-february-2017-ah7n9-china/en/› (5) Rotman, B. Proc. Natl. Acad. Sci. U. S. A. 1961, 47, 1981–1991. (6) Holzmeister, P.; Acuna, G. P.; Grohmann, D.; Tinnefeld, P. Chem. Soc. Rev. 2014, 43, 1014–1028. (7) Neuman, K. C.; Nagy, A. Nat. Methods 2008, 5, 491–505. (8) Mathwig, K.; Albrecht, T.; Goluch, E. D.; Rassaei, L. Anal. Chem. 2015, 87, 5470–5475. (9) Dinh, T. L.; Ngan, K. C.; Shoemaker, C. B.; Walt, D. R. Forensic Toxicol 2017, 35, 179–184. (10) Rissin, D. M.; Kan, C. W.; Song, L.; Rivnak, A. J.; Fishburn, M. W.; Shao, Q.; Piech, T.; Ferrell, E. P.; Meyer, R. E.; Campbell, T. G.; Fournier, D. R.; Duffy, D. C. Lab Chip. 2013, 13, 2902–2911. (11) Ma, F.; Li, Y.; Tang, B.; Zhang, C. Y. Acc. Chem. Res. 2016, 49, 1722–1730. (12) Zhang, C. Y.; Yeh, H. C.; Kuroki, M. T.; Wang, T. H. Nat. Mater. 2005, 4, 826–831. (13) Wang, L. J.; Yang, Y.; Zhang, C. Y. Anal. Chem. 2015, 87, 4696–4703. (14) Fan, F-RF.; Bard, A. J. Science 1995, 267, 871–874. (15) Xiao, X.; Bard, A. J. J. Am. Chem. Soc. 2007, 129, 9610–9612. (16) Fernando, A.; Parajuli, S.; Alpuche-Aviles, M. A. J. Am. Chem. Soc. 2013, 135, 10894‒10897. (17) Perera, N.; Karunathilake, N.; Chhetri, P.; Alpuche-Aviles, M. A. Anal. Chem. 2015, 87, 777‒784. (18) Zhou, C. H.; Wu, Z.; Chen, J. J.; Xiong, C.; Chen, Z.; Pang, D. W.; Zhang, Z. L. Chem. Asian 27



J. 2015, 10, 1387–1393. (19) Wu, Z.; Zhou, C. H.; Chen, J. J.; Xiong, C.; Chen, Z.; Pang, D. W.; Zhang, Z. L. Biosens.

Bioelectron. 2015, 68, 586−592. (20) Liu, S.; Wei, W.; Wang, Y.; Fang, L.; Wang, L.; Li, F. Biosens. Bioelectron. 2016, 80, 208–214. (21) Vogelstein, B.; Kinzler, K. W. Digital PCR. Proc. Natl. Acad. Sci. U. S. A. 1999, 96, 9236–9241. (22) Wang, T.; Zhang, M.; Dreher, D. D.; Zeng, Y. Lab Chip. 2013, 13, 4190–4197. (23) Kim, S. H.; Iwai, S.; Araki, S.; Sakakihara, S.; Iino, R.; Noji, H. Lab Chip. 2012, 12, 4986–4991. (24) Wu, Z.; Zhou, C. H.; Pan, L. J.; Zeng, T.; Zhu, L.; Pang, D. W.; Zhang, Z. L. Anal. Chem.

2016, 88, 9166–9172. (25) Young, S. L.; Kellon, J. E.; Hutchison, J. E. J. Am. Chem. Soc. 2016, 138, 13975–13984. (26) Hu, J.; Xie, M.; Wen, C. Y.; Zhang, Z. L.; Xie, H. Y.; Liu, A. A.; Chen, Y. Y.; Zhou, S. M.; Pang, D. W. Biomaterials 2011, 32, 1177–1184. (27) Wen, C. Y.; Hu, J.; Zhang, Z. L.; Tian, Z. Q.; Ou, G. P.; Liao, Y. L.; Li, Y.; Xie, M.; Sun, Z. Y.; Pang, D. W. Anal. Chem. 2013, 85, 1223−1230. (28) Ghosh, S. K.; Pal, A.; Kundu, S.; Nath, S.; Pal, T. Chem. Phys. Lett. 2004, 395, 366−372. (29) Guan, Z.; Zou, Y.; Zhang, M.; Lv, J.; Shen, H.; Yang, P.; Zhang, H.; Zhu, Z.; Yang, C. Y.

Biomicrofluidics 2014, 8, 014110−014123. (30) Zhang, X.; Zhang, B.; Miao, W.; Zou, G. Anal. Chem. 2016, 88, 5482−5488. (31) Zhou, C. H.; Zhao, J. Y.; Pang, D. W.; Zhang, Z. L. Anal. Chem. 2014, 86, 2752−2759. (32) Paunescu, D.; Mora, C. A.; Querci, L.; Heckel, R.; Puddu, M.; Hattendorf, B.; Günther, D.; Grass, R. N. ACS Nano 2015, 9, 9564−9572. (33) Qu, B.; Chu, X.; Shen, G.; Yu, R. Talanta 2008, 76, 785–790. (34) Fan, J.; Cui, D.; Lau, S.; Xie, G.; Guo, X.; Zheng, S.; Huang, X.; Yang, S.; Yang, X.; Huo, Z.; Yu, F.; Lou, J.; Tian, L.; Li, X.; Dong, Y.; Zhu, Q.; Chen, Y. BMC Infect. Dis. 2014, 14, 541–549. (35) Dong, S.; Zhao, R.; Zhu, J.; Lu, X.; Li, Y.; Qiu, S.; Jia, L.; Jiao, X.; Song, S.; Fan, C.; Hao, R. Z.; Song, H. B. ACS Appl. Mater. Interfaces. 2015, 7, 8834–8842. 28


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Digital Single Virus Electrochemical Enzyme-Linked Immunoassay for

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