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Cellular Beacons-mediated Counting for Ultrasensitive Detection of Ebola Virus on an Integrated Micromagnetic Platform Shao-Li Hong, Ya-Nan Zhang, Ya-Hua Liu, Man Tang, Dai-Wen Pang, Gary Wong, Jianjun Chen, Xiangguo Qiu, George F. Gao, Wenjun Liu, Yuhai Bi, and Zhi-Ling Zhang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b00513 • Publication Date (Web): 24 May 2018 Downloaded from http://pubs.acs.org on May 24, 2018

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Analytical Chemistry

Cellular Beacons-mediated Counting for Ultrasensitive Detection of Ebola Virus on an Integrated Micromagnetic Platform Shao-Li Hong,† Ya-Nan Zhang,† Ya-Hua Liu,† Man Tang,† Dai-Wen Pang,† Gary Wong, ‡,§ Jianjun Chen,ǁ Xiangguo Qiu,⊥ George F. Gao, ‡,§ Wenjun Liu, § Yuhai Bi, *‡, §

†

Zhi-Ling Zhang*, †

Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of

Education), College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072, China ‡

Shenzhen Key Laboratory of Pathogen and Immunity, State Key Discipline of

Infectious Disease, Shenzhen Third People’s Hospital, Shenzhen 518112, China §

CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of

Microbiology, Center for Influenza Research and Early-warning (CASCIRE), Chinese Academy of Sciences, Beijing 100101, China ǁ

Key Laboratory of Special Pathogens and Biosafety, Wuhan Institute of Virology,

Chinese Academy of Sciences, Wuhan 430071, China ⊥

Special Pathogens Program, Public Health Agency of Canada, Winnipeg, Manitoba,

Canada Corresponding Author Zhi-Ling Zhang, Email: [email protected]. Tel: 0086-27-68756759. Fax: 0086-27-68754067. Yuhai Bi, Email: [email protected]. Tel: 0086-10-64806013. Fax: 0086-10-64806247. 1

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Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract: Ebola virus (EBOV) disease is a complex zoonosis that is highly virulent in humans, and has caused many deaths. Highly sensitive detection of EBOV is of great importance to early-stage diagnosis for increasing the probability of survival. Herein, we established a cellular beacon-mediated counting strategy for ultrasensitive EBOV assay on a micromagnetic platform. The detection platform that was assisted by on-demand magnetic field manipulation showed high integration, and enhanced complex sample pretreatment ability by magnetophoretic separation and continuous flow washing. Using cellular beacons (i.e., fluorescent cells) with superior optical property as reporters, each cellular beacon as a fluorescence tracking unit was used to quantify the EBOV by counting the numbers of individual fluorescence signal on the micromagnetic platform. This method achieves in high sensitivity with a detection limit as low as 2.6 pg/mL, and the detection limit had little difference in a complex matrix. In addition, it had excellent specificity and good reproducibility. These results indicate that this method proposes an ultrasensitive detection strategy for early diagnosis of disease.

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EBOV is one kind of hemorrhagic fever virus, and has caused 24 epidemics in Africa since they were first identified in 1976 in Zaire and Sudan.1 The recent outbreak, to date being the 2013–2016, spread quickly into several African countries with a 90% fatality rate.2-3 World Health Organization (WHO) has declared EBOV disease epidemic as Public Health Emergency of International Concern.4 An early diagnosis of EBOV is critical for preventing the spread of the disease and enabling an earlier diagnosis for more effective treatment when it is an outbreak without warning.5 Traditionally,

enzyme-linked

immunosorbent

assays

(ELISAs)

and

reverse-transcriptase polymerase chain reactions (RT-PCR) have been applied to the detection of EBOV-specific proteins or nucleic acids.6-8 However, standard ELISA assays and RT-PCR for detection of biomarkers require large sample, complex sample pretreatment and low sensitivity.9-12 Therefore, there is still need to develop highly sensitive EBOV detection methods for early diagnosis. In recent years, many sensitive assays were developed for EBOV detection based on measuring the intensity of bulk ensemble signals, such as upconversion nanoparticles fluorescence sensors,13 electroluminescent nanospheres approaches,14 and platinum nanoparticles measurement technique.15 Compared with those methods by measurement of ensemble signals, the detection based on counting strategy is the new frontier in biomarker analysis and has shown great potential of ultrasensitive detection due to high signal-to-noise ratio, which can achieve single nanoparticles or molecules detection level.16-18 So far, fluorescence has been the most well-established and wide technique used for counting strategy. Over the years, the fluorescence counting

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strategy has been used in the detection of protein,19-20 DNA,21-22 microRNA,23 cocaine24 and pathgens.25-26 In order to perform the fluorescence counting measurements, the target molecules are commonly labeled with fluorescent tags, such as organic dyes and fluorescent semiconductor nanoparticles, i.e., quantum dots (QDs). However, accurate counting of the positive fluorescence signals still remains a challenge due to the background fluorescence interference, which usually requires special fluorescence resonance energy transfer (FRET) system or complicated and low throughput optical systems. Thereby, new fluorescent reporters that can overcome this problem are urgently needed for highly sensitive detection of pathogens using fluorescence counting techniques. Herein, we utilized cellular beacons as reporters for fluorescence-mediated counting techniques. Cellular beacons - fluorescent cells - were transformed from Staphylococcus aureus (S. aureus) cells according to our previous work, in which synthesized quantum dots. Compared with organic fluorophores and QDs, single cellular beacon was more reliable and fluorescence-enabled easier distinction against background. With simple modification, each cellular beacon was transformed into fluorescent nanobioprobe for labeling the target molecules.25 In this work, we harness the cellular beacon-mediated counting strategy on an integrated micromagnetic platform to achieve highly sensitive EBOV detection. Immunomagnetic nanospheres (IMNs) were obtained by functionalizing magnetic nanospheres (MNs) with the EBOV antibodies, and used to the specifically identify and capture the virus from sample. The sample was then treated via magnetic separation and enrichment.

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Fluorescent nanobioprobes as the fluorescence tracking units were reacted with magnetic tagged EBOV to form a sandwich complex to quantify the EBOV concentration by counting the nanobioprobe numbers on a detection zone. The unique advantages of our proposed strategy can be listed as follows: First, the detection process showed high integration, which could reduce the contact of high risk EBOV to protect the detection personnel. Second, complex sample could be directly loaded for detection. Third, the cellular beacons, due to reliable and fluorescence-enabled easy distinction against background, could be used for quantifying the EBOV based on the counting-mediated detection strategy on the platform. The assay can quantitatively detect EBOV with a linear relationship between 5 pg/mL and 100 pg/mL, yielding a detection limit of 2.6 pg/mL. In addition, the assay was robust with high specificity and good reproducibility. These results demonstrated that this method provided a promising strategy to use a cellular beacons-mediated counting strategy for ultrasensitive detection of pathogens. EXPERIMENTAL SECTION Reagents and Instruments. Branched poly(ethylene imine) (PEI, MW 25 kDa and MW 750 kDa), tetraethyl orthosilicate (TEOS), (3-aminopropyl)triethoxysilane (APTES), N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC), polyvinylpyrrolidone

(PVP-k30),

bovine

serum

albumin

(BSA)

and

N-hydroxysuccinimide (NHS) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Inactivated EBOV whole virion and horse antibody against EBOV glycoprotein were supplied by Prof. Xiangguo Qiu. Alexa Fluor 488-conjugated

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AffiniPure goat anti-horse IgG was purchased from Jackson ImmunoResearch Laboratories, Inc. AZ50XT (PR) and developer AZ400K were obtained from AZ Electronic Materials (AZ Electronic Materials Corp., USA). SU-8 2050 photoresists and developer were purchased from MicroChem (MicroChem Corp., USA). Polydimethylsiloxane (PDMS) and curing agent were purchased from GE (GE Toshiba Silicones Co., Ltd., Japan). Nanonickel powder (50 nm, 99.9%) was obtained from DeKeDaoKing (Beijing, China). Microscope cover glasses were purchased from CITOGLAS (Citotest Labware Manufacturing CO., Ltd., China). Array of handmade miniature bars guiding magnetism was purchased from BDE (Guangdong, China). Ultrapure water (18.2 MΩ·cm) was obtained with a Millipore Milli-Q system. Fluorescence images were recorded with a CCD camera (Nikon DS-Ri1) mounted on an inverted fluorescence microscope (TiU, Nikon,Japan). TEM images were obtained by a FEI Tecnai G220 TWIN electron microscope. DLS measurements were performed on a Malvern Zetasizer Nano ZS instrument. Design and Fabrication of the Micromagnetic Platform. The detection platform integrated the basis of our previous magnetic field manipulation methods.27-33 First, the layer of nickel powder@PDMS was fabricated, which the SU-8 2050 photoresist was spin-coated on a clean silicon wafer to fabricate an array of 40 µm height, 50 µm width and 50 µm length mold. Then nickel powder (50 nm) was filled into the mold and pressed by the head of dropper to enhance the nickel powder compact, and the redundant nickel powder was removed. After that, a layer of thin PDMS was spin-coated on the nickel powders and kept at 75 ℃ for 1 hour, fabricating the nickel

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pillars. On the other hand, handmade miniature bars guiding magnetism and two miniature permanent magnets were placed on the silicon wafer and poured into PDMS. After baking for 4 h, the cured PDMS layer was peeled off the surface of silicon wafer. Finally, the above two layers of PDMS were bound together with aid of oxygen plasma, and baked at 75 °C for 30 min, then peeled off the photoresist mold. They formed the layer of magnetic manipulation. Second, the fluidic channel was fabricated by using the same process of soft lithography. The layer of magnetic manipulation was linked to the fluidic channel with PDMS (10:1), and then kept at 75 ℃ for 1 hour. Lastly, they were peeled off the photoresist mold, and bound to a clean slide glass with aid of oxygen plasma to generate the integrated micromagnetic platform. Modification of the Microchannel in Detection Zone. The fluidic channel was hydrophilic after being treated by oxygen plasma. Then the detection zone of fluidic channel was modified with 3-aminopropyl triethoxysilane (APTES) to generate ammonia group for 12 h.34 After reaction, ethanol was used to wash the microchannel for removing the unreacted APTES, and the chip was kept at 75 ℃ for 10 min. Subsequently, the WGA was conjugated with ammonia group via cross-link amines with the carboxylic acid groups of WGA. The length of the detection zone was 4 mm, and the width was 142 µm which was fit for CCD screen size. The modified chip was store for detection. Fabrication of the MNs. The MNs were fabricated by LBL (layer-by-layer) method. First, the poly (styrene/acrylamide) copolymer nanospheres (Pst-AAm-COOH) were

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fabricated by means of emulsifier-free polymerization method. Subsequently, PEI (low MW 25 kDa) was coated on the surface of Pst-AAm-COOH nanospheres as a foundation layer to react with nano-γ-Fe2O3 in hexanol. And then the second layer was formed similarly, where another kind of PEI (high MW 750 kDa) was kept on coating on the surface of the nanospheres in PBS and then attach to an additional layer of nano-γ-Fe2O3 in hexanol. After four times repeating, five-layer magnetic nanosphere was assembled successfully. Outer shell of silica was coated on the surface

of

the

magnetic

nanospheres

by

a

seeded-growth

method.

35

Amino-terminated silica-coated magnetic nanospheres were purified with centrifuge and then dispersed into N, N-dimethylformamide (DMF) containing succinic anhydride to form carboxyl group for further reactions lastly. Preparation of Immunomagnetic Nanospheres. Carbodiimide chemistry was used to cross-link amines of the antibody with the carboxylic acid groups on the surface of MNs. Approximately 40 µL of MNs was activated in 10 mM EDC and 5 mM NHS in 400 µL of PBS (0.01 M pH=6.8) at room temperature with gentle shaking for 30 min. Then, the activated MNs were separated by a magnetic scaffold and washed with PBS (0.01 M pH=7.2) three times. Subsequently, they were resuspended in 400 µL of PBS (0.01 M pH=7.2) to react with 24 µg of anti-EBOV antibody for about 4 h with continuous shaking at room temperature. After that, the IMNs were washed with PBS to remove surplus antibody and then stored in PBS (0.01 M pH=7.2) at 4℃ for use. Transforming Cellular Beacons into Fluorescent Nanobioprobes. The S. aureus cells were transformed into cellular beacons according to our previous work.25

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Namely, the S. aureus was cultured for 12 h with LB broth so that the cells were in the stationary phase. Then cells were incubated with Na2SeO3 solution for 12 h, and they were harvested by centrifugation at 4000 rpm for 5 min. Subsequently, they were transferred into fresh LB broth. CdCl2 solution was added to the medium, and the cells were cultured for 12 h to obtain the cellular beacons. After that, cellular beacons were centrifuged at 4000 rpm for 5 min, and washed three times with Tris-HCl buffer (0.1 M, pH=8.0), and then resuspended in the Tris-HCl buffer. The cellular beacons with an optical density (OD ) of 1.5 were heated in a 65 ℃ water bath for 30 min, which allows the entrance of the cells into a nonculturable state (no colony formation when such treated cells were plated in the LB agar plate). Then cellular beacons were then modified with antibody as follows: the cellular beacons with an OD of 1.5 (a final volume of 100 µL) were incubated with 2 µL of monoclonal anti-EBOV antibody (0.59 mg/mL) for 4 h at 37 ℃ with shaking at 150 rpm. After incubating, the cells were washing three times by centrifugation at 4000 rpm for 5 min, and blocked with 1% BSA (w/v) for 30 min at 37 ℃. The cellular beacons were transformed into fluorescent nanobioprobes, and stored for use. Ultrasensitive Assay of EBOV by Counting Strategy. The 200 µL EBOV sample was incubated with 20 µL IMNs for 30 min in the Eppendorf Tube, and then transferred into the syringe. Subsequently, the sample was pumped into the microchannel at the rate of 5 µL/min, where the IMNs-virus was separated from the sample with the aid of magnetic force. Next, the magnetic tagged immune complex was stored in a reservoir, and 2 µL fluorescent nanobioprobes were added up to the

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reservoir and reacted with the complex for 30 min. Once the reaction was finished, the immune sandwich complex was inhaled into the enrichment zone to be captured by the nickel pillars at the rate of 10 µL/min, and the unreacted fluorescent nanobioprobes were stringently washed away chip by continuous flow. After washing with buffer, the external magnetic field was removed and the immune complex was further inhaled into the detection zone with the help of six-way valve, at which the fluorescent nanobioprobes were captured by the WGA modified microchannel and counted the numbers by IPP software. RESULTS AND DISCUSSION Characterization of the Fluorescent Nanobioprobes and IMNs. As shown in Figure 1A, cellular beacons were transformed from S. aureus cells. Briefly, the S. aureus cells were co-incubation of Na2SeO3 to reduce Na2SeO3 to organoselenium compounds, and then CdCl2 solution was added into the cell culture. Cd precursor formed by intracellular detoxification of Cd2+ can precisely react with the intracellularly reduced selenium and endogenous biomolecules containing mercapto groups to synthesize CdS0.5Se0.5 quantum dots. These fluorescent cells were defined as cellular beacons. After incubating with EBOV antibody, the cellular beacons were transformed into fluorescent nanobioprobes through the Fc region of antibodies specifically binding protein A of cellular beacons surface. To verify modification of antibody, the Alexa Fluor 488-conjugated AffiniPure goat anti-horse IgG was incubated with nanobioprobes. As shown in Figure 1B-D, the green fluorescence of Alexa Fluor 488

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exhibited on fluorescent nanobioprobes, while no green fluorescence was observed on the cellular beacons (Figure 1E-G). It illustrated that the fluorescent nanobioprobes were successfully coated with EBOV antibody. Meanwhile, the fluorescence intensity distribution of nanobioprobes was further determined under UV excitation (Figure 1D). The result showed that fluorescent nanobioprobes possessed highly fluorescent uniformity and had an average hydrodynamic diameter of 835 nm (Figure S1). On the other side, MNs were modified with antibody and characterized by dynamic light scattering (DLS). TEM image showed that the MNs were uniform in size with a diameter of about 380 nm (Figure S2A, B). After modification of antibody, MNs were transformed into IMNs, which the hydrodynamic size was significantly changed from 444 nm to 471 nm (Figure 1H, I), indicating that the antibody was successfully modified on the surface of MNs, too. The result could be further verified by immunofluorescence (Figure S2C-E).

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Figure 1. (A) Schematic illustration for the synthesis of fluorescent nanobioprobes. (B-D) Microscopic images of fluorescent nanobioprobes（Bright field, Green, UV）. (E-G) Microscopic images of cellular beacons（Bright field, Green, UV）. (H) Hydrodynamic size of the MNs. (I) Hydrodynamic size of the IMNs. Integrated Detection Platform. The detection platform consisted of separation, enrichment and detection zones (Figure 2). IMNs were mixed with the sample to generate the magnetic tagged immune complex. Then the sample was pumped into the chip, where the immune complex was transferred from the sample stream to the buffer stream in the separation zone. The detail separation process and performance were 12


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shown in supporting information section S.3. To determine whether EBOV could be captured by IMNs and separated from complex sample in this zone, a fluorescence microscopy analysis was done. The separated complex was stained by DiI which was a kind of lipophilic membrane dye, and then was observed under a fluorescence microscope. As shown in Figure S4A, many red fluorescence spots, which indicated the stained EBOV, could be observed after IMNs incubating with EBOV sample, while no obvious fluorescence signal was shown by using MNs incubating with EBOV sample (Figure S4B). TEM further confirmed that the EBOVs were specially captured by IMNs (Figure S4C). These results indicated that the EBOV could be specifically separated from the complex sample in the zone. After separation, the magnetic tagged immune complex was incubated with fluorescent nanobioprobes, and then flowed into the enrichment zone. The feasibility of fluorescent nanobioprobes as reporters was confirmed in Figure S5. Then the immune sandwich complex was trapped in the enrichment zone (Figure S6A-C), where the unreactive nanobioprobes were further isolated. Finally, the immune sandwich complex was counted in the wheat germ agglutinin (WGA) modified detection

zone,

through

which

WGA

could

specifically

bind

with

N-acetylglucosamine of cellular beacons surface. Figure S7 showed that WGA was modified on detection zone and bound with cellular beacons. Meanwhile, the platform, assisted by a six-way valve, helped the exchange of different zones to enhance its integration. Therefore, the detection steps from the sample-in to results-out could be completed in this platform, and its photograph was shown in Figure S8.

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Figure 2. Schematic illustration for the integrated detection platform. High Integration Based on On-demand Magnetic Manipulation. The integrated detection platform mainly depended on the precisely on-demand magnetic manipulation. In the separation zone, a miniature guiding magnetic bar was used to generate a magnetic force in the lateral direction of microchannel for magnetophoretic separation. The magnetic field gradients within the zone were numerically calculated by using finite element analysis (Comsol Multiphysics 4.0), and the detail calculation process was described in supporting information section note S1. The calculation results showed that the separation zone provided a uniform magnetic force to perform the stable magnetophoretic separation of the sample (Figure 3A, B). And the separation process was clearly tracked by using the fluorescence magnetic nanoshperes which not only had the same magnetic force magnitude with the MNs but also provided fluorescence signal according to our previous work (Figure 3C). 14


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Compared with separation zone, a strong magnetic force was necessary for the enrichment zone to trap the immune sandwich complex, where nickel power pillars were filled into this zone because nickel had a relatively high magnetic permeability (µr ≈ 200). As shown in Figure 3D, E, strong magnetic field gradient was created within the enrichment zone. The calculations of |(B·▽)B| demonstrated that the short-range gradient had a magnitude of 2 × 104 T/m within 50 µm in the platform, which the magnetic force (Fmag) was on the order of nano-Newtons and sufficient to trap and wash the immune sandwich complex in the continuous flow condition. The results were further proved by the experiment (Figure 3F).

Figure 3. (A) Numerical simulation of the magnetic field distribution in the separation zone. (B) Calculations result of |(B·▽) B| vs the distance along the Y-axis direction. (C) Fluorescence microscopic images of magnetophoretic separation. (D) Numerical simulation of the magnetic field distribution in the enrichment zone. (E) Calculations result of |(B·▽) B| vs the distance along the Y-axis direction in the enrichment zone. (F) Microscopic image of MNs patterns.

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Enhancing Complex Sample Pretreatment Ability. To investigate the applicability of this integrated platform in complex sample, the assay was tested in different matrixes (PBS, saliva, blood). As shown in Figure 4A, compared with conventional magnetic scaffold for sample pretreatment, using this platform had the higher positive signals and less signal differences between PBS and mimic complex matrixes in the EBOV positive sample. The above results confirmed that the magnetophoretic separation in the separation zone aided the purification of target virus and made the detection more sensitivity. On the other side, the blank sample showed lower negative signals and had little difference using this method (Figure 4B), which illustrated that continuous flow washing in the enrichment zone reduced the nonspecific binding of nanobioprobes. These results illustrated that the platform with on-demand magnetic manipulation enhanced complex sample pretreatment ability.

Figure 4. Histogram for the detected fluorescent nanobioprobes number using the on-demand magnetic manipulation and magnetic scaffold in different matrixes. (A) Positive (with 500 pg/mL EBOV). (B) Negative (without EBOV). Error bars correspond to standard deviation (n = 3). Ultrasensitive Assay of EBOV. Based on the excellent complex sample pretreatment ability and cellular beacons-mediated counting strategy, a sensitive immunosensor 16


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was constructed for EBOV detection. The immune sandwich complex was arranged in a detection zone. Compared with nanobioprobes trapped in enrichment zone, the detection zone could avoid the layer-by-layer stack of immune sandwich complex to increase about 4~fold number of fluorescent dots for ensuring the accuracy of counting (Figure 5A). And this design made the nanobioprobes arrange in a particular zone to improve the counting efficiency than previous time consuming random statistics using the nanobioprobes.25 As shown in Figure 5 C-H, the number of fluorescent nanobioprobes in the detection zone increased gradually with the increasing concentration of the target virus (5, 10, 20, 50, 100, 500 pg/mL). A linear relationship was obtained in a wide range of 5 to 100 pg/mL with a linear correlation coefficient (R2) of 0.99 (Figure 5B). The detection limit was calculated to be 2.6 pg/mL in PBS, and corresponded to 4.2 pg/mL in saliva (Figure S9). Herein, the LOD was estimated by the IUPAC standard method (LOD=yblank+3×SDblank, where yblank is the average number of fluorescent nanobioprobes at zero and SDblank is the standard deviation of the blank measurements).

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Figure 5. (A) Histogram of fluorescent nanobioprobes number in different zones. (B) Linear response for EBOV detection; concentration range of 5-100 pg/mL in the buffer. (C-I) Fluorescence images of the samples at different concentrations (5, 10, 20, 50, 100 and 500 pg/mL, respectively). The scale bar is 20 µm. Error bars correspond to standard deviation (n = 3). Compared with previous works about detection of inactive EBOV or recombinant EBOV particles,14, 36-38 this strategy showed better analytical performance (Table 1). Possible explanations may be as follows: (1) the integrated detection platform can take advantage of magnetophoretic separation and continuous flow washing to enhance complex sample pretreatment ability and decrease the nonspecific adsorption;

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(2) in compared with the conventional ensemble fluorescence measurements, the fluorescence counting strategy owned significant advantages for improving sensitivity, and the cellular beacon with superior optical property could be directly used for counting to make the strategy easy and achieve highly sensitive detection on the micromagnetic platform. All these results suggest that this strategy has a very promising prospect in the early-stage detection for pathogens. Table 1. Summary of Analytical Performance of EBOV Particle Detection of Different Methods method ELISA Immunofluorescence Electroluminescent assay Test strip Celluar beacons@IMNs

target inactivated Ebola recombinant baculovirus inactivated Ebola recombinant filovirus inactivated Ebola

detection limit

ref

qualitative qualitative 5.2 pg/mL 160.0 ng/mL 2.6 pg/mL

37 36 14 38

current method

Specificity, Precision and Reproducibility of the Counting Strategy-mediated Assay. To investigate the specificity of the method, 1.0 µg/mL avian influenza virus (H1N1, H9N2) and 1.0 µg/mL porcine pseudorabies virus (PrV) were selected as negative control. As shown in Figure 6, the numbers of fluorescent nanobioprobes in EBOV (0.5 ng/mL) sample were conspicuously higher than those of negative controls, which suggested that the method had a good specificity. The intra-assay and inter-assay variabilities were used to further evaluate the reproducibility and precision of this detection method. The intra-assay variability was the variability of the same sample (5 pg/mL EBOV) analyzed in parallel five times with the same batch of IMNs. The interassay variability was tested in the same way but using five different batches of IMNs. The intra-assay and interassay variabilities were calculated by the

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coefficient of variability (CV% = SD/mean) of the parallel results. As shown in Table S1, intra-assay CV and interassay CV of the method were calculated to be 4.9 and 6.7%, respectively, which confirmed the high reproducibility and good precision of the assay.

Figure 6. Histograms of the detected fluorescent nanobioprobes number using different viruses for analysis of the detection specificity. Error bars correspond to standard deviation (n = 3). CONCLUSIONS In summary, we had successfully designed an ultrasensitive cellular beacons-mediated counting strategy for quantifying EBOV on a micromagnetic platform. The on-demand magnetic manipulation performed different detection steps for high integration, and enhanced complex sample pretreatment ability by using the multiple physical fields controlling (i.e., magnetic and fluid fields). Coupled with cellular beacon-mediated counting strategy on the micromagnetic platform, this method made the fluorescence counting techniques easy and achieved a high sensitivity. Finally, EBOV could be detected as low as 2.6 pg/mL, and detection limit had little effect on complex matrix. In addition, the assay showed high specificity and good 20


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reproducibility. In this way, we believe that this method may open up new avenues for the ultrasensitive detection of pathogens toward early-stage detection. ASSOCIATED CONTENT Supporting Information Figure S1, fluorescence intensity distribution and the hydrodynamic size of fluorescent nanobioprobes. Figure S2, characterization of MNs and IMNs. Figure S3, characterization of magnetic separation. Figure S4, EBOV captured by IMNs. Figure S5, feasibility of fluorescent nanobioprobes as reporters on the micromagnetic platform. Figure S6, the immune sandwich complex captured by the enrichment zone. Figure S7, WGA modified on detection zone and bound with cellular beacons. Figure S8, controlling process of six-way valve and photograph of the chip. Figure S9, linear response for EBOV detection in saliva. Note S1, numerical simulation of the on-demand magnetic manipulation. Table S1, intra- and interassay variability of this method. Corresponding Author Zhi-Ling Zhang, Email: [email protected]. Tel: 0086-27-68756759. Fax: 0086-27-68754067.

Yuhai Bi, Email: [email protected]. Tel: 0086-10-64806013. Fax: 0086-10-64806247.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGEMENTS

This work was supported by the National Natural Science Foundation of China (21475099, 21775111, and 8161101193), National Key R & D Program of China (2016YFE0205800),

National

Science

and

Technology

Major

Project

(2016ZX10004222). Y.B. is supported by the Youth Innovation Promotion Association of Chinese Academy of Sciences (CAS) (2017122). We are grateful to Ling-Ling Wu for her support in fluorescence magnetic nanoshperes.

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Cellular Beacons-mediated Counting for ... - ACS Publications

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