Ebola Virus Aptamers: From Highly Efficient Selection to Application

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Ebola Virus Aptamers: From Highly Efficient Selection to Application on Magnetism-controlled Chips Shao-Li Hong, Meng-Qi Xiang, Man Tang, Dai-Wen Pang, and Zhi-Ling Zhang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b04623 • Publication Date (Web): 11 Feb 2019 Downloaded from http://pubs.acs.org on February 11, 2019

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Analytical Chemistry

Ebola Virus Aptamers: From Highly Efficient Selection to Application on Magnetism-controlled Chips Shao-Li Hong, Meng-Qi Xiang, Man Tang, Dai-Wen Pang, 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

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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: Aptamers of Ebola virus (EBOV) offer a powerful means for the prevention and diagnostics. Unfortunately, few of aptamers for EBOV are discovered yet. Herein, assisted by magnetism-controlled selection chip to strictly manipulate selection conditions, a highly efficient aptamer selection platform for EBOV is proposed. With the high-stringency selection conditions of rigorous washing, minuscule amount of magnetic beads manipulating and real-time evaluation of the selection effectiveness, the selection performance of platform was improved significantly. Only by three rounds of selection, the high-performance aptamers for EBOV GP and NP proteins were obtained simultaneously, with dissociation constants (Kd) in nanomolar range. The aptamer was further applied to the detection of EBOV successfully, with a detection limit of 4.2 ng/mL. The whole detection process that consisted of sample mixing, separation and signal acquisition was highly integrately conducted in a magnetism-controlled detection chip, showing high biosafety and making it promising for point-of-care detection. The method may open up new avenues for prevention and control of EBOV.

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Ebola virus (EBOV) is one kind of hemorrhagic fever virus with a fatality rate up to 90%.1,2 EBOV has posed a great threat to human health and life and brought devastating effects on social and economic development.3 There are five species identified for EBOV: Zaire, Bundibugyo, Sudan, Reston and Tai Forest.4 EBOV is endemic throughout the year, without obvious seasonal differences, and can be transmitted from animals-to-animals,5 animals-to-persons6, and persons-to-persons.7 The EBOV early detection plays a key role in stopping the transmission of the disease, which usually uses traditional affinity reagents like antibodies.8-10 However, the antibody, due to “batch-to-batch variation” and expensive11, has shown a deficiency in the response to the outbreak of the EBOV. Therefore, the discovery of new affinity reagents for EBOV is of vital importance. Aptamers as next affinity reagents have attracted a lot of attention, because they are chemically synthesized, stable, and economical.12-17 Nowdays, thousands of aptamers have been generated against a wide range of targets, including small molecules18,19, proteins20,21, viruses22, bacteria23, whole cells15, etc. Nonetheless, the aptamers for EBOV are still lacking.24,25 One of the main factors is that the selection efficiency remains a bottleneck for aptamer discovery, despite that many selection methods have developed to accelerate aptamers discovery, including traditional filter-binding assays26, affinity flow cytometry,27 chromatography,28 capillary electrophoresis (CE)29,30 and magnetic beads.31,32 To further gain insights into those experimental factors that can influence aptamers selection efficiency, a number of researchers have developed theoretical models and performed numerical simulations of the selection 3



process.33-35 These studies show that two important parameters of influencing the selection performance are the concentration of unbound aptamers ([A]) and concentration of unbound target ([S]). According to the dissociation constants (Kd=[A][S]/[AS]) value of aptamers, it becomes clear that minimized Kd is kept and selection efficiency is improved as [S] → 0 and as [A] → 0.36 Based on these theories, the best-performing aptamers will be found by using the smallest feasible amount of target and stringently removing unbound aptamers.13 Despite these factors theoretically offer superior on selection efficiency, it is difficult for practical manipulation that requires delicate handling of a minuscule amount of target molecules without loss and removing unbound aptamers. Therefore, new selection techniques that can stringently control these conditions are urgently needed for highly efficient discovery of EBOV aptamer. Microfluidic techniques improve new insights to accelerate aptamers discovery, since a number of physical manipulation at the microscale can be easily handled while it would be difficult for conventional macroscale technologies.37-41 In our previous work, magnetism-controlled chip was used to develop a multifunctional screening platform.42 This platform could improve the selection efficiency via trapping magnetic nanospheres on the microscale. On the previous basis, we further exploited the magnetism-controlled chip to strictly manipulate the parameters that affect the selection efficiency. Highly efficient aptamer discovery was obtained through these sides. First, as low as 5 × 104 magnetic beads were manipulated to improve the selection performance, which was 40-fold less than previous work.36,43 Second, 4


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continuous flow washing was used to eliminate unbound nucleic acids. Third, dual selection zones were designed for screening GP protein and NP protein for EBOV simultaneously, of which GP protein is a type I transmembrane protein that is used as an antigen for the detection, and NP is nucleoprotein that contributes to subtype analysis for EBOV.44 By these high-stringency selection conditions, the selection efficiency was improved significantly. After three rounds of selection, high-affinity aptamers for EBOV were achieved with dissociation constants in nanomolar range. In addition, the selected GP aptamer could be further used to biosensor for EBOV on a highly integrated magnetism-controlled detection chip. The detection limit of this method was 4.2 ng/mL. This platform, from aptamer discovery to application, opens up new avenues for prevention and control of infectious diseases.

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EXPERIMENTAL SECTION Reagents and Instruments. Protein (NP, zaire) of 95% purity was synthesized by Sino Biological (China). Xiangguo Qiu kindly provided the EBOV glycoprotein, horse antibody against EBOV glycoprotein and goat antibody against EBOV nucleoprotein. Bovine serum albumin (BSA) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Dynabeads M-270 Carboxylic Acid was purchased from Invitrogen (Life technologies,USA). PCR Mix was purchased from TransGen (Beijing TransGen Biotech Co., Ltd., China). ssDNA marker was purchased from cnbioruler.cn Co., Ltd.. TBE-Urea Sample Buffer was purchased from Sangon Biotechnology (Shanghai) Co., Ltd., China). AZ50XT and AZ9260 photoresist (PR) was obtained from AZ Electronic Materials (AZ Electronic Materials Corp., USA). Poly (dimethylsiloxane) (PDMS) used for chip was brought from GE (Toshiba Silicones Co., Ltd., Japan). Ultrapure water (18.2 MΩ·cm) generated by a Millipore water-purification system (Synergy UV, Millipore, USA) was used to buffer solutions. All of nucleotides (nt) used in the experiment was synthesized from Sangon Biotechnology (Shanghai) Co., Ltd., China). A quantum dots 605 streptavidin conjugate (SA-QDs) was purchased from Wuhan Jiayuan Quantum Dots Co., Ltd. Binding buffer (20 mM HEPES, 150 mM NaCl, 2mM KCl, 2 mM MgCl2 and 2 mM CaCl2 (pH 7.4) ). Portable optical fiber spectrometer was purchased from QE65000, Ocean Optics. Fluorescence microscope (TiU, Nikon, Japan). Dynamic light scattering instrument (Malvern Zetasizer Nano ZS). Charge coupled device (CCD) camera used for 6


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microscope images was purchased from Nikon (Nikon DS-Ri1). Indium tin oxide (ITO) glass with a resistance of 10 Ω was purchased from LaiBao (LaiBao Hi-Tech Co.,Ltd., China). Amicon Ultra 0.5 mL 10K (Millipore, USA). Fabrication

of

the

Magnetism-controlled

Chips.

The

fabrication

of

magnetism-controlled selection chip was mainly based on our previous work.42 Namely, the silicon wafers and AZ50XT photoresist assisted by soft lithography were used to fabricate the fluidic microchannel. 50 µm × 50 µm nickel patterns were fabricated via photoresist (AZ9260) and electroplating process on ITO glass. Finally, fluidic microchannel was bound with nickel patterns on the ITO by plasma. To the magnetism-controlled detection chip, the fluidic channel and nickel patterns were fabricated according to the above description. Then, handmade miniature bars, which guided the magnetism, were located beside the fluidic channel of the separation zone. And then PDMS prepolymers were poured into a silicon wafer and cured. The cured PDMS with microchannels of detection was bound with nickel patterns to form the magnetism-controlled detection chip. Selection Procedures. The initial DNA library was consisted of a central random region of 60 nt flanked by 2 specific 20 nt as primer-binding sites for PCR (5’-AlexaAGCAGCACAGAGGTCAGATG(N60)CCTATGCGTGCTACCGTGAA-3’), where Alexa Fluor was labeled the forward primer (FP) and biotin was labeled the reverse primer (RP). 200 pmol DNA library was added into the binding buffer and denatured by heating at 95 °C for 10 min, immediately snapped cooling down to 0 °C for keeping 10 min, and then laid at room temperature for another 5 min. The library was 7



incubated with GP protein in 2.5 h, and then pumped into the selection chip where the GP protein-coated magnetic beads were trapped by GP selection unit. The unbound ssDNA library was collected in a reservoir, and the NP protein-coated magnetic beads were added to the reservoir for 2.5 h. After incubating, they were inhaled into the NP selection unit, and 2 µL/min washing buffer was injected into the microchannel. The selection process was being real-time evaluation by the fluorescence signal acquisition with a fiber optical spectrometer. Finally, the magnetic beads with ssDNA in the different zones were separately collected. The collected ssDNA-bound magnetic beads were further amplified through PCR, and the PCR program was described in our previous work42. The PCR products were resolved on 20% denaturing polyacrylamide gel electrophoresis (PAGE) to find out the optimal amplification condition. After PCR using the optimal condition, PCR products were removed to electrophoresis on a 20% denature PAGE to further confirm successful amplification of the DNA fragment. The successful amplification products were labeled with biotin, and they were reacted with streptavidin coated magnetic nanospheres for 1 h at room temperature. After magnetic separation, the separated PCR products were treated by NaOH solution (0.1 M) to release the single stranded DNA, and the Alexa-labeled sense ssDNA strands were store in NaOH solution and desalted by Ultra-4 Centrifugal Filter Devices (10K) for the next selection or characterization. Selection Effectiveness Evaluation of Each Round. To characterize the selection effectiveness, 50 pmol ssDNA of each round was incubated with protein-coated 8


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magnetic beads (MBs) for 2 h. After incubation, they were flowed into selection chip and washing buffer was flowed into the microchannel to washing the unreacted ssDNA, and the fluorescence intensities were acquired by a fiber optical spectrometer equipped on an inverted fluorescence microscope. Characterization of Candidate Aptamers. After selection with three rounds, the amplification products were sent to be cloned. All the colonies were sequenced and analyzed by the software to get the candidate aptamers. The candidate aptamers were further characterized to get the average dissociation constant (Kd). To abtain the Kd values, FAM (6-carboxyfluorescei) labeled candidate aptamers pools were diluted to a series of different concentrations and heat-treated according to the above selection method. These treated candidate aptamers solutions were incubated with target-proteins coated MBs for 2 h at the room tempreture. Then washing buffer was used to remove the unreacted ssDNA. The Kd values were then calculated by non-linear regression analysis based on the one-site saturation equation Y = Bmax X/ (Kd +X). 37 To further characterize the specificity of the candidate aptamers, FAM-labeled candidate aptamers were incubated with target proteins and control proteins (such as BSA, Avian influenza hemagglutinin (HA), human serum albumin (HSA) ). Then the fluorescence intensities bound with different proteins were measured. In addition, the secondary structure of candidate aptamers was further simulated with Mfold software on the website. Sensitive Assay of Aptamer-mediated EBOV Detection. The selected aptamer 9



could be further used for EBOV assay on a detection chip. 200 µL sample containing EBOV was mixed and incubated with 200 µL Aptamer modified magnetic nanospheres in the mixing zone of detection chip with a rate of 2.5 µL/min, where magnetic nanospheres (MNs) were fabricated by according to our previous work45. Subsequently, 5 µL/min reaction sample was flowed into the separation zone of detection chip, at which magnetic-tagged immune complex could be separated from the sample and stored in a reservoir. Then biotin-labeled aptamer (2 µL) was incubated with immune complex for 30 min in the reservoir. After reaction, the complex was inhaled and trapped into the detection zone of detection chip at the flow rate of 10 µL/min, and stringently washed by a continuous flow. After washing, the streptavidin labeled quantum dots (SA-QDs) were flowed into the detection zone and reacted with complex to acquire the detection signal. RESULTS AND DISCUSSION Design of Magnetism-Controlled Selection Chip. The magnetism-controlled selection chip was designed, including GP protein selection zone and NP protein selection zone (Figure 1), where had a high magnetic field gradient generated by nickel pillars patterns. Magnetic beads (MBs) could be trapped and delicately arranged in a cross array in the selection zones (Figure S1A-B). The target proteins were immobilized on the MBs surface via chemical coupling. After the GP and NP protein modification, the hydrodynamic size had changed to 3.10 µm and 3.15 µm compared with 2.88 µm before modification. Meanwhile, the Zeta （ζ） potential had also changed from -28.9 mV to -45.4 mV and -51.5 mV (Figure S2). These 10


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results indicated GP and NP proteins were successfully modified. To verify that the native active conformations of proteins were preserved after modification, the MBs coated with proteins were incubated with the monoclonal antibody and fluorescence labeled-IgG. As shown in Figure S3 and Figure S4, MBs coated with proteins displayed high-fluorescence signals after incubation, while MBs without proteins didn’t show fluorescence signal obviously. These results indicated that the native active conformations kept well after modification. Based on the fluorescence signals, the number of protein molecules per MBs was further measured by fluorescence assay, with the amount of 1.55×104 (Figure S5).

Figure 1. Schematic illustration for the magnetism-controlled selection chip. Overview of Magnetism-Controlled Selection Process. The selection process was mainly performed by the magnetism-controlled selection chip. As shown in scheme 1, the initial library of ssDNA was incubated with NP protein coated MBs, and then they were flowed into selection chip and trapped by the NP selection zone (step 1). The 11



part of unbound nucleic acids went through the GP selection zone to conduct GP protein selection (step 2). The GP and NP selection zones exchanged as the selection round increased, which could play a role of negative selection to reduce nonspecific binding ssDNA. To guide the selection process, the selection was in-situ monitored by a fiber optical spectrometer at the same time (step 3). After incubating, high rigorous washing was performed in the two selection zones to eliminate the weakly and nonspecifically bound ssDNA (step 4). The ssDNA bound with proteins were washed out of selection chip and collected for PCR amplification with Alexa Fluor- and biotin-labeled primers (step 5). Subsequently, PCR products were denatured to generate an evolved ssDNA library (step 6). One part was used for next round selection, and the other part was serviced for evaluating the enrichment (step 7). Once the enrichment signal didn’t change obviously, the selection would stop and the PCR products were chosen for cloning and sequenced (step 8).

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Scheme 1. Overview of Selection Process on Magnetism-controlled Selection Chip. High-Stringency Selection Condition. The magnetism-controlled selection chip enables us to precisely manipulate a number of physical manipulation for EBOV aptamer discovery. To apply high-stringency selection condition, minimal MBs were used to control the EBOV proteins and incubated with the random ssDNA library for offering higher selection pressure and reducing background binding (Figure S6). The partition efficiency (PE) with different numbers of MBs were significantly increased with the decrease of MBs (Figure 2A), and the value of PE was up to (1.0±0.3) × 107 at the 5×104 MBs, which increased about 10-fold compared with the current best methods, indicating less background adsorption30,36. Meanwhile, the binding percent — the ratio of target proteins-bound aptamers to the initial library of ssDNA — was dramatically decreased with the reduction of the amount of MBs, which demonstrated 13



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the selection pressure increased using a small amount of MBs (Figure 2B). To determine the minimum amount of MBs, proteins coated MBs with different numbers were used to incubate with ssDNA library, and then the MBs were collected to PCR. As shown in Figure 2C, the minimum number of MBs that amplified the correct size of the DNA fragment was 5×104, thus the MBs number used in this selection was settled. After the ssDNA library incubating with the GP and NP coated MBs, high rigorous washing was imposed on target proteins-bound ssDNA. Most of weakly or nonspecifically bound ssDNA could be removed. During the washing, the fluorescence intensity change was in-situ monitored. Most of weakly or nonspecifically bound ssDNA were removed within 9 min (Figure 2D). Compared with

the

conventional

selection

method

for

separating

ssDNA,

the

magnetism-controlled chip could take advantage of continuous flow to improve the selection pressure.

14


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Figure 2. (A) Partition efficiency of different amount of magnetic beads. (B) Binding percent of different amount of magnetic beads. (C) PAGE electrophoretogram results: (M) 50 bp marker. (line 1) PCR product of 104 magnetic beads. (line 2) PCR product of 5×104 magnetic beads. (line 3) PCR product of 105 magnetic beads. (control) PCR product of H2O. (D) Fluorescence intensity in four different washing times. Error bars indicate the standard deviation of three experiments. Real-time Evaluation of the Selection Effectiveness. To accelerate the aptamer discovery, the enrichment effectiveness was real-time evaluation by a fiber optical spectrometer during the selection process. Figure 3A showed that the enrichment signal of GP-specific aptamers was enhanced with the selection round increasing, and the intensity increased slowly after three rounds of selection. Therefore, the selection was stopped at this time. Similarly, the enrichment for NP protein was slightly increased after three rounds of selection and could terminate the selection (Figure 3B). Therefore, selection products for GP and NP were sequenced to reveal the 15



binding performance after three rounds of selection. In this work, real-time evaluation of enrichment effectiveness had enhanced the selection process control which could further improve the selection efficiency.

Figure 3. (A) Fluorescence intensity of each round selection toward GP protein. (B) Fluorescence intensity of each round selection toward NP protein. Affinity and Specificity of the Selected Sequences for EBOV. After three rounds of selection, all of the colonies were picked out and sequenced, which contained 28 sequences for GP protein and 26 sequences for NP protein. These sequences were found to distribute into several families according to their homology (Figure S7). For GP sequences, three sequences named GP-G02, GP-D01, and GP-B03, which was generated from different families, were chosen to determine their affinities (Table S1). Figure 4A-C showed that the Kd values of GP-G02, GP-D01, and GP-B03 were 14.9 ± 4.8, 4.1 ± 0.9, and 10.4 ± 2.6 nM, respectively. GP-D01 aptamer with the lowest Kd was incubated with other proteins (such as BSA, HAS, HA and NP) to further investigate its selectivity. Figure S8A showed that the intensity of GP-D01 bound with GP protein was higher than the other four proteins. On the other hand, three sequences (NP-D01, NP-C04, and NP-D02) for NP protein from different 16


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families were chosen. As shown in Figure 4D-F, the Kd values of NP-D01, NP-C04, and NP-D02 were 76..1 ± 10.9, 8.1 ± 2.4, and 41.3 ± 9.5 nM, respectively. NP-C04 aptamer with the lowest Kd was also further used to analyze its selectivity. Figure S8C showed that NP-C04 had an obvious specificity. To understand the structural differences between the aptamers, we modeled the GP-D01 and NP-C04 sequences using the mfold software (Figure S8B and D).

Figure 4 (A-C) Determination of the dissociation constants for GP protein with GP-G02, GP-D01, GP-B03. (D-F) Determination of the dissociation constants for NP protein with NP-D01, NP-C04, NP-D02. Sensitive Assay of EBOV on a Magnetism-controlled Detection Chip. The selected aptamer for GP protein was further used for the sensitive detection of EBOV. Highly integrated detection platform could improve the safety of the detection personnel by reducing contact of high-risk EBOV, thus a detection chip was designed, consisted of mixing, separation and detection zones (Figure 5A). In this detection chip, the aptamer of GP modified magnetic nanospheres (MNs) and sample were injected into 17



the chip and reacted in the mixing zone, generating the magnetic tagged complexes. The effectiveness of the mixing zone was shown in Figure S9-10. Then, the magnetic tagged complexes were transferred from the sample stream through magnetophoretic separation in the separation zone of detection chip. After separation, the magnetic tagged complexes were reacted with biotin-labeled aptamer in the reservoir. And then the complex was incubated with streptavidin modified quantum dots (SA-QD) in the detection zone, where they were enriched and directly acquired the detection signal via a fiber optical spectrometer.

Figure 5 (A) Schematic diagrams of highly integrated magnetism-controlled detection chip. (B-G) Fluorescence microscopic images of different concentrations of samples (5.0, 10.0, 40.0, 100.0, 150.0 and 200.0 ng/mL, respectively). (I) Linear response for Ebola virus detection; concentration range of 5.0-150.0 ng/mL in the buffer. Error bars indicate the standard deviation of three experiments. The scale bar is 50 µm. 18


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On the basis of the highly integrated detection chip, the detection process from the sample in to the results out had been achieved. As shown in Figure 5 B−G, fluorescence intensity of different EBOV concentrations was obtained (5.0, 10.0, 40.0, 100.0, 150.0 and 200.0 ng/mL). Figure 5H showed that the fluorescence intensity increased linearly range from 5.0−150.0 ng/mL, with a detection limit (LOD) of 4.2 ng/mL. Compared with previous work on the detection of EBOV9,10, using the detection chip made the detection process high integration and reduced the contact with high-risk EBOV, further improving the biosafety for detection personnel. Meanwhile, the EBOV aptamer was chemical synthesis and inexpensive compared with antibody, making a promising future in point-of-care diagnostics. Robustness of the Aptamer-Mediated Assay. The specificity of the detection was further investigated. As negative controls, 1.0 μg/mL avian influenza virus (H9N2) and three malaria patient samples (P. falciparum, P. ovale, and P. vivax) were chosen to test the specificity of this assay. These viruses can cause the symptoms of fever like EBOV. As shown in Figure 6, 5.0 ng/mL EBOV sample had a higher fluorescence intensity compared with the negative controls, which showed a good specificity using the method. Good precision and reproducibility are important for further application of this method, too. Here, 5.0 ng/mL EBOV sample was tested by using the same batch of MNs-Aptamer to analyze the intra-assay variabilities. And five different batches of MNs-Aptamer were used to detect the EBOV sample to obtain the interassay variability. The values of 3.7 and 6.5% (intra- and interassay) were shown in Table S1, respectively. These results indicated that this aptamer-mediated assay had good precision and the well reproducibility. 19



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Figure 6 Histograms of the specificity of this method. Error bars indicate the standard deviation of three experiments. CONCLUSIONS In summary, we have successfully achieved the selection and application of the EBOV aptamers based on the magnetism-controlled chips. Benefiting from high-stringency conditions, this method improved the selection efficiency. It generated separation efficiency up to (1.0±0.3) × 107, which was exceeded about 10 times than the previous methods. Nonspecific absorption was removed by continuous flow washing within 9 min, and the selection process was real-time evaluation, further enhancing the selection control. Using this method, the high-affinity and -specificity aptamers for GP and NP proteins were discovery within three rounds of selection, whose dissociation constants were 4.1 ± 0.9 and 8.1 ± 2.4 nM respectively. The selected aptamer for GP protein was further successfully applied to the detection of EBOV, with a detection limit of 4.2 ng/mL. And the detection showed high integration

which

could

perform

the

sample-to-result

detection

on

the

magnetism-controlled detection chip, showing high biosafety. To conclude, this 20


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method may improve a promising strategy for prevention and control of infectious diseases. ASSOCIATED CONTENT Supporting Information Figure S1, characterization of selection chip. Figure S2, characterization of target proteins coated MBs. Figure S3-4, characterization of native active conformations of target proteins. Figure S5, the amount of protein modified with the MBs. Figure S6, different numbers of MBs controlled by chip. Figure S7, sequences of significantly homologous. Figure S8, specificity and secondary structure analysis. Figure S9, photograph of highly integrated magnetism-controlled chip. Figure S10, microscopic image of fluidic flow in highly integrated magnetism-controlled chip using the blue ink tracking. 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. Notes The authors declare no competing financial interest. ACKNOWLEDGEMENTS This work was supported by the National Natural Science Foundation of China (21775111, 21475099) and the National Science and Technology Major Project of China (2018ZX10301405). References 21



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(1) Gire, S. K.; Goba, A.; Andersen, K. G.; Sealfon, R. S.; Park, D. J.; Kanneh, L.; Jalloh, S.; Momoh, M.; Fullah, M.; Dudas, G.; Wohl, S.; Moses, L. M.; Yozwiak, N. L.; Winnicki, S.; Matranga, C. B.; Malboeuf, C. M.; Qu, J.; Gladden, A. D.; Schaffner, S. F.; Yang, X., et al. Genomic surveillance elucidates Ebola virus origin and transmission during the 2014 outbreak. Science 2014, 345, 1369-1372. (2)

WHO

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Ebola Virus Aptamers: From Highly Efficient Selection to Application

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