Zwitterion Hybrid ... - ACS Publications

Subscriber access provided by University of South Dakota

Biological and Environmental Phenomena at the Interface

Gold Nanoparticles with Ligand/Zwitterion Hybrid Layer for Individual Counting of Influenza A H1N1 Subtype Using Resistive Pulse Sensing Yukichi Horiguchi, Tatsuro Goda, Akira Matsumoto, Hiroaki Takeuchi, Shoji Yamaoka, and Yuji Miyahara Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b01586 • Publication Date (Web): 22 Aug 2018 Downloaded from http://pubs.acs.org on August 23, 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 32 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

Langmuir

Gold Nanoparticles with Ligand/Zwitterion Hybrid Layer for Individual Counting of Influenza A H1N1 Subtype Using Resistive Pulse Sensing Yukichi Horiguchi,1 Tatsuro Goda,1 Akira Matsumoto,1 Hiroaki Takeuchi, 2 Shoji Yamaoka,2 Yuji Miyahara1* 1

Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University (TMDU),

2-3-10 Kanda-Surugadai, Chiyoda, Tokyo 101-0062, Japan

2

Department of Molecular Virology, Graduate School of Medical and Dental Sciences, Tokyo

Medical and Dental University (TMDU), 1-5-45 Yushima, Bunkyo, Tokyo 113-8510, Japan

*Corresponding author: Yuji Miyahara, e-mail address: [email protected]

ABSTRACT Resistive pulse sensing (RPS) is an analytical technique for detecting particles with nano- to micrometer diameters, such as proteins, viruses, and bacteria. RPS is a promising tool for diagnosis as it can analyze the characteristics of target particles individually from ion current blockades as pulse waveforms. However, it is difficult to discriminate analog targets because

ACS Paragon Plus Environment

1

Langmuir 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

RPS merely provides physical information such as size, shape, concentration, and charge density of the analyte. Influenza A virus, which is 80–120 nm in diameter, has various subtypes, demonstrating the diversity of virus characteristics. For example, highly pathogenic avian influenza infections in humans are recognized as an emerging infectious disease with high mortality rates compared with human influenza viruses. Distinguishing human from avian influenza using their differing biological characteristics would be challenging using RPS. To develop a highly selective diagnostic system for infectious diseases, we combined RPS with molecular recognition. Gold nanoparticles (GNPs) that have human influenza A (H1N1 subtype) virus-specific sialic acid receptors on the surface were prepared as a virus label for RPS analysis. A sulfobetaine and sialic acid (ligand) hybrid surface was formed on the GNPs for the suppression of non-specific interaction. The results show a size change of viruses derived from specific interactions with GNPs. In contrast, no size shift was observed when non-specific sialic acid receptor-immobilized GNPs were used. Detection of viruses by individual particle counting could be a new facet of diagnosis.

INTRODUCTION Resistive pulse sensing (RPS) is an emerging technique for the measurement of small analytes within the nano- to micrometer size range.1-2 When target particles pass through a membrane pore, the transmembrane ion current changes due to the exclusion of ions and the change can be used to calculate the characteristics of the analyte. The advantage of RPS is that the various analyte particles are measured individually, rendering it an ultra-trace analytical method. For example,


2

Page 2 of 32

Page 3 of 32 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

Langmuir

the sequence information of a single nucleic acid can be obtained by pore sequencing using membrane transport proteins (direct, non-amplification process).3-4 Artificial nano- and micropore membranes have been also developed to detect various kinds of analyte such as nucleic acids,5 peptides,6 proteins,7-8 whole bacteria,9 whole viruses,10 and extracellular vesicles.11 In addition, RPS can precisely calculate the size distribution of polydisperse particles, which cannot be measured by dynamic light scattering (DLS), more rapidly than transmission electron microscopy (TEM) methods.12 RPS is useful for virus detection because viruses conform to specific sizes within individual species, for example, influenza A virus has a diameter of 80–120 nm,13 and picornaviruses such as enteroviruses have a diameter of 30 nm.14 If a virus such as influenza can be individually discriminated, sufficiently early diagnosis for prevention of epidemic diseases may become a reality. However, it is difficult to discriminate between analog targets such as human and avian influenza virus because RPS merely provides physical information such as size, shape, concentration, and charge density of the analyte. Recently, highly pathogenic avian influenza (HPAI) such as the H5N1 influenza A subtype has been recognized as an emerging epidemic disease with high mortality rates in humans,15-17 however the physical characteristics of this virus are almost identical to other less pathogenic subtypes of influenza A virus. It has been reported that interphyletic types of influenza virus that can infect both avians and humans can be created by mutation,18-19 and several kinds of H5N1 influenza A subtype have this potential.20 An adaptation of highly pathogenic avian influenza virus for humans may therefore be a threat to our


3

Langmuir 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

health.17 To apply the RPS system to influenza virus detection, a molecular recognition technique can be combined with RPS to obtain an additional layer of particle selectivity. In this report, we propose RPS with engineered virus-binding nanoparticles to improve specificity while retaining current sensitivity compared with conventional diagnostic techniques (Figure 1). Human and avian influenza viruses have different hemagglutinin (HA) proteins, responsible for sialic acid (N-acetylneuraminic acid, Neu5Ac) recognition, resulting in selective infection for each host (Figure 1a). For example, the HA of a human influenza A virus recognizes the Neu5Acα(2-6)Gal moiety on the human cell membrane, while avian influenza A viruses recognize the Neu5Acα(2-3)Gal moiety on the membrane of avian cells.19 We prepared Neu5Acα(2-6)Galβ(1-4)GlcNAc (6′SLN)-immobilized gold nanoparticles (GNPs) to investigate the sensing performance by specific binding to the influenza A H1N1 subtype (Figure 1b). However, specific interaction between a virus and GNP has the potential to be disturbed by non-specific interactions. For example, conventional label-free biosensing techniques such as quartz crystal microgravimetry (QCM) and surface plasmon resonance (SPR) are disturbed by non-specific adhesion of impurities in an analyte solution because they simply detect changes in physical quantities on the surface due to adsorbed molecules, including unwanted adsorption of impurities.21-22 If GNPs are dispersed in biological fluid, non-specific binding of impurities such as protein on the GNP surface will prevent specific recognition. To suppress non-specific interactions, a sulfobetaine-terminated molecule (sulfobetaine3-undecane thiol, SB-SH) was co-immobilized on the gold surface (Figure 1b). Zwitterion-containing molecules such as sulfobetaine, carboxybetaine, and phosphobetaine are known to prevent nonspecific


4

Page 4 of 32

Page 5 of 32 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

Langmuir

interactions,23-27 which is useful for the preparation of non-fouling surfaces. Figure 1c shows an illustration of RPS measurement after molecular recognition. If the virus is recognized by the GNPs, the virus particles are coated with GNPs prior to RPS measurement, which results in a change of blockade magnitude (Figure 1d). The sensing performance of this system was evaluated using whole human influenza A virus (H1N1 subtype, PR8 strain), cultivated in chicken embryos and MDCK cells.

EXPERIMENTAL SECTION Materials and equipment. 11-mercaptoundecanoic acid (MUA) and stabilized gold nanoparticle suspensions in citrate buffer (20 and 60 nm diameters) were purchased from Sigma-Aldrich Co. LLC St. Louis, MO, Neu5Acα(2-6)Galβ(1-4)GlcNAc-β-ethylamine

U.S.A. (6′-sialyl-N-acetyllactosamine-β-ethylamine,

6′SLN-β-ethylamine)

Neu5Acα(2-3)Galβ(1-4)GlcNAc-β-ethylamine

and

(3′-sialyl-N-acetyllactosamine-β-ethylamine,

3′SLN-β-ethylamine) were purchased from Tokyo Chemical Industry Co., Ltd., Tokyo, Japan. 4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride n-hydrate (DMT-MM) was purchased

from

Wako

Pure

Chemical

Industries,

Ltd.,

Osaka,

Japan.

N-(11-Mercaptoundecyl)-N,N-dimethyl-3-ammonio-1-propanesulfonate (Sulfobetaine3-undecanethiol, SB-SH) was purchased from Dojindo Laboratories, Kumamoto, Japan. These chemicals were used as received. Dialysis was carried out using Spectra/Por®


5

Langmuir 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

Dialysis Membrane (Biotech CE Tubing, MWCO: 3.5 kD and 12–14 kD, Spectrum Laboratories, Inc., Rancho Dominguez, CA). The size distribution of influenza A H1N1 in solution was measured using a qNano nanoparticle analyzer (Izon Science Ltd., Christchurch, New Zealand). CPC100B (genuine standard nanoparticle solution, mode size: 110 nm, mean size: 110 nm, raw concentration: 1.0 × 1013 particles/mL, Izon Science Ltd., Christchurch, New Zealand) was used as calibration for qNano. Human influenza A virus subtype H1N1 (A/PR/8/34) was cultivated in chicken embryos or Madin-Darby canine kidney (MDCK) cells. The collected virus solutions were detoxified using 0.05% formaldehyde solution. The HA titer of the obtained influenza A virus subtype H1N1 solution was confirmed using a hemagglutination assay. The original titers of the virus solutions cultivated in chicken embryos and MDCK cells were 256 and 64 HAU, respectively (Figure S1 and S2). The absorption spectra were measured using UV-VIS Spectrophotometer V-670 (JASCO Corporation, Tokyo, Japan)

Preparation of MUA and SB-SH co-immobilized GNP (MUA/SB-GNP). Twenty milliliters of citrate-stabilized GNP (20 nm, 1 OD, 6.5 × 1011 particles/mL) was mixed with 1 mL of 2 mM SB-SH solution in 0.1 M NaOH and 1 mL of 2 mM MUA solution in 0.1 M NaOH (the final concentration of thiol molecules was 192 µM) and the mixture was stirred for 48 h. The solution was dialyzed three times against 500 mL of pure water to remove citrate and excess free thiol molecules (MWCO: 3.5 kD, 3 h, 3 h, and 15 h, respectively). The obtained solution was concentrated to 2 mL using ultrafiltration and stored at 4°C. As a control experiment, SB-GNPs were prepared using the same procedure without MUA.


6

Page 6 of 32

Page 7 of 32 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

Langmuir

Preparation of sialic acid immobilized GNP (6′SLN/SB-GNP). One milliliter of MUA/SB-GNP solution was mixed with 0.25 mL of 8 mM 6′SLN-β-ethylamine solution. Two hundred and fifty microliters of 8 mM DMT-MM solution was then added and the solution was stirred for 3 h at RT. After the condensation reaction, the solution was stored at 4°C overnight. The solutions were dialyzed against 500 mL of pure water to remove excess 6′SLN-β-ethylamine and byproducts (MWCO: 12–14 kD, 3 h, 3 h and 15 h, respectively) and the obtained 6′SLN/SB-GNPs were stored at 4°C. As a control experiment, 3′SLN/SB-GNPs were prepared using the same procedure with 3′SLN-β-ethylamine.

RPS measurement of Influenza A virus subtype H1N1 (cultivated in chicken embryos) with 6′SLN/SB-GNP Viruses and 6′SLN/SB-GNPs were mixed dispersed in 1/3 PBS solution. 45 µL of each solution were mixed together then stirred using vortex mixer for 1 min. After a few minutes, the particle size was measured by qNano. The final concentrations of virus and 6′SLN/SB-GNP were 8.6×108 and 4.3×1011 particles/mL, respectively (1 HAU virus solution). A pore membrane (NP100) was placed on a fluid cell of qNano then ion current was confirmed by 1/3 PBS solution. After the conduction of the device, 1/3 PBS solution on the fluid cell was replaced with virus-GNP mixed solution. The applied pressure, stretch and voltage were 1 kPa, 47.01 mm and 1.5 V, respectively. For the calculation of particle size, genuine standard nanoparticle solution (CPC100B, mode size: 110 nm, mean size: 110 nm, raw concentration: 1.1 × 1013 particles/mL) was measured for


7

Langmuir 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

calibration. RPS measurements of viruses with SB-GNPs and viruses with 3′SLN/SB-GNPs were also performed under the same conditions as control experiments.

RPS measurement of Influenza A virus subtype H1N1 (cultivated in MDCK cells) with 6′SLN/SB-GNP The measurement procedure using qNano was same as describe above. The final concentrations of virus and 6′SLN-GNP were 7.7 × 108 and 1.9 × 1011 particles/mL, respectively (1 HAU virus solution). RPS measurement of viruses with 3′SLN/SB-GNPs was also performed under the same conditions as a control experiment.

RESULTS AND DISCUSSION Confirmation of GNP surface modification. A schematic illustration of GNP surface modification is shown in Figure 2. Surface immobilization using the gold-sulfur bond is a well known technique for tethering molecules because of the interaction strength (47 kcal/mol).28 In addition, alkanethiol molecules form a self-assembled monolayer (SAM) on gold surfaces, which contributes to the immobilization stability. In the case of citrate-stabilized GNPs, the adsorbed citrate molecules are exchanged gradually with thiol molecules after mixing (Figure 3a). Figure 1a shows the extinction spectra of GNPs before and after molecular exchange. The initial GNPs showed a surface plasmon peak at


8

Page 8 of 32

Page 9 of 32 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

Langmuir

522 nm, while the peak shifted towards the infrared region after the immobilization of thiol molecules as a result of the reflective index change at the gold surface due to the SAM layer. Furthermore, the extinction in the ultraviolet region increased after the immobilization of the thiol molecules. This change is attributed to the absorption of MUA and SB-SH molecules (Figure S3 and S4). The increase in particle size introduced by the SAM layer may also contribute to the extinction increases because the scattering intensity of the particle increases multiplicatively with size. Based on these findings, we concluded that thiol molecules were successfully immobilized on the gold surfaces. Absorption spectra are useful for evaluating the dispersion of metal nanoparticles. When the surface exchange proceeded in 91 µM thiol solution, a different peak derived from plasmon coupling appeared as a shoulder (Figure 3a, blue line, red arrow), which indicates that the GNPs were partially aggregated in solution. The plasmon coupling of GNPs are conventional technique to check the aggregation of GNPs in the solution. When monodispersed GNPs form dimer, a different peak appears in the infrared side.29 This technique is widely used for colorimetric biosensor.30 Although the Au–S bond is stronger than physical adsorption of citrate, citrate molecules also bind strongly to gold surfaces as a citrate layer.31 The insufficient exchange reaction might lead to the aggregation of GNPs after dialysis. To increase the efficacy of the surface exchange reaction, the same reaction was carried out in 182 µM thiol solution. The absorption spectrum showed no plasmon coupling peak (Figure 3a, green line), indicating that the GNPs were monodisperse. Based on this result we established the sialic acid immobilization


9

Langmuir 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 10 of 32

procedure. 6′SLN-β-ethylamine was conjugated to the –COOH surface using DMT-MM, a well-known coupling agent. Figure 3b shows the absorption spectra of GNPs before and after sialic acid immobilization. The plasmon peak of the modified GNPs was almost the same, and the extinction in the ultraviolet region increased (Figure 3b, red line). This extinction increase results from 6′SLN-β-ethylamine because it has absorption in the ultraviolet region (Figure S5). The adsorption

spectrum

was

measured

after

dialysis

to

completely

remove

the

free

6′SLN-β-ethylamine and the byproducts of the condensation reaction, indicating that sialic acid was tethered to the GNP surface via MUA molecules.

Analysis of virus particles using RPS. Virus particles were studied using a standard RPS analysis. The depth of the current change (blockade of ion current) and time taken to traverse the pore membrane (duration time) were measured for each particle to analyze characteristics such as size, shape, and charge density (Figure 4a). For example, the depth of the current change can be attributed to particle size because the volume of ion exclusion depends on the size of the particle. In this study, all of the RPS measurements were performed using qNano, known as a commercially available particle analyze system. The qNano system is called tunable RPS (TRPS), because a pore membrane for RPS measurement is made of polyurethane, which can be controlled a pore size by mechanical stretching.32 TRPS system can also calculate the size distribution of the particles quickly and precisely like a TEM image,12, 33 and the zeta potential of the particles can be also measured


10

Page 11 of 32 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

Langmuir

individually.34-36 For the above reasons, qNano is widely used for the analysis of biological nanoparticles.12, 37-38 An important feature of this product is that the pore shape is conical and the size of pores are not defined precisely, because pores are prepared by penetration using tungsten needle.39 Therefore, size standard particles should be also measured as calibration data to calculate size of target particles. The case of this study, genuine standard nanoparticle solution (CPC100B, mode size: 110 nm, mean size: 110 nm, raw concentration: 1.0 × 1013) was measured for calibration. The obtained plot data (blockade magnitude and duration time) then converts this to a histogram. Figure 4b and 4c show the results for influenza A H1N1 subtype cultivated in chicken embryos and MDCK cells. One dot represents one virus particle and the sum of the plots was converted to histograms. Both peak sizes were ~100 nm, which is in agreement with a previous report.13 The case of the virus cultivated in chicken embryo, the size distribution width is wide (Figure 4b), which is not agree with the size range of 80−120 nm.13 On the other hand, previous reports including TEM images shows that virus particles have various shape, and some virus particles were larger than 120 nm.40-41 Therefore, the size range of influenza virus is not determined precisely even though the majority of the size range is 80−120 nm. In contrast, the size distribution of virus cultivated in MDCK cell is narrow compare with the case of chicken embryo. According to these result, the size range of obtained influenza virus depends on an environmental condition for cultivation. RPS measurement is therefore a useful technique for precise measurement of particle characteristics.


11

Langmuir 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

1 HAU virus samples were used for the measurements. Virus titers such as HAU do not represent the physical quantity of viruses present, but are a significant aid in clinical situations for understanding the infectivity of the virus solution. RPS is also a helpful technique for converting virus titers into physical quantities.

RPS measurements of influenza A H1N1 subtype cultivated in chicken embryos with GNPs After evaluation of the obtained GNPs and viruses, molecular recognition experiments between the virus and GNPs were carried out. Figure 5a shows the distribution histogram of viruses using RPS measurements. The mixture of virus and sulfobetaine-covered GNP (SB-GNP) showed no change in size distribution compared with the original virus solution (Figure 5a, green). The obtained plot data of “Virus + SB-GNP” (Figure 5b) is also almost same as that of “Virus only” (Figure 4b), because SB-GNPs have no ligand for the virus. By comparison, specific sialic acid and SB co-immobilized GNPs (6′SLN/SB-GNPs) were attached to virus surfaces and a size distribution shift was observed (Figure 5a and Figure 5c, purple). As a control experiment, the measurement of viruses with non-specific sialic acid and SB co-immobilized GNPs (3′SLN/SB-GNPs) was also carried out and no distribution shift was observed (Figure 5a and Figure 5d, turquoise). The merged plot data of Figure 5c and 5d can understand visually that whole of the virus plot was shifted to large side by the molecular recognition (Figure 5e). In our previous reports, densely immobilized Neu5Acα(2-6)Gal- surface can detect Influenza A virus subtype H1N1 selectively.21 It has been reported that the result of the dissociation constant (KD) between recombinant hemagglutinin derived from influenza A virus and sialic acid receptor was


12

Page 12 of 32

Page 13 of 32 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

Langmuir

6.10 ×10-9 M.42 The multipoint binding may also improve the affinity stronger. For above reasons, the size shift derived from the molecular recognition was observed. The important point is that molecular recognition was not observed without SB co-immobilized at the interface. In the preliminary experiment, 6′SLN-GNPs and 3′SLN-GNPs were added to virus solutions and both size distributions shifted (Figure S6). As mentioned previously, the collected virus solution contains significant levels of impurities. When the GNP and virus solutions were mixed together, the impurities may have bound to the GNPs leading to loss of dispersion stability, inducing the non-specific interaction between GNPs and viruses. On the other hand, this non-specific interaction was not observed when 3′SLN/SB-GNP was used (Figure 5). This difference indicated that SB-SH molecule contributes the suppression of non-specific interaction. The role of the ligand/zwitterion hybrid surface on the GNPs is therefore to maintain the dispersion stability to monitor the specific interactions.

RPS measurements of influenza A H1N1 subtype cultivated in MDCK cells with GNPs In the previous section, molecular recognition between 6′SLN/SB-GNP and influenza A H1N1 subtype cultivated in chicken embryos was measured by RPS. Cultivation of influenza virus using chicken embryos is the most conventional technique for clinical use, such as in vaccine production. However, it has been reported that cultivation of human influenza virus in egg cells has the potential to induce an influenza virus mutation called “egg adaptation”. If an egg adaptation occurs, the human influenza virus is able to bind to an avian cell surface.43 The appearance of interphyletic influenza virus in other host animals has been reported, and it is


13

Langmuir 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

understood that an adaptation of highly pathogenic avian influenza virus for human cells may be a threat to our health.17 Our experimental results showed that egg adaptation had not occurred because no interaction between the virus and 3′SLN/SB-GNPs was observed. However, the same experiment using influenza A H1N1 subtype cultivated in MDCK cells was also performed to confirm the specific interactions. According to a previous report, adaptation does not occur for viruses cultivated in MDCK cells.43 Figure 6 shows the result of RPS measurement. The size distribution of the influenza virus shifted after mixing with 6′SLN/SB-GNPs (Figure 6a and 6b, red), in contrast, the significant distribution shift was not observed in case of 3′SLN/SB-GNPs (Figure 6a and 6b, blue). To understand the specific binding of the GNPs on the virus visually, the merged plot data of Figure 6b and 6c are shown in Figure 6d. The distribution change by the specific interaction of GNP was clearer than that in case of the virus derived from chicken embryo, which may be a result of the different conditions, such as host cells. Based on these results we concluded that the virus size distribution shift was a result of molecular recognition. As a reference experiment, RPS measurement was also carried out using a different size of GNPs. 6′SLN/SB-GNPs were prepared using GNPs with 60 nm diameters, and were then applied to virus recognition experiments. If particle size increases, the blockade of the ion current will also increase because the number of excluded ions depends on particle volume. However, results showed that the size of the shift was similar to that for the 20 nm GNPs even though the diameter of the GNPs was tripled (Figure 6a and 6e, green). This observation can be explained by the binding efficacy of the GNPs with a virus. If the size of the GNPs is increased, the number of


14

Page 14 of 32

Page 15 of 32 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

Langmuir

binding sites on a virus for the large bulky particles will decrease. As a result, a conjugate of a virus and large GNPs has numerous gaps compared with conjugates with small GNPs where packing is more efficient. In addition, over 20 times more Au atoms were required as a result of the larger particle size. It is therefore not reasonable to use large particles for molecular recognition from the point of view of both binding efficacy and cost. Note that the unattached free GNPs were not counted by RPS measurement, because the both 20 and 60 nm GNPs were out of the range from the RPS measurements. To understand this, typical detection signals of 100 and 80 nm virus were shown in Figure S13. The case of a virus with 100 nm in diameter, the obtained signal is observed clearly (Figure S13a). On the other hand, a signal of virus with 80 nm in diameter is slightly higher than the baseline noise (Figure S13b), and the height of this signal is almost half compared with the case of 100 nm. This result can be explained the volume ratio of each particle, because magnitude of a signal depends on an exclusion of ions. For example, the volume ratio of 100, 80 and 60 nm particle is 1.000 : 0.512 : 0.216. Therefore, the signals of small GNPs were buried under the baseline noise. 1 HAU virus solution was used for the detection of viruses and the concentration of virus particles was sufficient to obtain a histogram to illustrate the specific interactions from the distribution shift. In a previous report, avian or human influenza could be positively distinguished from human influenza using a conventional immunochromatographic test (ICT) at >32 HAU.20 For ICTs, large amounts of viruses are required because trapped and labeled viruses must be determined by the naked eye. In contrast, RPS can monitor virus particles by individual counting, which allows for the precise detection of trace amounts of viruses.


15

Langmuir 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

One of a breakthrough in this study is that the diversity of the influenza virus affects the results of molecular recognition. According to the results of Figure 5 and 6, the size distribution shift of viruses cultivated in chicken embryo was ~20 nm, while the shift of viruses cultivated in MDCK cells was ~40 nm. For the molecular recognition experiment, high concentration of GNPs solution was applied to virus particles in order to cover virus surfaces completely. The numerical ratio of virus and GNP was about 1 : 500 for viruses cultivated in chicken embryo and 1 : 250 for viruses cultivated in MDCK cells, respectively. Thus, the difference of the shift amount is not derived from the insufficient number of GNPs. One of a possibility is that this difference may be derived from the replication condition in host cells, because the replicated RNA and proteins were combined then dissociated with cell membrane as envelope.44 Namely, the viral characteristics such as density of viral protein depends on an environment for replication. It is also reported that influenza hemagglutinin sometimes shows mutations such as antigenic shift and antigenic drift at a replication process,45 which may change the affinity strength between the hemagglutinin and sialic acid receptor. Thus, the characteristics of the replicated virus are not constant even though the subtype of influenza A virus is the same. From above the reasons, it is difficult to calculate an expected shift amount for the quantitative analysis. On the other hand, all of the influenza A virus H1N1 subtype have more or less hemagglutinins for sialic acid receptor. Therefore, a counted particle rate of a shifted group can be used for quantitative analysis in principle, because there is a correlation between counted particle rate and concentration.12 One of a problem is clogging of a pore when a sample solution


16

Page 16 of 32

Page 17 of 32 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

Langmuir

was measured, because biological fluid contains proteins and some other impurities. If a pore clogging were happened, a passing particle rate will change result in a shift from a correct value. A serious clogging pore sometimes interrupt a measurement. The development of a devices for RPS measurement will contribute to a precision for quantitative analysis. In this study, several hundreds of virus particles were measured to obtain histograms for the determination of specific interactions, however, RPS is a continually developing technology. It has been reported that ultrathin nano-sized pores can read particle shape like a scanner.46 If the conjugation of viruses and GNPs can be read by this technology, in future it would be possible to detect the specific interaction by counting only a few conjugates. RPS measurement combined with a molecular recognition technique is therefore a promising way of improving the performance of biosensing.

CONCLUSIONS We have demonstrated RPS measurement of influenza A H1N1 subtype particles using specific sialic acid-engineered GNPs for virus labeling. The ligand/zwitterion hybrid interface of the GNPs plays an important role in suppressing the unwanted conjugation of GNPs to effectively detect specific interactions. The advantage of RPS measurement is that trace amounts of virus can be monitored, which could contribute to an improvement in sensitivity. This technique will be an appropriate tool for future developments in diagnosis.


17

Langmuir 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

ASSOCIATED CONTENT The Supporting information is available free of charge on the ACS Publication website at DOI: (XXXXXXXXX). Supporting methods Supplementary Figures S1–S13 (PDF)

AUTHOR INFORMATION Corresponding Author *E-mail (Y. Miyahara): [email protected] ORCID Yukichi Horiguchi: 0000-0002-6748-9643 Tatsuro Goda: 0000-0003-2688-8186 Notes The authors declare no competing financial interests.

ACKNOWLEDGMENTS This work was supported by the Impulsing Paradigm Change through Disruptive Technologies (ImPACT) Miyata Program on the “Ultra high-speed multiplexed sensing system beyond evolution for the detection of extremely small amounts of substances” (launched 2014, funded by the Council for Science, Technology and Innovation, headed by the Prime Minister of Japan)


18

Page 18 of 32

Page 19 of 32 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

Langmuir

from the Japan Science and Technology (JST) Agency. We thank Sarah Dodds, PhD, from Edanz Group (www.edanzediting.com/ac) for editing a draft of this manuscript.


19

Langmuir 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 CAPTIONS Figure 1. Conceptual rendering of H1N1 virus detection using the resistive pulse sensing system. (a) Schematic illustration of human influenza A virus. The virus was detoxified using 0.05% formalin solution. (b) The design of H1N1 influenza virus-recognizable gold nanoparticles (6′SLN/SB-GNP). Neu5Acα(2-6)Galβ(1-4)GlcNAc-β-ethylamine (6′SLN-β-ethylamine), the specific binding receptor for human influenza virus H1 hemagglutinin, was immobilized on 11-mercaptoundecaonic acid immobilized GNPs via a condensation reaction. (c) Illustration of virus detection using resistive pulse sensing. The H1N1 virus was measured after binding with 6′SLN/SB-GNP. (d) Prospective result using this system. If a specific interaction occurred between 6′SLN/SB-GNPs and H1N1 viruses, the formation of conjugates would result in a size distribution shift, allowing for detection of influenza virus.

Figure 2. Schematic illustration of GNP surface modification. SB-SH and MUA were co-immobilized via the Au–S bond, then 6′SLN-β-ethylamine was conjugated to the –COOH groups using a coupling agent. Figure 3. (a) Extinction spectra of initial citrate stabilized GNPs (black) and GNPs following the co-immobilization of SB-SH and MUA in 91 (blue) or 182 (green) µM thiol solution (MUA/SB-GNPs). Extinction spectra were measured after dialysis to remove the unbound free thiol molecules. (b) Extinction spectra of MUA/SB-GNPs (green) and 6′SLN/SB-GNPs (red). Extinction spectra were measured after dialysis to remove the unbound free 6′SLN-β-ethylamine and byproducts.

Figure 4. RPS measurement of virus solutions. (a) An illustration of typical pulses obtained by RPS measurement. Blockade magnitude represents the size of a particle, and duration time represents the total time for its passage through the pore membrane. (b) The result for influenza A H1N1 cultivated in chicken embryos (1 HAU, 8.6 × 108 particles/mL). (c) The measurement data for influenza A H1N1 cultivated in MDCK cells (1 HAU, 7.7 × 108 particles/mL).


20

Page 20 of 32

Page 21 of 32 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

Langmuir

Figure 5. (a) Size histograms for influenza A H1N1 cultivated in chicken embryos (orange: virus only (n=984), green: viruses with SB-GNPs (n=994), purple: viruses with 6′SLN/SB-GNPs (n=698), turquoise: viruses with 3′SLN/SB-GNPs (n=564)). The vertical scales of these histograms (population (%)) are normalized by mode peak. (b) – (d) Individual virus plot data of “Virus + SB-GNP”, “Virus + 6′SLN/SB-GNPs” and “Virus + 3′SLN/SB-GNPs”. (e) Merged plot data of (c) and (d). The whole of RPS measurement results were shown in Figure S7-S9.

Figure 6. Size histograms for influenza A H1N1 cultivated in MDCK cells (brown: virus only (n=1294), red: viruses with 6′SLN/SB-GNPs (20 nm, n=634), blue: viruses with 3′SLN/SB-GNPs (20 nm, n=655), green: viruses with 6′SLN/SB-GNPs (60 nm, n=574). The vertical scales of these histograms (population (%)) are normalized by mode peak. (b) – (d) Individual virus plot data of “Virus + 6′SLN/SB-GNP (20 nm)”, “Virus + 6′SLN/SB-GNP (20 nm)” and “Virus + 6′SLN/SB-GNP (60 nm)”. (e) Merged plot data of (c) and (d). The whole of RPS measurement results were shown in Figure S10-S12.

REFERENCES (1)

Bayley, H.; Martin, C. R., Resistive-Pulse SensingFrom Microbes to Molecules. Chem.

Rev. 2000, 100, 2575-2594. (2)

Bayley, H.; Braha, O.; Gu, L. Q., Stochastic Sensing with Protein Pores. Adv. Mater.

2000, 12, 139-142. (3)

Cherf, G. M.; Lieberman, K. R.; Rashid, H.; Lam, C. E.; Karplus, K.; Akeson, M.,

Automated forward and reverse ratcheting of DNA in a nanopore at 5-A precision. Nat Biotech 2012, 30, 344-348. (4)

Manrao, E. A.; Derrington, I. M.; Laszlo, A. H.; Langford, K. W.; Hopper, M. K.;

Gillgren, N.; Pavlenok, M.; Niederweis, M.; Gundlach, J. H., Reading DNA at single-nucleotide resolution with a mutant MspA nanopore and phi29 DNA polymerase. Nat Biotech 2012, 30, 349-353. (5)

Harrell, C. C.; Choi, Y.; Horne, L. P.; Baker, L. A.; Siwy, Z. S.; Martin, C. R.,

Resistive-Pulse DNA Detection with a Conical Nanopore Sensor. Langmuir 2006, 22, 10837-10843.


21

Langmuir 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

(6)

Yusko, E. C.; Johnson, J. M.; Majd, S.; Prangkio, P.; Rollings, R. C.; Li, J.; Yang, J.;

Mayer, M., Controlling protein translocation through nanopores with bio-inspired fluid walls. Nat Nano 2011, 6, 253-260. (7)

Wharton, J. E.; Jin, P.; Sexton, L. T.; Horne, L. P.; Sherrill, S. A.; Mino, W. K.; Martin,

C. R., A Method for Reproducibly Preparing Synthetic Nanopores for Resistive-Pulse Biosensors. Small 2007, 3, 1424-1430. (8)

Takakura, T.; Yanagi, I.; Goto, Y.; Ishige, Y.; Kohara, Y., Single-molecule detection of

proteins with antigen-antibody interaction using resistive-pulse sensing of submicron latex particles. Appl. Phys. Lett. 2016, 108, 123701. (9)

Yu, A. C. S.; Loo, J. F. C.; Yu, S.; Kong, S. K.; Chan, T.-F., Monitoring bacterial

growth using tunable resistive pulse sensing with a pore-based technique. Appl. Microbiol. Biotechnol. 2014, 98, 855-862. (10)

Uram, J. D.; Ke, K.; Hunt, A. J.; Mayer, M., Submicrometer Pore-Based

Characterization and Quantification of Antibody–Virus Interactions. Small 2006, 2, 967-972. (11)

Böing, A. N.; van der Pol, E.; Grootemaat, A. E.; Coumans, F. A. W.; Sturk, A.;

Nieuwland, R., Single-step isolation of extracellular vesicles by size-exclusion chromatography. J. Extracell. Vesicles 2014. (12)

Akpinar, F.; Yin, J., Characterization of vesicular stomatitis virus populations by

tunable resistive pulse sensing. J. Virol. Methods 2015, 218, 71-76. (13)

Stanley, W. M., The Size of Influenza Virus. The Journal of Experimental Medicine

1944, 79, 267-283. (14)

Smyth, M. S.; Martin, J. H., Picornavirus uncoating. Molecular Pathology 2002, 55,

214-219. (15)

Yuen, K. Y.; Chan, P. K. S.; Peiris, M.; Tsang, D. N. C.; Que, T. L.; Shortridge, K. F.;

Cheung, P. T.; To, W. K.; Ho, E. T. F.; Sung, R.; Cheng, A. F. B., Clinical features and rapid viral diagnosis of human disease associated with avian influenza A H5N1 virus. The Lancet 1998, 351, 467-471. (16)

Subbarao, K.; Klimov, A.; Katz, J.; Regnery, H.; Lim, W.; Hall, H.; Perdue, M.;

Swayne, D.; Bender, C.; Huang, J.; Hemphill, M.; Rowe, T.; Shaw, M.; Xu, X.; Fukuda, K.; Cox, N., Characterization of an Avian Influenza A (H5N1) Virus Isolated from a Child with a Fatal Respiratory Illness. Science 1998, 279, 393-396.


22

Page 22 of 32

Page 23 of 32 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

Langmuir

(17)

Peiris, J. S. M.; de Jong, M. D.; Guan, Y., Avian Influenza Virus (H5N1): a Threat to

Human Health. Clin. Microbiol. Rev. 2007, 20, 243-267. (18)

Yamada, S.; Suzuki, Y.; Suzuki, T.; Le, M. Q.; Nidom, C. A.; Sakai-Tagawa, Y.;

Muramoto, Y.; Ito, M.; Kiso, M.; Horimoto, T.; Shinya, K.; Sawada, T.; Kiso, M.; Usui, T.; Murata, T.; Lin, Y.; Hay, A.; Haire, L. F.; Stevens, D. J.; Russell, R. J.; Gamblin, S. J.; Skehel, J. J.; Kawaoka, Y., Haemagglutinin mutations responsible for the binding of H5N1 influenza A viruses to human-type receptors. Nature 2006, 444, 378-382. (19)

Sawada, T.; Fedorov, D. G.; Kitaura, K., Role of the Key Mutation in the Selective

Binding of Avian and Human Influenza Hemagglutinin to Sialosides Revealed by Quantum-Mechanical Calculations. J. Am. Chem. Soc. 2010, 132, 16862-16872. (20)

Watanabe, Y.; Ito, T.; Ibrahim, M. S.; Arai, Y.; Hotta, K.; Phuong, H. V. M.; Hang, N.

L. K.; Mai, L. Q.; Soda, K.; Yamaoka, M.; Poetranto, E. D.; Wulandari, L.; Hiramatsu, H.; Daidoji, T.; Kubota-Koketsu, R.; Sriwilaijaroen, N.; Nakaya, T.; Okuno, Y.; Takahashi, T.; Suzuki, T.; Ito, T.; Hotta, H.; Yamashiro, T.; Hayashi, T.; Morita, K.; Ikuta, K.; Suzuki, Y., A novel immunochromatographic system for easy-to-use detection of group 1 avian influenza viruses with acquired human-type receptor binding specificity. Biosens. Bioelectron. 2015, 65, 211-219. (21)

Horiguchi, Y.; Goda, T.; Matsumoto, A.; Takeuchi, H.; Yamaoka, S.; Miyahara, Y.,

Direct and label-free influenza virus detection based on multisite binding to sialic acid receptors. Biosens. Bioelectron. 2017, 92, 234-240. (22)

Hai, W.; Goda, T.; Takeuchi, H.; Yamaoka, S.; Horiguchi, Y.; Matsumoto, A.;

Miyahara, Y., Specific Recognition of Human Influenza Virus with PEDOT Bearing Sialic Acid-Terminated Trisaccharides. ACS Appl. Mater. Interfaces 2017, 9, 14162–14170. (23)

Holmlin, R. E.; Chen, X.; Chapman, R. G.; Takayama, S.; Whitesides, G. M.,

Zwitterionic SAMs that Resist Nonspecific Adsorption of Protein from Aqueous Buffer. Langmuir 2001, 17, 2841-2850. (24)

Yuan, J.; Mao, C.; Zhou, J.; Shen, J.; Lin, S. C.; Zhu, W.; Fang, J. L., Chemical

grafting of sulfobetaine onto poly(ether urethane) surface for improving blood compatibility. Polym. Int. 2003, 52, 1869-1875. (25)

Zhang, Z.; Zhang, M.; Chen, S.; Horbett, T. A.; Ratner, B. D.; Jiang, S., Blood

compatibility of surfaces with superlow protein adsorption. Biomaterials 2008, 29, 4285-4291.


23

Langmuir 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

(26)

Chang, Y.; Shu, S.-H.; Shih, Y.-J.; Chu, C.-W.; Ruaan, R.-C.; Chen, W.-Y.,

Hemocompatible Mixed-Charge Copolymer Brushes of Pseudozwitterionic Surfaces Resistant to Nonspecific Plasma Protein Fouling. Langmuir 2010, 26, 3522-3530. (27)

Kitano, H.; Suzuki, H.; Matsuura, K.; Ohno, K., Molecular Recognition at the Exterior

Surface of a Zwitterionic Telomer Brush. Langmuir 2010, 26, 6767-6774. (28)

Felice, R. D.; Selloni, A., Adsorption modes of cysteine on Au(111): Thiolate,

amino-thiolate, disulfide. J. Chem. Phys. 2004, 120, 4906-4914. (29)

Sönnichsen, C.; Reinhard, B. M.; Liphardt, J.; Alivisatos, A. P., A molecular ruler

based on plasmon coupling of single gold and silver nanoparticles. Nat. Biotechnol. 2005, 23, 741. (30)

Rosi, N. L.; Mirkin, C. A., Nanostructures in Biodiagnostics. Chem. Rev. 2005, 105,

1547-1562. (31)

Park, J.-W.; Shumaker-Parry, J. S., Structural Study of Citrate Layers on Gold

Nanoparticles: Role of Intermolecular Interactions in Stabilizing Nanoparticles. J. Am. Chem. Soc. 2014, 136, 1907-1921. (32)

Kozak, D.; Anderson, W.; Grevett, M.; Trau, M., Modeling Elastic Pore Sensors for

Quantitative Single Particle Sizing. J. Phys. Chem. C 2012, 116, 8554-8561. (33)

Anderson, W.; Kozak, D.; Coleman, V. A.; Jämting, Å. K.; Trau, M., A comparative

study of submicron particle sizing platforms: Accuracy, precision and resolution analysis of polydisperse particle size distributions. J. Colloid Interface Sci. 2013, 405, 322-330. (34)

Kozak, D.; Anderson, W.; Vogel, R.; Chen, S.; Antaw, F.; Trau, M., Simultaneous Size

and ζ-Potential Measurements of Individual Nanoparticles in Dispersion Using Size-Tunable Pore Sensors. ACS Nano 2012, 6, 6990-6997. (35)

Weatherall, E.; Willmott, G. R., Applications of tunable resistive pulse sensing. Analyst

2015, 140, 3318-3334. (36)

Willmott, G. R., Tunable Resistive Pulse Sensing: Better Size and Charge

Measurements for Submicrometer Colloids. Anal. Chem. 2018, 90, 2987-2995. (37)

Vogel, R.; Willmott, G.; Kozak, D.; Roberts, G. S.; Anderson, W.; Groenewegen, L.;

Glossop, B.; Barnett, A.; Turner, A.; Trau, M., Quantitative Sizing of Nano/Microparticles with a Tunable Elastomeric Pore Sensor. Anal. Chem. 2011, 83, 3499-3506.


24

Page 24 of 32

Page 25 of 32 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

Langmuir

(38)

Blundell, E. L. C. J.; Mayne, L. J.; Billinge, E. R.; Platt, M., Emergence of tunable

resistive pulse sensing as a biosensor. Analytical Methods 2015, 7, 7055-7066. (39)

Sowerby, S. J.; Broom, M. F.; Petersen, G. B., Dynamically resizable nanometre-scale

apertures for molecular sensing. Sensors Actuators B: Chem. 2007, 123, 325-330. (40)

Ruigrok, R. W.; Wrigley, N. G.; Calder, L. J.; Cusack, S.; Wharton, S. A.; Brown, E.

B.; Skehel, J. J., Electron microscopy of the low pH structure of influenza virus haemagglutinin. EMBO J. 1986, 5, 41-49. (41)

Fujiyoshi, Y.; Kume, N. P.; Sakata, K.; Sato, S. B., Fine structure of influenza A virus

observed by electron cryo-microscopy. EMBO J. 1994, 13, 318-326. (42)

Takahashi, T.; Kawagishi, S.; Masuda, M.; Suzuki, T., Binding kinetics of sulfatide

with influenza A virus hemagglutinin. Glycoconjugate J. 2013, 30, 709-716. (43)

Gambaryan, A. S.; Tuzikov, A. B.; Piskarev, V. E.; Yamnikova, S. S.; Lvov, D. K.;

Robertson, J. S.; Bovin, N. V.; Matrosovich, M. N., Specification of Receptor-Binding Phenotypes

of

Influenza

Virus

Isolates

from

Different

Hosts

Using

Synthetic

Sialylglycopolymers: Non-Egg-Adapted Human H1 and H3 Influenza A and Influenza B Viruses Share a Common High Binding Affinity for 6′-Sialyl(N-acetyllactosamine). Virology 1997, 232, 345-350. (44)

Neumann, G.; Noda, T.; Kawaoka, Y., Emergence and pandemic potential of

swine-origin H1N1 influenza virus. Nature 2009, 459, 931. (45)

Carrat, F.; Flahault, A., Influenza vaccine: The challenge of antigenic drift. Vaccine

2007, 25, 6852-6862. (46)

Sou, R.; Makusu, T.; Yuhui, H.; Kazumichi, Y.; Akihide, A.; Takanori, M.; Masateru,

T.; Tomoji, K., Rapid structural analysis of nanomaterials in aqueous solutions. Nanotechnology 2017, 28, 155501.


25

Langmuir 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. Conceptual rendering of H1N1 virus detection using the resistive pulse sensing system. (a) Schematic illustration of human influenza A virus. The virus was detoxified using 0.05% formalin solution. (b) The design of H1N1 influenza virus-recognizable gold nanoparticles (6′SLN/SB-GNP). Neu5Acα(26)Galβ(1-4)GlcNAc-β-ethylamine (6′SLN-β-ethylamine), the specific binding receptor for human influenza virus H1 hemagglutinin, was immobilized on 11-mercaptoundecaonic acid immobilized GNPs via a condensation reaction. (c) Illustration of virus detection using resistive pulse sensing. The H1N1 virus was measured after binding with 6′SLN/SB-GNP. (d) Prospective result using this system. If a specific interaction occurred between 6′SLN/SB-GNPs and H1N1 viruses, the formation of conjugates would result in a size distribution shift, allowing for detection of influenza virus. 282x200mm (300 x 300 DPI)


Page 26 of 32

Page 27 of 32 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

Langmuir

Figure 2. Schematic illustration of GNP surface modification. SB-SH and MUA were co-immobilized via the Au–S bond, then 6′SLN-β-ethylamine was conjugated to the –COOH groups using a coupling agent. 223x117mm (300 x 300 DPI)


Langmuir 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) Extinction spectra of initial citrate stabilized GNPs (black) and GNPs following the coimmobilization of SB-SH and MUA in 91 (blue) or 182 (green) µM thiol solution (MUA/SB-GNPs). Extinction spectra were measured after dialysis to remove the unbound free thiol molecules. (b) Extinction spectra of MUA/SB-GNPs (green) and 6′SLN/SB-GNPs (red). Extinction spectra were measured after dialysis to remove the unbound free 6′SLN-β-ethylamine and byproducts. 340x156mm (300 x 300 DPI)


Page 28 of 32

Page 29 of 32 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

Langmuir

Figure 4. RPS measurement of virus solutions. (a) An illustration of typical pulses obtained by RPS measurement. Blockade magnitude represents the size of a particle, and duration time represents the total time for its passage through the pore membrane. (b) The result for influenza A H1N1 cultivated in chicken embryos (1 HAU, 8.6 × 108 particles/mL). (c) The measurement data for influenza A H1N1 cultivated in MDCK cells (1 HAU, 7.7 × 108 particles/mL). 451x514mm (300 x 300 DPI)


Langmuir 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 5. (a) Size histograms for influenza A H1N1 cultivated in chicken embryos (orange: virus only (n=984), green: viruses with SB-GNPs (n=994), purple: viruses with 6′SLN/SB-GNPs (n=698), turquoise: viruses with 3′SLN/SB-GNPs (n=564)). The vertical scales of these histograms (population (%)) are normalized by mode peak. (b) – (d) Individual virus plot data of “Virus + SB-GNP”, “Virus + 6′SLN/SBGNPs” and “Virus + 3′SLN/SB-GNPs”. (e) Merged plot data of (c) and (d). The whole of RPS measurement results were shown in Figure S7-S9. 349x202mm (300 x 300 DPI)


Page 30 of 32

Page 31 of 32 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

Langmuir

Figure 6. Size histograms for influenza A H1N1 cultivated in MDCK cells (brown: virus only (n=1294), red: viruses with 6′SLN/SB-GNPs (20 nm, n=634), blue: viruses with 3′SLN/SB-GNPs (20 nm, n=655), green: viruses with 6′SLN/SB-GNPs (60 nm, n=574). The vertical scales of these histograms (population (%)) are normalized by mode peak. (b) – (d) Individual virus plot data of “Virus + 6′SLN/SB-GNP (20 nm)”, “Virus + 6′SLN/SB-GNP (20 nm)” and “Virus + 6′SLN/SB-GNP (60 nm)”. (e) Merged plot data of (c) and (d). The whole of RPS measurement results were shown in Figure S10-S12. 352x202mm (300 x 300 DPI)


Langmuir 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

TOC graphic 270x124mm (300 x 300 DPI)


Page 32 of 32

Zwitterion Hybrid ... - ACS Publications

Recommend Documents