Self-Assembly of Antigen Proteins into Nanowires Greatly Enhances

Subscriber access provided by Kaohsiung Medical University

Biological and Medical Applications of Materials and Interfaces

Self-assembly of antigen proteins into nanowires greatly enhances the binding affinity for high efficiency target capture Dong Men, Juan Zhou, Wei Li, Cuihua Wei, Yuanyuan Chen, Kun Zhou, Ying Zheng, Ke Xu, Zhi-Ping Zhang, and Xian-En Zhang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b12511 • Publication Date (Web): 02 Nov 2018 Downloaded from http://pubs.acs.org on November 3, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 25 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

ACS Applied Materials & Interfaces

Self-Assembly of Antigen Proteins into Nanowires Greatly Enhances the Binding Affinity for High Efficiency Target Capture Dong Men,*, †, ‡ Juan Zhou,†, ‡ Wei Li,# Cui-Hua Wei,† Yuan-Yuan Chen,┴ Kun Zhou,† Ying Zheng,† Ke Xu,‖ Zhi-Ping Zhang,† Xian-En Zhang*,§ †State

Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, PR China

§National

Key Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China #College

of life sciences, Hubei University, Wuhan 430062, China

┴Huazhong

‖CAS

Agricultural University, Wuhan 430070, China

Key Laboratory of Molecular Virology and Immunology, Institut Pasteur of Shanghai,

Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai 200031, China KEYWORDS: self-assembly, protein nanowire, polyvalent interaction, binding affinity, sensitive immunoassay

ACS Paragon Plus Environment

1

ACS Applied Materials & Interfaces 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

Page 2 of 25

ABSTRACT: High-efficiency target capture is an essential prerequisite for sensitive immunoassays. However, the current available immunoassay approaches are subject to deficient binding affinities between predator-prey molecules that greatly restrict the target capture efficiency and immunoassay sensitivity. Herein, we present a new strategy through the selfassembly of antigen proteins into nanowires to enhance the binding affinity between an antigen and antibody. Through the genetic fusion of antigen proteins (e.g., HIV p24) with the yeast amyloid protein Sup35 self-assembly domain, specific antigen nanowires (Ag nanowires) were constructed and demonstrated a remarkable enhancement in binding affinity compared with that of the monomeric antigen molecule. The Ag nanowires were further combined with magnetic beads to form a 3D magnetic probe based on a seed-induced self-assembly strategy. Taking advantage of both the strong binding affinity and the rapid magnetic separation and enrichment capacity, the specific 3D magnetic probe achieved a 100-fold improvement in detection sensitivity within a significantly shorter period of 20 min over that of the conventional ELISA method.

1. INTRODUCTION Rapid and sensitive immunoassays are of great importance for the control and prevention of infectious disease,1 including the early pathogen screening of epidemics,2 field detection of foodborne diseases,3 and point of care diagnostics.4 It is generally known that efficient target capture is an essential prerequisite for all immunoassay-based methods. In this principle, the binding affinity or the interaction between a specific antigen and antibody intrinsically determines the detection sensitivity and time consumption.5 However, the current available immunoassay techniques, typified by enzyme-linked immunosorbent assays (ELISAs), are mainly reliant on the immobilization of proteins on a two-dimensional (2D) surface through physical adsorption or


2

Page 3 of 25 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


chemical crosslinking.6 Among these procedures, the anchoring of proteins usually harbors a low surface density and randomly orientated arrangements. This greatly reduces the availability and the bioactivity of capture proteins leading to an inherent deficiency in binding affinity to the target molecules, thereby restricting the detection sensitivity and prolonging capture time. Alternatively, signal enhancement approaches have been employed to improve the sensitivity by using transducers (e.g., fluorescence, electrochemical signals, chemiluminescence, and surfaceenhanced Raman scattering),7-12 assembling multiple tagged molecules for a single target,13 or by combining with nanoprobes,8 nanoparticles,14-15 and new techniques.16 Nevertheless, those technologies also utilize the aforementioned methods to immobilize the proteins randomly, making efficient target capture challenging. Therefore, the exploration of novel immobilization methods to improve the binding affinity for high-efficiency target capture would be a reasonable approach to achieve sensitive immunoassay. In biological systems, there are substantial periodical nanostructures that can partake in synergistic polyvalent interactions different from those of the individual components, making important contributions to multiple biological events, including ligand-acceptor binding, molecular recognition, pathogen inhibition, cellular interactions and so forth.17-19 Inspired by the polyvalent interactions in biology, the polyvalent presentation of functional ligands on artificial scaffolds has been designed to construct analogous periodic nanostructures, and as expected, these nanostructures have demonstrated outstanding performances that are similar to the natural biological interactions.20-22 More importantly, the polyvalent interactions have been confirmed to enhance the binding affinity for the target molecules by orders of magnitude compared with those of monovalent interactions.23-24 Therefore, we conceive the employment of polyvalent interactions as a feasible strategy to enhance the binding affinity between antigen and antibody


3


Page 4 of 25

molecules, thereby breaking through the inherent limitations in sensitivity and timeliness of immunoassays. Utilizing the unique features of a symmetrical structure, controllable self-assembly and available genetic modification, periodic biological nanostructures have shown special superiority in the polyvalent presentation of functional ligands.21 As a representative, the yeast protein Sup35 derived from Saccharomyces cerevisiae, which can be self-assembled into nanowire structures, offers a good periodic scaffold for the display of functional proteins in a high surface density with controllable orientation and preserved bioactivity.25 Here, we propose to construct Ag nanowires based on the self-assembly of antigen proteins that genetically fused with the Sup35 protein (Sup35-Ag) into nanowires (Scheme 1A), on which the antigen proteins were organized with a high density and specific spatial arrangement. Through altering the selfassembly monomers by means of the genetic fusion of specific antigens with the Sup35 selfassembly domain, this stratagem can be employed for the 3D immobilization of various antigen proteins with desirable functions. Furthermore, the above Ag nanowires were decorated on a magnetic bead to harvest the 3D magnetic probe using a seeding-induced self-assembly method (Scheme 1B).26 It should be emphasized that the employment of magnetic microspheres here not only endowed a large surface to support the in situ self-assembly of Ag nanowires but also enabled a rapid immunoassay because of the convenient magnetic separation and enrichment processes.


4

Page 5 of 25 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


Scheme 1. Schematic illustrations of constructing (A) Ag nanowires for 3D immobilization of antigen proteins (TEM image shows Sup35-based nanowire scaffold); (B) 3D magnetic probe based on a seeding-induced self-assembly stratagem. 2. EXPERIMENTAL SECTION Preparation of biotinylated seeds and Ag nanowires. The biotinylated seeds and Ag nanowires were prepared according to our previous procedure with certain modifications.25 The prion domain of Sup35, a linker peptide (2  GGGGS), GFP, p24 gene were cloned in order into the pET28 vector to give pET28-Sup35-p24 plasmid. The fusion protein of Sup35-p24 was expressed by transforming the pET28-Sup35-p24 plasmid into Escherichia coli BL21 by induction with isopropyl -D-1-thiogalacto-pyranoside (IPTG). The biotinylated Sup35 fusion protein was expressed by co-transforming pET28-Sup35-BAP with pCDFDuet-BirA into Escherichia coli BL21 by induction with IPTG. The obtained fusion proteins were kept in assembly buffers (20 mM Tris-HCl, 150 mM NaCl, pH 8.6) at 4 C for 48 hours to generate nanowires, and the biotinylated seeds were prepared through ultrasonication of the corresponding nanowires in an ice bath.


5


Page 6 of 25

Binding affinity measurement between monomeric antigen/Ag nanowire and antibody. The binding affinity between Ag nanowire and antibody was measured using an Octet RED 96 instrument. Firstly, the anti-p24 antibody was diluted with the buffer (0.1% BSA, 0.02% tween20, assembly buffer) and then immobilized onto the anti-murine IgG Fv biosensor at a concentration of 5 g/mL. The entire testing process includes: baseline with buffer only (0-120 seconds), antibody loading (120-720 seconds), second baseline to rid away any excess antibody (720-1020 seconds), Ag nanowire association (1020-1620 seconds), and Ag nanowire dissociation (1620-2220 seconds). Association process was measured by immersing the antibody-loaded biosensors into Ag nanowires at different concentrations of 80 nM, 40 nM, 20 nM, 10 nM, 5 nM, 2.5 nM, 1.25 nM and 0 nM (buffer), respectively. The concentration of the nanowire is defined as the mole of antigen molecules per unit volume. Dissociation process was measured after moving the sensors into buffers. the monomeric p24 protein at different concentrations of 80, 40, 20, 10, 5, 2.5, 1.25 and 0 nM (buffer), were used to replace the above Ag nanowire to measure the interactions between p24 antigen and anti-p24 antibody under the same conditions. Meanwhile, the binding affinities between the Sup35-p24 nanowires and a nonspecific antibody (anti-Zika antibody), and that between the Ag nanowires and serum proteins were tested on the Octet RED96 system. The kinetic parameters were calculated according to a nonlinear global fitting using the Octet data processing software (version 3.43 data simulation) with a mass transport model. Preparation of 3D magnetic probe. The streptavidin-functionalized magnetic bead (3.5 L, 10 mg/mL, Dynabeads™ M-280 Streptavidin) were reacted with excess of biotinylated seeds (5 L, 2 mg/mL) in PBS buffer (1 mL) at 37 C for 45 min. The solution was purified through magnetic separation to remove the unreacted protein under a magnetic shelf for 90 s followed by


6

Page 7 of 25 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


washing with PBS buffer twice. Subsequently, enough of Sup35-p24 monomers (7.5 L) were added into the solution for reacting 15-30 min at room temperature. The final solution was purified through magnetic separation followed by washing with PBS buffer four times, and then resuspended in PBS (100 L) for further use. The 3D magnetic probe was characterized by SEM, steady-state fluorescence spectroscopy and laser-scanning confocal microscopy LSCM. Detection of anti-p24 antibody by 3D magnetic probe. The detection of anti-p24 antibodies by 3D magnetic probe was simply conducted in a 1.5 mL Eppendorf tube. The anti-p24 antibody with gradient dilutions (100 L, 10-fold dilution from 1 g/mL to 1 pg/mL), enzyme-labeled secondary antibody and the 3D magnetic probe solutions were mixed together and reacted for 5 min, respectively. The unreacted proteins were removed through magnetic separation under a magnetic shelf for 90 s followed by washing with PBS buffer 4 times. TMB (200 L) was added into the solutions and reacting at 37 C for 10 min. Finally, the sulfuric acid (50 L, 2 M) was added to terminate the reaction, and the results were measured at 450 nm on a microplate spectrophotometer. As a control, excess of biotinylated p24 proteins was reacted with streptavidin-coated magnetic bead (3.5 L, 10 mg/mL, Dynabeads™ M-280 Streptavidin) in PBS buffer (1 mL) at 37 C for 45 min, and then the product was purified by magnetic separation to yield M-p24 complex. Anti-p24 antibody with gradient dilutions (100 L, 10-fold dilution from 1 g/mL to 1 pg/mL), enzyme-labeled secondary antibody, and M-p24 complex were added into the tube and reacted for 5 min. After magnetic separation, washing with PBS buffers and TMB coloration, the final enzymatic signal was recorded on a microplate spectrophotometer.

3. RESULTS AND DISCUSSION


7


Page 8 of 25

The p24 protein was genetically fused with Sup35 self-assembly domains to construct Sup35p24 nanowires. The successful expression of the fusion proteins was confirmed by SDS-PAGE (Figure 1A). The Western blot analysis obtained by staining the product with the anti-p24 antibody also verified the successful fusion of the p24 gene and the preserved biological function of the p24 antigen (Figure 1B). After the in vitro self-assembly process, a mass of uniform linear structures was observed in the transmission electron microscopy image (Figure 1C), suggesting the fusion of the p24 gene cannot disturb the self-assembly behavior of the Sup35 domain. In addition, the specificity of the obtained Sup35-p24 nanowire was assessed based on an indirect ELISA approach, through the immobilization of the nanowire on a microplate in place of the monomeric antigen molecule to measure the antibody reactivity. Such Ag nanowires were employed to capture antibody molecules, and with the addition of an enzyme-labeled secondary antibody, the specific antigen-antibody interactions could be reflected by the enzymatic signals. As shown in Figure 1D, strong enzymatic signals were detected only in the presence of the antip24 antibody, while no clear enzymatic signals were detected with the addition of non-specific antibodies including the anti-Zika antibody and anti-LASV antibody. Similarly, no evident enzymatic signals were detected for the control groups with the addition of the PBS buffer, health human serum and BSA. These results clearly demonstrate that the Ag nanowires preserved the excellent specificity of the antigen-antibody interactions without cross-reactivity.


8

Page 9 of 25 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


Figure 1. (A) SDS-PAGE analysis of the Sup35-p24 fusion protein; (B) The Western blot analysis of Sup35-p24 stained with the anti-p24 antibody; (C) TEM image of the Sup35-p24 nanowires; (D) The specificity measurement of the Sup35-p24 nanowires, the concentration of antibodies and proteins both were 1 g/mL. To further define the molecular interactions between the Sup35-p24 nanowire and the corresponding anti-p24 antibody, binding affinity measurements were conducted on an Octet RED96 system based on the principle of biolayer interferometry using fiberoptic sensors. Briefly, the anti-p24 antibody was loaded onto the biosensors to attract the nanowires. The biosensors were immersed with Sup35-p24 nanowires at different concentrations and then transferred into buffer solutions to acquire association and dissociation curves (Figure 2A and Figure S1). In contrast, the binding affinity between the monomeric p24 antigen and anti-p24 antibody was also measured under the same conditions (Figure 2B and Figure S2). The kinetic parameters of the association rate constant (Kon), dissociation rate constant (Kdis) and equilibrium


9


Page 10 of 25

dissociation constant (KD) were calculated based on the acquired curves (Figure 2C). The Kon value was 1.11E+05 (Ms)-1 for the Sup35-p24 nanowire, which was 6-folder lower than that of 6.65E+05 (Ms)-1 for the monomeric p24 molecule, indicating the association rate of monomeric p24 antigen is faster than the nanowire. Notably, Kdis was 8.35E-10 s-1 for the nanowire, which was orders of magnitude smaller than that of 2.01E-07 s-1 for the monomeric p24 molecule, supporting the notion that the Ag nanowire binding is significantly stronger than the monomeric molecular binding. The KD values were determined by the Kdis values versus the Kon values, independent of the Ag nanowire concentration. The KD value of the Sup35-p24 nanowire and anti-p24 antibody was calculated to be 7.49E-15 M, and the same was calculated to be 3.02E-13 M for the monomeric p24 antigen and anti-p24 antibody. Evidently, the Ag nanowire provides a significant enhancement in binding affinity compared with the monomeric antigen molecule. This is probably because the polyvalent display of antigen molecules can induce synergetic polyvalent interaction, which is much stronger than the corresponding monovalent interaction. In addition, the binding affinity between the Sup35-p24 nanowire and anti-Zika antibody, and that between the Sup35-p24 nanowire and serum proteins were tested on the Octet RED96 system (Figure S3). Nearly no responses can be detected either for the anti-Zika antibody or serum proteins, and the KD was calculated to be 2.26E-08 M and 1.96E-08 M for the anti-zika antibody and serum proteins respectively. The low binding affinity to the nonspecific antibody and serum proteins further support the good specificity of the Ag nanowire. Based on the results above, Ag nanowires are confirmed to possess strong binding affinity for highly efficient capture capability, making them hold great potential for highly sensitive antibody detection.


10

Page 11 of 25 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


Figure 2. Association and dissociation curves for the affinity measurements between the (A) anti-p24 antibody and different concentrations of Sup35-p24 nanowires and (B) anti-p24 antibody and different concentrations of monomeric p24 proteins. (C) The kinetic binding constants of the Sup35-p24 nanowire and monomeric p24 antigen to the anti-p24 antibody. The concentration of the anti-p24 antibody was 5 g/mL, and the concentrations of Sup35-p24 nanowires and the monomeric p24 antigens were 80 nM, 40 nM, 20 nM, 10 nM, 5 nM, 2.5 nM, and 1.25 nM, defined as mole of p24 antigen per unit volume. To overcome the weaknesses of multi-step operations and time-consuming processes of conventional immunoassays, the 3D magnetic probe was constructed by combing the Ag nanowires with magnetic beads. In this section, biotinylated seeds played the crucial roles of attaching to streptavidin-coated magnetic microspheres through biotin-streptavidin interactions, and then, promoting a seed-induced self-assembly process to harvest the 3D magnetic probe (Scheme 1B). The biotinylated seeds were first prepared by the genetic fusion of a biotin


11


Page 12 of 25

acceptor peptide (BAP) with the Sup35 self-assembly domain to yield biotinylated nanowires, and subsequently, ultrasonication was used to separate the nanowires. The Western blotting analysis through staining the fusion protein with streptavidin-labeled horseradish peroxidase (SA-HRP) certified the successful biotinylation of the Sup35 protein (Figure 3A). Clearly, the TEM image revealed that these seeds possessed a rather short linear structure with the length mainly distributed in a range of 20-50 nm (Figure 3B and Figure S4). The Sup35-p24 monomers were employed as building blocks for the in situ growth of the Ag nanowires on the magnetic beads. The obtained 3D magnetic probe was investigated by scanning electron microscopy (SEM), steady-state fluorescence spectroscopy and laser scanning confocal microscopy (LSCM). The SEM images showed that the surface of the magnetic microspheres changed from rough to smooth upon the growth of the Ag nanowires (Figures 3C-F). To exhibit the coverage of nanowires on the magnetic bead surface more intuitively, the green fluorescent protein (GFP) gene was inserted into Sup35-p24 to achieve fluorescent Ag nanowires. The fluorescence spectroscopy demonstrated that the fluorescent intensity of the 3D magnetic probe gradually increased with prolonged growth time (Figure 3G). In addition, LSCM clearly showed bright green fluorescence around the magnetic beads (Figures 3H-J), while no fluorescence could be observed for the pristine magnetic beads (Figures 3K-M), further supporting the successful assembly of Ag nanowires on the surface of the magnetic microspheres.


12

Page 13 of 25 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


Figure 3. (A) Western blotting analysis of biotinylated Sup35 (Sup35-BAP) by staining with SA-HRP. (B) A TEM image of the biotinylated seeds. SEM images of the pristine magnetic


13


Page 14 of 25

beads (C-D) and 3D magnetic probe (E-F). (G) The variation of the fluorescence intensity with increased growth time of the nanowires. (H-J) Bright-field images and fluorescence images of the 3D magnetic probe (H, bright field; I, emission at 488 nm; J, overlay) and the pristine magnetic beads (K, bright field; L, emission at 488 nm; M, overlay). Utilizing the two advantages of enhanced binding affinity and rapid magnetic separation and enrichment, the 3D magnetic probe was employed for the rapid and sensitive detection of the anti-p24 antibody based on a 3D detection mode. The detection principle is depicted in Figure 4A. A mass of target antibodies was first captured by the 3D magnetic probe based on the specific antigen-antibody interactions, and with the subsequent addition of an enzyme-labeled secondary antibody, the target proteins were detected based on the enzymatic signals. As a control, biotinylated p24 proteins were expressed and then decorated on the surface of the magnetic beads through biotin-streptavidin interactions to harvest magnetic bead-p24 complexes (M-p24). It should be emphasized that excess of biotinylated p24 proteins were added into streptavidin-coated magnetic microspheres solutions to make sure maximized amount of p24 molecules on the surface of the magnetic microspheres. Based on the theoretical calculation that the magnetic bead per milligram is capable to bind 650-900 pmoles of biotinylated proteins, the entire amount of antigen molecules is at least 22.75 pmol in the M-p24 system. Similarly, in the 3D magnetic probe case, excesses of biotinylated Sup35-p24 seeds and enough of Sup35-p24 monomers were reacted with streptavidin-coated magnetic microspheres solutions. The assembly of antigen molecules into nanowires undoubtedly led to more antigen molecules in the 3D magnetic probe system. More importantly, in both cases the amount of antigen molecules is greatly more than the amount of the antibody molecules for the detection, even for the highest concentration of antibody molecule at 1 g/mL that was determined to be 0.66 pmole (the


14

Page 15 of 25 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


molecular weight of antibody was supposed to be 150 KDa). The detection of the anti-p24 antibody proceeded by using the 3D magnetic probe, the M-p24 and the conventional ELISA method, respectively (Figure 4B). As a result, the utilization of the 3D magnetic probe achieved the lowest detection limit of 0.1 ng/mL, which was improved by more than two orders of magnitude compared with that of 10 ng/mL for the M-p24 and conventional ELISA methods. Allowing for the adequate amounts of p24 proteins to capture anti-p24 antibody both in the Mp24 complex and in the 3D magnetic probe systems, the enhancement of binding affinity is exactly responsible for the improvement of detection sensitivity. Furthermore, by using the current 3D detection approach, the entire analytic process was completed within 20 minutes. This greatly shortens the detection time compared with the conventional ELISA method that usually involves tedious manipulations and hours of expense. Therefore, the 3D magnetic probe clearly demonstrates its capacity as a sensitive immunoassay in a short period of time, and it might be a smart platform for the assay of other proteins through altering the specific ligands on the nanowires.


15


Page 16 of 25

Figure 4. (A) Schematic principles of antibody detection based on the M-p24 (top) and 3D magnetic probe (bottom). (B) Comparing the detection of the anti-p24 antibody using the 3D magnetic probe, M-p24, and conventional ELISA methods. Periodic nanostructures with repeating modules are ideal scaffolds for the presentation of functional ligands in an ordered arrangement possessing novel properties superior to the single component, eventually is profitable for the advanced functions.20, 27 Enlightened by the periodic nanostructures in biology that display synergistic polyvalent effects to enhance specific binding, a variety of periodic scaffolds including those based on polypeptides,28 oligonucleotides,29 polymers30-31 and assembled nanoparticles32 have been designed for the study of multivalent interactions. It is worth noting that the periodic protein nanowires selected in this research benefit from special advantages. The formation of the periodic Ag nanowires is driven by a selfassembly process, and such a simple and mild strategy enables the good retention of protein activity. In addition, this periodic protein nanostructure is convenient to display a diversity of antigen proteins with a high density, controlled orientation and specific spatial arrangement based on genetic manipulation technologies (e.g., Sup35-Zika nanowires and Sup35-LASV nanowires in Figure S5). As shown in Figure S6, the enhanced binding affinity in the protein nanowire is also approved with Sup35-Zika nanowire/anti-Zika antibody binding pairs. The KD value of Sup35-Zika nanowire and anti-Zika antibody was 2.24E-10 M, and the KD value was 2.29E-09 M for the monomeric Zika protein and the anti-Zika antibody. It is worth to further study the mechanism of this phenomenon and investigate whether it is a general strategy by using at least a dozen of interaction protein pairs. Learning from the polyvalent interactions in biology, we speculate that the self-assembly of antigen proteins into nanowires creates polyvalent interactions significantly stronger than the monovalent antigen-antibody interactions


16

Page 17 of 25 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


and is responsible for the dramatic enhancement in binding affinity toward the specific antibody molecules. Furthermore, we suppose that in conventional immunoassays several target molecules are unable to be captured due to the intrinsic deficiency in binding affinity between antigen and antibody interactions. Different from our previous reports involving the integration of a mass of signal molecules (e.g., fluorescent proteins or enzymes) on nanowires for the enhancement of detection sensitivity,26,

33-34

the current stratagem that displays antigen molecules on the

nanowires focuses on addressing the intrinsic deficiency in target capture that restricts immunoassay sensitivity. Notably, the 3D magnetic probe enables rapid target capture and detection within a few minutes, which usually takes a few hours of manipulation for conventional methods based on a 2D surface. Therefore, the 3D magnetic probe not only improves the opportunity for target attachment, and accelerates the separation and enrichment process but also assists to form a stable complex product. Taking all these features, such a 3D magnetic probe can serve as a novel platform for the rapid and sensitive immunoassay of various proteins with significant potential for early diagnosis, emergency treatment and field-based testing. Furthermore, the proposed method can conveniently collaborate with signal enhancement approaches or new detection equipment to further increase the sensitivity.

4. CONCLUSIONS In conclusion, we developed a 3D immunoassay platform for rapid and highly sensitive antibody detection through the self-assembly of antigen proteins into nanowires on magnetic beads. The polyvalent presentation of antigens on the nanowires is critical for the improvement in the binding affinity between specific antigen-antibody interactions. Using the 3D magnetic probe, the detection limit achieves 0.1 ng/mL within 20 min, which is substantially shorter than hours of


17


Page 18 of 25

testing and improved by more than 100-fold in sensitivity compared with the conventional ELISA method. The current approach possesses the merits of extreme simplicity, rapidity, high sensitivity, and good flexibility for the immobilization of different functional ligands, and thus, might be further extended to the early detection of specific protein markers and fast on-site diagnosis.

ASSOCIATED CONTENT Supporting Information. Affinity measurements, length distribution of biotinylated seeds, SDS-PAGE of Sup35-LASV and Sup35-Zika fusion proteins, TEM images of Sup35-LASV nanowires and Sup35-Zikananowires. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author * Tel.: +86 27 87197671; +86 10 64888148. E-mail: [email protected]; [email protected]. Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ‡These authors contributed equally. Notes Any additional relevant notes should be placed here. ACKNOWLEDGMENT


18

Page 19 of 25 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


This work was supported by the National Natural Science Foundation of China (Grant 9152742, 31771098), the Key Research Program of the Chinese Academy of Science (Grant ZDRW-ZS2016), the Strategic Priority Research Program of the Chinese Academy of Science (Grant XDP030501), Youth Innovation Promotion Association of CAS (Grant 2014308), CAS Key Laboratory of Special Pathogens and Biosafety, Chinese Academy of Sciences, the Open Research Fund Program of Wuhan National Bio-Safety Level 4 Lab of CAS (NBL2017011), Collaborative Research Grant KLMVI-OP-201603 of CAS Key Laboratory of Molecular Virology & Immunology, Institut Pasteur of Shanghai, Chinese Academy of Sciences. We thank Dr. Ding Gao in the core facility and the technical support of Wuhan Institute of Virology, for assistance with binding affinity measurements. We thank Dr. Yuanyuan Chen of Institute of biophysics, Chinese Academy of Sciences, for valuable discussion. The Zika antibody is kind gifts from Prof. Bing Sun’s lab in Institut Pasteur of Shanghai, Chinese Academy of Sciences. REFERENCES 1.

Mabey, D.; Peeling, R. W.; Ustianowski, A.; Perkins, M. D. Diagnostics for the Developing World. Nat. Rev. Microbiol. 2004, 2, 231-240.

2.

Yu, X.; Xia, Y.; Tang, Y.; Zhang, W.-L.; Yeh, Y.-T.; Lu, H.; Zheng, S.-Y. A Nanostructured Microfluidic Immunoassay Platform for Highly Sensitive Infectious Pathogen Detection. Small 2017, 13, 1700425.

3.

Valderrama, W. B.; Dudley, E. G.; Doores, S.; Cutter, C. N. Commercially Available Rapid Methods for Detection of Selected Food-borne Pathogens. Crit. Rev. Food Sci. Nutr. 2016, 56, 1519-1531.


19


4.

Page 20 of 25

Kozel, T. R.; Burnham-Marusich, A. R. Point-of-Care Testing for Infectious Diseases: Past, Present, and Future. J. Clin. Microbiol. 2017, 55, 2313-2320.

5.

Welch, N. G.; Scoble, J. A.; Muir, B. W.; Pigram, P. J. Orientation and Characterization of Immobilized Antibodies for Improved Immunoassays. Biointerphases 2017, 12, 02D301.

6.

Jung, Y.; Jeong, J. Y.; Chung, B. H. Recent Advances in Immobilization Methods of Antibodies on Solid Supports. Analyst 2008, 133, 697-701.

7.

Li, B.; Yu, Q.; Duan, Y. Fluorescent Labels in Biosensors for Pathogen Detection. Crit. Rev. Biotechnol. 2015, 35, 82-93.

8.

Fu, X.; Chen, L.; Choo, J. Optical Nanoprobes for Ultrasensitive Immunoassay. Anal. Chem. 2017, 89, 124-137.

9.

Chikkaveeraiah, B. V.; Bhirde, A. A.; Morgan, N. Y.; Eden, H. S.; Chen, X. Electrochemical Immunosensors for Detection of Cancer Protein Biomarkers. ACS Nano 2012, 6, 6546-6561.

10. Zhou, Y.; Zhang, Y.; Lau, C.; Lu, J. Sequential Determination of Two Proteins by Temperature-triggered Homogeneous Chemiluminescent Immunoassay. Anal. Chem. 2006, 78, 5920-5924. 11. Pei, Y.; Wang, Z.; Zong, S.; Cui, Y. Highly Sensitive SERS-based Immunoassay with Simultaneous Utilization of Self-assembled Substrates of Gold Nanostars and Aggregates of Gold nanostars. J. Mater. Chem. B 2013, 1, 3992-3998.


20

Page 21 of 25 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


12. Hwang, J.; Lee, S.; Choo, J. Application of a SERS-based Lateral Flow Immunoassay Strip for the Rapid and Sensitive Detection of Staphylococcal Enterotoxin B. Nanoscale 2016, 8, 11418-11425. 13. Men, D.; Zhang, T.-T.; Hou, L.-W.; Zhou, J.; Zhang, Z.-P.; Shi, Y.-Y.; Zhang, J.-L.; Cui, Z.-Q.; Deng, J.-Y.; Wang, D.-B.; Zhang, X.-E. Self-Assembly of Ferritin Nanoparticles into an Enzyme Nanocomposite with Tunable Size for Ultrasensitive Immunoassay. ACS Nano 2015, 9, 10852-10860. 14. Farka, Z.; Jurik, T.; Kovar, D.; Trnkova, L.; Skladal, P. Nanoparticle-Based Immunochemical Biosensors and Assays: Recent Advances and Challenges. Chem. Rev. 2017, 117, 9973-10042. 15. Tang, D.; Cui, Y.; Chen, G. Nanoparticle-based Immunoassays in the Biomedical Field. Analyst 2013, 138, 981-990. 16. Dong, J.; Ueda, H. ELISA-type Assays of Trace Biomarkers Using Microfluidic Methods. Wiley Interdiscip. Rev.: Nanomed. Nanobiotechnol. 2017, 9, e1457. 17. Mammen, M.; Choi, S. K.; Whitesides, G. M. Polyvalent Interactions in Biological Systems: Implications for Design and Use of Multivalent Ligands and Inhibitors. Angew. Chem., Int. Ed. 1998, 37, 2755-2794. 18. Vorup-Jensen, T. On the Roles of Polyvalent Binding in Immune Recognition: Perspectives in the Nanoscience of Immunology and the Immune Response to Nanomedicines. Adv. Drug Delivery Rev. 2012, 64, 1759-1781.


21


Page 22 of 25

19. Lauster, D.; Glanz, M.; Bardua, M.; Ludwig, K.; Hellmund, M.; Hoffmann, U.; Hamann, A.; Boettcher, C.; Haag, R.; Hackenberger, C. P. R.; Herrmann, A. Multivalent PeptideNanoparticle Conjugates for Influenza-Virus Inhibition. Angew. Chem., Int. Ed. 2017, 56, 5931-5936. 20. Varner, C. T.; Rosen, T.; Martin, J. T.; Kane, R. S. Recent Advances in Engineering Polyvalent Biological Interactions. Biomacromolecules 2015, 16, 43-55. 21. Kushner, A. M.; Guan, Z. Modular Design in Natural and Biomimetic Soft Materials. Angew. Chem., Int. Ed. 2011, 50, 9026-9057. 22. Bai, Y.; Nguyen, L.; Song, Z.; Peng, S.; Lee, J.; Zheng, N.; Kapoor, I.; Hagler, L. D.; Cai, K.; Cheng, J.; Chan, H. Y. E.; Zimmerman, S. C. Integrating Display and Delivery Functionality with a Cell Penetrating Peptide Mimic as a Scaffold for Intracellular Multivalent Multitargeting. J. Am. Chem. Soc. 2016, 138, 9498-9507. 23. Dubacheva, G. V.; Araya-Callis, C.; Volbeda, A. G.; Fairhead, M.; Codee, J.; Howarth, M.; Richter, R. P. Controlling Multivalent Binding through Surface Chemistry: Model Study on Streptavidin. J. Am. Chem. Soc. 2017, 139, 4157-4167. 24. Duret, D.; Grassin, A.; Henry, M.; Jacquet, T.; Thoreau, F.; Denis-Quanquin, S.; Coll, J.L.; Boturyn, D.; Favier, A.; Charreyre, M.-T. "Polymultivalent" Polymer-Peptide Cluster Conjugates for an Enhanced Targeting of Cells Expressing alpha(v)beta(3) Integrins. Bioconjugate Chem. 2017, 28, 2241-2245.


22

Page 23 of 25 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


25. Glover, J. R.; Kowal, A. S.; Schirmer, E. C.; Patino, M. M.; Liu, J. J.; Lindquist, S. SelfSeeded Fibers Formed by Sup35, the Protein Determinant of [PSI+], a Heritable Prion-like Factor of S. cerevisiae. Cell 1997, 89, 811-819. 26. Men, D.; Guo, Y.-C.; Zhang, Z.-P.; Wei, H.-p.; Zhou, Y.-F.; Cui, Z.-Q.; Liang, X.-S.; Li, K.; Leng, Y.; You, X.-Y.; Zhang, X.-E. Seeding-Induced Self-assembling Protein Nanowires Dramatically Increase the Sensitivity of Immunoassays. Nano Lett. 2009, 9, 2246-2250. 27. Nie, Z.; Petukhova, A.; Kumacheva, E. Properties and Emerging Applications of Selfassembled Structures Made from Inorganic Nanoparticles. Nat. Nanotechnol. 2010, 5, 1525. 28. Rudra, J. S.; Ding, Y.; Neelakantan, H.; Ding, C.; Appavu, R.; Stutz, S.; Snook, J. D.; Chen, H.; Cunningham, K. A.; Zhou, J. Suppression of Cocaine-Evoked Hyperactivity by Self-Adjuvanting and Multivalent Peptide Nanofiber Vaccines. ACS Chem. Neurosci. 2016, 7, 546-552. 29. Gaddes, E. R.; Gydush, G.; Li, S.; Chen, N.; Dong, C.; Wang, Y. Aptamer-Based Polyvalent Ligands for Regulated Cell Attachment on the Hydrogel Surface. Biomacromolecules 2015, 16, 1382-1389. 30. Salvado, M.; Reina, J. J.; Rojo, J.; Castillon, S.; Boutureira, O. Topological Defects in Hyperbranched Glycopolymers Enhance Binding to Lectins. Chem. - Eur. J. 2017, 23, 15790-15794.


23


Page 24 of 25

31. Sletten, E. T.; Loka, R. S.; Yu, F.; Nguyen, H. M. Glycosidase Inhibition by Multivalent Presentation of Heparan Sulfate Saccharides on Bottlebrush Polymers. Biomacromolecules 2017, 18, 3387-3399. 32. Guo, Y.; Nehlmeier, I.; Poole, E.; Sakonsinsiri, C.; Hondow, N.; Brown, A.; Li, Q.; Li, S.; Whitworth, J.; Li, Z.; Yu, A.; Brydson, R.; Turnbull, W. B.; Poehlmann, S.; Zhou, D. Dissecting

Multivalent

Lectin-Carbohydrate

Recognition

Using

Polyvalent

Multifunctional Glycan-Quantum Dots. J. Am. Chem. Soc. 2017, 139, 11833-11844. 33. Men, D.; Zhou, J.; Li, W.; Leng, Y.; Chen, X.; Tao, S.; Zhang, X.-E. Fluorescent Protein Nanowire-Mediated Protein Microarrays for Multiplexed and Highly Sensitive Pathogen Detection. ACS Appl. Mater. Interfaces 2016, 8, 17472-17477. 34. Men, D.; Zhang, Z.-P.; Guo, Y.-C.; Zhu, D.-H.; Bi, L.-J.; Deng, J.-Y.; Cui, Z.-Q.; Wei, H.P.; Zhang, X.-E. An Auto-biotinylated Bifunctional Protein Nanowire for Ultra-sensitive Molecular Biosensing. Biosens. Bioelectron. 2010, 26, 1137-1141.


24

Page 25 of 25 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


Table of Contents

A rapid and sensitive 3D immunoassay platform was constructed through self-assembling of antigen protein into nanowires. The polyvalent presentation of antigen protein on the nanowires enables a significant enhancement of binding affinity to the target antibody, and the 3D magnetic probe achieves a 100-fold improvement of sensitivity within a much shorter period of time as compared with the conventional ELISA.


25

Self-Assembly of Antigen Proteins into Nanowires Greatly Enhances

Recommend Documents