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Fluorescent protein nanowire-mediated protein microarrays for multiplexed and highly sensitive pathogen detection Dong Men, Juan Zhou, Wei Li, Yan Leng, Xinwen Chen, Sheng-ce Tao, and Xian-En Zhang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b04786 • Publication Date (Web): 17 Jun 2016 Downloaded from http://pubs.acs.org on June 26, 2016

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ACS Applied Materials & Interfaces

Fluorescent protein nanowire-mediated protein microarrays for multiplexed and highly sensitive pathogen detection Dong Men,†,#,‡,* Juan Zhou,†,‡ Wei Li,ǁ Yan Leng,† Xinwen Chen,† Shengce Tao,┴ and 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 #

ǁ

Nursing College, Henan University, Kaifeng 475004, China

College of life sciences, Hubei University, Wuhan 430062, China

┴

Shanghai Center for Systems Biomedicine, Key Laboratory of Systems Biomedicine (Ministry of Education), Shanghai Jiao Tong University, Shanghai 200240, China

KEYWORDS: self-assembly, fluorescent protein, protein nanowire, protein microarray, pathogen detection, multiplexed detection

ACS Paragon Plus Environment

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ABSTRACT: Protein microarrays are powerful tools for high-throughput and simultaneous detection of different target molecules in complex biological samples. However, the sensitivity of conventional fluorescence-labeling protein detection methods is limited by the availability of signal molecules for binding to the target molecule. Here, we built a multi-functional fluorescent protein nanowire (FNw) by harnessing self-assembly of yeast amyloid protein. The FNw integrated a large number of fluorescent molecules, thereby enhancing the fluorescent signal output in target detection. The FNw were then combined with protein microarray technology for detecting proteins derived from two pathogens including influenza virus (hemagglutinin 1, HA1) and human immunodeficiency virus (p24 and gp120). The resulting detection sensitivity achieved a 100-fold improvement over a commercially available detection reagent.

1. INTRODUCTION Over the past decade, the outbreaks of emerging and re-emerging infectious diseases have caused widespread panic and heavy socio-economic losses.1,2 Rapid pathogen screening is one of the most important tools for controlling the spread of infectious diseases.3-5 Compared with commonly used pathogen protein detection methods, such as enzyme-linked immunosorbent assays (ELISAs),6 electrochemistry,7 label-free optical methods,8 and nanowire-based fieldeffect transistors,9 protein microarrays offer outstanding advantages in terms of high-throughput and parallel analytical capacity, making it an attractive approach for meeting the requirements of rapid and multiplexed analysis.10-12 Despite wide application, the protein microarray technique remains challenged by the limited sensitivity due to the low available quantity of signal molecules for binding to the target molecules.13 Therefore, signal enhancing approaches to improve the sensitivity of protein microarrays is of great significance for highly sensitive pathogen detection in complex systems.


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The amyloid protein, which exists across many species, has the unique ability to self-assemble into a fibril-like nanostructure.14 In combination with gene fusion techniques, the amyloid protein can be engineered as a fusion protein comprising a series of functional biomolecules including enzymes,15,16 metal-binding peptides,17 cell adhesion molecules18 and fluorescent proteins,19 resulting in the production of a protein nanowire with the desired functionalities. Based on the previous studies that the yeast protein Sup35 isolated from Saccharomyces cerevisiae can be self-assembled into amyloid fibers,20 we have constructed a series of fusion proteins based on Sup351-61 (the prion domain of Sup35 containing the first 61 amino acid residues of Sup35), which has been considered as a suitable building block for the assembly of functional nanostructures.21-23 In addition, the Sup35-based functional protein nanowires were successfully employed in the construction of molecular biosensors, and the capacity of these nanowires was successfully demonstrated through the dramatic enhancement of ELISA sensitivity.21-23 Therefore, we proposed that the combination of functional protein nanowires and protein microarray technology would have significant potential for highly sensitive and multiplexed pathogen detection. In this study, we prepared multi-functional protein nanowires based on the self-assembly of Sup35 and the genetic fusion of green fluorescent protein (GFP) and a biotin acceptor peptide (BAP). The fusion protein Sup35-GFP (Figure 1A) can be selected as a backbone to generate the FNw due to the self-assembly process of Sup35 domain. The fusion protein Sup35-BAP can be transformed into biotinylated Sup35 through co-expression of Sup35-BAP and biotin-protein ligase (BirA enzyme) in vivo in the presence of biotin (Figure 1B). As shown in Figure 1C, the preparation of the FNw contains two steps: (i) self-assembling Sup35-GFP seeds and Sup35-GFP monomers at the molar ratio of 1:4 to harvest FNw; and followed by (ii) self-assembling with


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biotinlyated Sup35 monomers to functionalize the FNw with biotin molecules. As a proof-ofconcept, the FNw enriched with GFP molecules is able to provide significant signal amplification and sensitivity enhancement. In addition, the FNw functionalized with biotin molecules can be used to attach biotinylated antibodies on the microarray surface through the biotin-streptavidin-biotin interaction. By using three different antibodies (anti-HA1, anti-HIV-1 gp120 and anti-HIV-1 p24), the FNw-mediated protein microarray has been investigated for sensitive and parallel pathogen detection.

Figure 1. (A) Schematic structure of Sup35-GFP and Sup35-BAP. The fusion protein Sup35GFP was expressed by transforming pET28-Sup35-GFP plasmid (Sup351-61, a linker peptide and GFP were cloned in order into pET28 vector to give pET28-Sup35-GFP) into the E. coli BL21, and inducing with IPTG. The fusion protein Sup35-BAP was expressed by transforming pET28Sup35-BAP plasmid (Sup351-61, a linker peptide and a BAP tag were cloned in order into pET28


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vector to give pET28-Sup35-BAP) into the E. coli BL21, and inducing with IPTG. (B) Autobiotinylation of the Sup35-BAP through co-expression of Sup35-BAP and BirA in the E. coli BL21 in the presence of biotin. The BAP tag was biotinylated by BirA during Sup35-BAP expression. (C) Preparation of FNw based on the self-assembly of Sup35 domain between Sup35-GFP seeds and Sup35-GFP monomers, followed by the self-assembly with biotinylated Sup35-BAP. 2. EXPERIMENTAL SECTION Preparation of FNw. The FNw was prepared according to the previous literature.23 Briefly, the prion domain of Sup35, a linker peptide (2×GGGGS) and GFP were cloned in order into pET28 vector to give pET28-Sup35-GFP. The fusion protein Sup35-GFP was expressed by transforming the pET28-Sup35-GFP plasmid into the E. coli BL21, by induction with isopropyl β-D-1-thiogalactopyranoside (IPTG). Similarly, the prion domain of Sup35, a linker peptide (2×GGGGS), GFP and a BAP tag were cloned in order into pET28 vector to give pET28-Sup35BAP plasmid. The auto-biotinylated Sup35-BAP fusion protein was expressed by cotransforming pET28-Sup35-BAP with the gene of BirA (cloned in co-expression vector pCDFDuet to get pCDFDuet-BirA) into E. coli BL21, by induction with IPTG. The Sup35-GFP nanowire was prepared by regulating the molar ratio between Sup35-GFP seeds and Sup35-GFP monomers at 1:4, and harvested by centrifugation at 4 ºC. The FNw was prepared by the addition of biotinylated Sup35 monomer into the Sup35-GFP nanowire, followed by centrifugation and washing with TBS buffer for three times. The resulting FNw was collected and stored at 4 ºC. Preparation of biotinylated antibody. The biotinylation of four polyclonal antibodies (antiF1, anti-HA1, anti-p24 and anti-gp120 antibody) were conducted by using biotin labeling kits


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from Dojindo. The biotinylated antibodies were re-suspended in the TBS buffer (TBS buffer: 200 nM Tris-HCl (pH 7.4), 150 mM NaCl) and stored at 4 ºC. Preparation of FNw-mediated protein microarray. The printing of protein microarray was conducted on a SmartArrayTM-48 microarrayer (CapitaBio Corp.). For the function evaluation of FNw-mediated protein microarray, the negative control BSA and three pathogen proteins of HA1, HIV-1 p24 and HIV-1 gp120 with different concentrations were spotted on the blocks of the microarray (Figure S1). Each concentration of the pathogen proteins was spotted three times in parallel. For the preparation of FNw-mediated protein microarray for the detection of pathogen proteins, four capture antibodies of anti-F1 (F1 antigen from Yersinia pestis), anti-HA1, antiHIV-1 p24 and anti-HIV-1 gp120 (HIV related pathogen proteins and antibody purchased form Immune Technology Corp.) were spotted on the blocks of the microarrays (Figure S2). The antiF1 antibody spotted on the microarray was used as a negative control. In both protein microarrays, fluorescein isothiocyanate labeled ConA was used as a land mark. Function evaluation of FNw-mediated protein microarray. After the immobilization of BSA (negative control) and three pathogen proteins on the microarray and the blocking of the microarray with 5% TBSM (TBSM: TBS buffer dissolved with 0.5% milk), four biotinylated antibodies of anti-HA1, anti-HIV-1 p24, anti-HIV-1 gp120 and anti-F1 (1 µg/mL, 15~20 µL) were separately added into each block of the protein microarray and reacted for 75 min at 37 ºC. The streptavidin (1 µg/mL, 15~20 µL) diluted by TBSM buffer was added into each block and reacted for 45 min at room temperature. Then the as-prepared FNw was added into each block and reacted for another 45 min at room temperature, and followed by washing the microarray with TBST buffer for 5 times (TBST buffer: 200 nM Tris-HCl (pH 7.4), 150 mM NaCl, 0.1%(v/v) Tween-20). For the evaluation of multiplexed analytical capacity, the four mixed


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antibodies were added into the block spotted by the negative control BSA and three pathogen proteins. The results were observed by microarray scanner (GenePix 4200A) using the excitation of 635 nm and 488 nm. Detection of pathogen proteins by FNw-mediated protein microarray. The detection of pathogen proteins by the FNw-mediated protein microarray was based on a sandwich ELISA mode. After the immobilization of four antibodies on the microarray, the mixture of three pathogen proteins of HA1, HIV-1 p24, HIV-1 gp120 and BSA with different concentrations were added into the block of the protein microarrays. The concentration of each pathogen protein was diluted from 1000 ng/mL to 0.1 ng/mL (10-fold dilution) by using TBS buffer. Subsequently, the biotinylated recognizing antibodies, streptavidin and the FNw were added in order using the same manipulation for the function evaluation, and the results were observed by microarray scanner using the excitation of 635 nm and 488 nm.

3. RESULTS AND DISCUSSION The expression of biotinylated Sup35 and Sup35-GFP were analyzed by SDS-PAGE analysis (Figure 2A, Lanes 1 and 2), which indicated the relative purity of the two monomeric proteins. Further western blotting analysis by staining the product with streptavidin-labeled horseradish peroxidase and anti-GFP antibody confirmed the successful fusion of Sup35 with BAP (Figure 2A, Lanes 3 and 5) and with GFP (Figure 2A, Lanes 4 and 6). In addition, the FNw were evaluated by steady-state fluorescence spectroscopy, an inverted fluorescence microscope and transmission electron microscopy (TEM). Fluorescence spectroscopy illustrated the high fluorescence intensity of the FNw with peak emission at 510 nm which similar with wild-type GFP (Figure 2B). Obviously, the observation from the fluorescence microscope also supported


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the strong fluorescence signals of the FNw (Figure 2C). TEM image revealed that the FNw were uniformly linear in structure with the diameter of 14.8 ± 0.4 nm and the length mainly distributing in 300 - 400 nm (Figure 2D and Figure 2E).

Figure 2. (A) SDS-PAGE and western blot analysis of biotinylated Sup35 and Sup35-GFP. Lane 1, purified biotinylated Sup35; lane 2, purified Sup35-GFP; lane 3, purified biotinylated Sup35 stained with streptavidin-labeled horseradish peroxidase (SA-HRP); lane 4, purified biotinylated Sup35 stained with anti-GFP antibody; lane 5, purified Sup35-GFP stained with SA-HRP; lane 6, purified Sup35-GFP stained with anti-GFP antibody. (B) Fluorescence spectroscopy of the FNw. (C) Fluorescence image of the FNw. (D) TEM image of the FNw. (E) Length distribution histogram of FNw. Data presented was counted from a total of 570 nanowires randomly selected from TEM images. To evaluate their functionality, the FNw were employed as a signal label for examining the specificity of the antibody-antigen interactions. As shown in Figure 3A, the testing principle


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incorporating the FNw was similar to that of an indirect ELISA approach. The pathogen proteins immobilized on the microarray were captured by the corresponding specific biotinylated antibodies. Followed by the addition of streptavidin and the FNw, the FNw were conjugated to the antibodies through the biotin-streptavidin-biotin interaction, thereby indicating the specific antigen-antibody interactions based on the fluorescence emission from the FNw. As a proof of concept, the negative control (BSA) and three antigens of HIV-1 gp120, HIV-1 p24 were immobilized on the blocks of the microarrays. Followed by the addition of four antibodies into the four blocks of the microarrays, respectively, and with subsequent addition of streptavidin and the FNw into all the four blocks, the specific antigen-antibody interactions can be verified based on the fluorescent signals observed from the four blocks. As shown in Figure 3B, no fluorescent signals were observed for the first block with the addition of anti-F1 antibody. While intense fluorescent signals were observed for the HIV-1 gp120-spotted microarrays in the second block and the HIV-1 p24-spotted microarrays in the third block, with the addition of anti-gp120 antibody and anti-p24 antibody, respectively. Similarly, obvious fluorescent signals were observed for the HA1-spotted microarrays in the fourth block with the addition of anti-HA1 antibody. These results indicate that the FNw-mediated protein microarrays exhibit good specificity towards pathogen proteins through the use of the corresponding specific antibodies, without any antibody cross-reactivity being evident. Moreover, with the addition of the four mixed antibodies into the block of the microarrays, fluorescent signals can be observed in parallel for the three pathogen proteins (gp120, p24, HA1) spotted microarrays (Figure 3C). The fluorescent signals decreased with decreasing antigen concentrations, and again no signals can be observed for the BSA-spotted microarrays. Based on the above results, we concluded that the


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proposed FNw-mediated protein microarray platform is highly suitable for the specific and multiplexed detection of pathogen proteins in a complex system.

Figure 3. (A) Schematic illustration of the functional evaluation of the FNw-mediated microarray based on an indirect ELISA approach. (B) The orthogonal experiment results of the FNw-mediated microarray: no fluorescent signals for the BSA-spotted microarrays, in contrast, apparent fluorescent signals for the HIV-1 gp120, HIV-1 p24 and HA1-spotted microarrays with the addition of anti-gp120 antibody, anti-p24 antibody and anti-HA1 antibody respectively, demonstrating the specific antigen-antibody interactions without any antibody cross-reactivity being evident. Each antigen or BSA per block included three replicate spots. FITC-labeled ConA served as a spatial orientation marker (red dots) (C) The addition of four mixed antibodies with different concentrations (from down to up: 1000, 100, 10, 1, 0.1 and 0 ng/mL for each antibody) into the block spotted by BSA, HIV-1 gp120, HIV-1 p24 and HA1, indicating the occurrence of fluorescent signals in parallel for the HIV-1 gp120, HIV-1 p24 and HA1-spotted microarrays and no fluorescent signals for the BSA-spotted microarrays. Furthermore, the FNw was employed to detect the multiple pathogen proteins using a sandwich ELISA mode. As shown in Figure 4A, the specific antibody for capturing the corresponding antigen was first immobilized on the microarray. Following the addition of the


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mixed antigens, streptavidin, FNw and the same antibody in biotinylated form, the antigen was captured by the specific antibodies through a sandwich format. The sandwich formation was then detected by the fluorescent signal emitted by the FNw, due to the FNw binding to the biotinylated antibody via the biotin-streptavidin-biotin interaction. Using this principle, three pathogen proteins including HA1, HIV-1 p24 and HIV-1 gp120, were specifically detected (Figure 4B). By using anti-HA1, anti-HIV-1 p24 and HIV-1 gp120 antibody, respectively, the corresponding pathogen proteins were detected based on the observation of strong fluorescent signals on the microarray. In sharp contrast, no fluorescent signal was observed for the control, demonstrating the specificity of the FNw-mediated protein microarray in multiplex pathogen proteins detection. Quantitative analysis clearly showed that the fluorescent signal gradually decreased with decreasing pathogen proteins concentrations (Figures 4B-C). As a result, a detection limit of 1 ng/mL for HA1 (18.2 fM), 0.1 ng/mL for HIV-1 p24 (4.1 fM) and 0.1 ng/mL for HIV-1 gp120 (1.9 fM) was achieved, which comprises a more than 100-fold improvement in sensitivity compared with the commercial reagent SA-Cy5 (Figures 4D-F), supporting the employment of FNw as an effective method for improving the sensitivity of protein microarrays for multiplexed antigens detection.


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Figure 4. Schematic illustrations of the FNw-mediated protein microarray (A, left) and the SACy5-mediated protein microarray (D, left) for the detection of pathogen proteins based on a sandwich ELISA method. Detection of the negative control (BSA, column 1) and the three pathogen proteins including p24 (column 2), gp120 (column 3), and HA1 (column 4) at different antigen concentrations (1000, 100, 10, 1, 0.1 and 0 ng/mL from top to bottom; A and D, right). Antigen detection based on the FNw-mediated protein microarray (B) and the SA-Cy5-mediated protein microarray (E), both of which contained five replicate spots of each antigen per block


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with a 10-fold dilution of the pathogen proteins for each subsequent block. The detection was carried out with a mixture of the three antigens and BSA. FITC-labeled ConA served as a spatial orientation marker on both microarray types (red dots). (C) and (F) are the corresponding histograms for (B) and (E), respectively, demonstrating the variation of fluorescent signals with the change in pathogen proteins concentrations. For early detection of emerging infectious diseases, there are substantial requirements for the specific screening of pathogens at ultra-low concentrations in complex biological samples. The FNw-mediated protein microarray described in this study shows significant potential for the highly sensitive and simultaneous detection of multiple pathogen proteins. Compared with the fluorescent nanomaterials previously employed in super- or ultra-sensitive protein microarrays such as quantum dots,24 carbon nanotubes25 and fluorescent dye-doped nanoparticles,26 the FNw offer several advantages. First, the FNw are prepared by the self-assembly of engineered fluorescent proteins into a nanowire that can transfer hundreds of fluorophores to an antibodyantigen complex, thereby leading to higher protein microarray sensitivity. Second, the preparation of FNw entails the use of very mild conditions during self-assembly and in vivo biological modification. This not only mitigates the fluorescence quenching experienced by other conventional nanomaterial-labeling technologies, but also enables the coupling of different functional ligands together in a controlled orientation rather than via random chemical crosslinking. The use of controlled coupling further enhances the signal capacity of the nanowire-based method compared with other approaches, as it fully enables the polyvalent effect during biomolecule conjugation and binding.27 Furthermore, the diversity of biological molecules that can be incorporated into the nanowires endows these detection molecules with the capacity for multi-functionalization. For example, the GFP could be replaced with other


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fluorescent proteins to produce protein nanowires with different color fluorescent emissions, while the BAP tag could be substituted with a single-chain variable fragment for binding to a specific target. Taking all of these advantages into account, protein nanowires offer a very promising approach with which to investigate a variety of different research questions simply by changing the functional ligands along the protein nanowire. 4. CONCLUSIONS We have fabricated an FNw-mediated protein microarray that successfully enabled the highly sensitive and multiplexed detection of pathogen proteins based on a sandwich ELISA format. Detection sensitivities of as low as 1 ng/mL for HA1 (18.2 fM), and 0.1 ng/mL for both HIV-1 p24 (4.1 fM) and HIV-1 gp120 (1.9 fM) were achieved, which constitutes a greater than 100-fold improvement compared with the commercially available detection reagent, SA-Cy5. The proposed FNw can be a new labeling reagent with protein microarray platform for detection of other target proteins.

ASSOCIATED CONTENT Supporting Information. Immobilization of three pathogens or antibodies on the block of the microarray. 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


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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 The authors declare no competing financial interest. ACKNOWLEDGMENTS D.M was supported by the National Natural Science Foundation of China (No. 31200755) and the Youth Innovation Promotion Association of CAS (No. 2014308). X.E.Z was supported by the Chinese Academy of Sciences Key project (KJZD-EW-TZ-L04). J.Z was supported by the National Natural Science Foundation of China (Grant No. 81301324). Y.L was supported by the National Basic Research Program 973 (No. 2012CB721001). In addition, we thank Z. Chen and J. J. Chen for offering the influenza pathogen proteins and the antibodies. We thank R. F. Yang for offering the anti-F1 antibody. We also thank Dr. D. Gao, Ms. B. C. Xu and Ms. P. Zhang in the core facility and technical support of Wuhan Institute of Virology, CAS for assistance with TEM imaging. REFERENCES 1.

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Fluorescent Protein Nanowire-Mediated Protein ... - ACS Publications

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