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Biological and Medical Applications of Materials and Interfaces

Magnetic nanozyme-linked immunosorbent assay for ultrasensitive influenza A virus detection Sangjin Oh, Jeonghyo Kim, Van Tan Tran, Dong Kyu Lee, Syed Rahin Ahmed, Jong Chul Hong, Jaewook Lee, Enoch Y. Park, and Jaebeom Lee ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b02735 • Publication Date (Web): 29 Mar 2018 Downloaded from http://pubs.acs.org on March 29, 2018

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ACS Applied Materials & Interfaces

Magnetic nanozyme-linked immunosorbent assay for ultrasensitive influenza A virus detection Sangjin Oh1, Jeonghyo Kim1, Van Tan Tran1, Dong Kyu Lee1, Syed Rahin Ahmed2, Jong Chul Hong3, Jaewook Lee4, Enoch Y. Park4 *, Jaebeom Lee1, * 1

Department of Cogno-Mechatronics Engineering, Pusan National University, Busan, 46241,

Republic of Korea 2

BioNano Laboratory, School of Engineering, University of Guelph, Gulph, ON N1G 2W1,

Canada 3

Department of Otolaryngology, Head and Neck Surgery, College of Medicine, Dong-A

University, Busan, 49201 4

Research Institute of Green Science and Technology, Shizuoka University, 836 Ohya,

Suruga-ku, Shizuoka 422-8529, Japan

*Corresponding authors: E.Y. Park, Ph.D., E-mail: [email protected]; Jaebeom Lee, Ph.D., E-mail: [email protected];

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Abstract Rapid and sensitive detection of influenza virus is of soaring importance to prevent further spread of infections and adequate clinical treatment. Herein, an ultra-sensitive colorimetric assay called magnetic nano(e)zyme - linked immunosorbent assay (MagLISA) is suggested, in which silica-shelled magnetic nanobeads (MagNBs) and gold nanoparticles are combined to monitor influenza A virus up to femtogram per milliliter concentration. Two essential strategies for ultrasensitive sensing are designed, i.e., facile target separation by MagNBs and signal amplification by the enzyme-like activity of gold nanozymes (AuNZs). The enzymelike activity was experimentally and computationally evaluated, where the catalyticity of AuNZ was tremendously stronger than normal biological enzymes. In the spiked test, straightforward linearity was presented in the range of 5.0 × 10

-15 ~ -6

gmL-1, detecting

influenza virus A (New Caledonia/20/1999) (H1N1). The detection limit is up to 5.0 × 10-12 gmL-1 only by human eyes as well as up to 44.2 × 10-15 gmL-1 by a microplate reader, which is the lowest record to monitor influenza virus using ELISA based technology as far as we know. Clinically isolate human serum samples were successfully observed at the detection limit of 2.6 PFUmL-1. This novel MagLISA demonstrates, therefore, a robust sensing platform possessing the advances of fathomable sample separation, enrichment, ultrasensitive readout, and anti-interference ability may reduce the spread of influenza virus and provide immediate clinical treatment.

Keywords: Nanozyme, Magnetic nano(e)zyme-linked immunosorbent assay (MagLISA), magnetic force induced enrichment, three-dimensional (3D) immunoassay, point-of-care 2


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testing (POCT), Influenza virus

3



Introduction With the worldwide trend toward the transition to 'preventive medicine' from 'therapeutic medicine' accurate diagnostics is a growing demand. For example, an influenza virus that is one of the most common infectious diseases of the respiratory tract requires rapid, sensitive and selective diagnostic devices to prohibit further spread and also provide immediate clinical treatment.1 The only clinical symptom may not differentiate influenza from other respiratory viruses or bacteria so that various diagnostic tools have been developed and utilized in hospitals and laboratories, for example, viral culture, fluorescence immunoassay, and molecular diagnosis using a real-time polymerase chain reaction.2-5 In particular, rapid kit type portable sensors are recently prepossessing owing to its costeffectiveness and handiness. Commercially available rapid influenza diagnostic tests have been launched in clinical field; e.g., SASTM FluAlert Influenza A&B (SAS Institute, USA), 3MTM Rapid Detection Flu A+B Test (Response Biomedical Corp. for 3M Healthcare, USA), TRUFLU® (Meridian Bioscience, Inc., USA), Remel X/pect® Flu A&B (Thermos Fisher Scientific, Canada), Quickvue Influenza (Quidel Corp., USA). However, the sensitivity (usually, ~ µg per mL) eventuates inevitable impediment for prevenient diagnosis and treatment. 6-7 Latterly, novel ultrasensitive colorimetric sensors have been emerged owing to its uncomplicated design and excellent sensitivity. Xianyu et al. controlled the dispersion and aggregation of gold nanoparticles (AuNPs) to achieve sensitive colorimetric detection by using horseradish protein (HRP)-catalyzed oxidation of iodide and iodide-catalyzed oxidation of cysteine. In this strategy, the HRP-triggered cascade reaction was read by the naked eye with enhanced sensitivity.8 Peng et al. reported the detection of disease biomarkers with 4


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ADH-mediated gold growth on AuNPs to produce the color signal.9 More recently, Zhao et al. reported ultrasensitive plasmonic ELISA with the gold nanoclusters as the mimetic enzyme. It enabled to detect ultratrace breast cancer biomarker, CA15-3 by the colorimetric device.10 Yang et al. employed gold nanorods (AuNRs) in the ALP-mediated plasmonic ELISA. With an anisotropic nanocrystal of controlled geometry, the AuNRs are offering the potential for use in sensitive colorimetric nanosensor because it is highly sensitive to the changes in the local refractive index, resulting in LSPR changes.11 Enzymatic activity is the key parameter in this type of biosensor.12-17 Usually, enzymes are remarkably efficient to catalyze a moiety in specificity under biological conditions. They have been extensively used in various research fields including biosensor, pharmaceutical process, and food industry.18-21 Enzyme-linked immunosorbent assay (ELISA) is a popular example, which is an alternative, well-recognized, a serological diagnostic method for influenza detection.22 ELISA systems are expected to be useful for point-of-care testing (POCT) in resource-limited settings. Furthermore, enzyme-mediated signal amplification strategy has been widely employed by using natural enzyme label for discrete ultrasensitive colorimetric detection methods in the biomedical field. Nevertheless, most sensors with the same approach using enzymes were suffered from difficulty in preparation and purification as well as denaturation of bio-moieties during modification.23 In order to overwhelm the limitations of enzymatic signal amplification, artificial enzymes have been enticing alternatives with the development of nanotechnology; e.g., catalase, oxidase, peroxidase, superoxide dismutase, esterase, phosphatase and protease mimic.17,

24-28

Nanomaterials are immensely efficient catalysts because of their extremely large surface-tovolume ratio. In particular, AuNPs are considered as one of the artificial enzyme mimics, aka, 5



Au nanozymes (AuNZs), by virtue of their unique advantages of abrupt catalyticity, robust stability, and facile surface reformation.

20, 29-31

Even though ELISA is a typical method

performed in the all manner of biomedical application, its conventional sensitivity is unsatisfactory and inexorably enhanced to fit the environment of clinical diagnosis, in particular, for influenza monitoring.32 The clinical diagnosis environment is consistently debated owing to its complexity including diverse health background of patients, signaling drag via analogous bio-moieties, reliability, and insufficient target specimen, which may retard precision and accuracy. Disruptive approaches are required to recognize and separate target bio-moieties with sufficient efficiency and sensitivity. One essential modus operandi is called as magnetic nanobeads (MagNBs) based immunoassays. MagNBs that possess reliable biocompatibility and extraordinary spatial controllability have fascinated great interest for biochemical analysis in recent years.33-35 Their magnetic susceptibility has been widely utilized for the separation and enrichment of biomarkers such as bacteria, viruses, and cells by applying external magnetic fields, leading to being advantageous in exclusive specificity and reduction of total assay processing time.3641

The magnetic separation efficiency is influenced by the size and superparamagnetic

property of the MagNBs. The separation process using Fe3O4 NPs with the diameter of < 30nm can be protracted in the actual experiment, usually an overnight method for complete separation. On the other hands, Fe3O4 NPs above 30 nm size, performing ferromagnetism might expect an expeditious separation rate, but it is demanding to re-disperse after magnetic separation due to their remanent magnetization.42-43 Thus, a cluster type composite of Fe3O4 NPs has been proposed which is advantageous to increase its magnetization while its superparamagnetism is maintained. Furthermore, its size can be moderately controllable up to 6


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a few hundreds of nanometers, allowing proficient separation.44-45 Herein, a simple colorimetric assay aimed at ultrasensitive and exquisite detection of influenza virus is introduced, called MagNBs-based nano(e)zyme-linked immunosorbent assay (MagLISA), where newly synthesized cluster type of superparamagnetic MagNBs and AuNZs are utilized for peroxidase activity. The MagLISA merged three functional components to be a robust and essential sensing platform; i.e., MagNBs for biomarker enrichment, AuNZs for the peroxidase-like artificial catalyst, and anti-hemagglutinin (HA) monoclonal antibody (mAb) for specific recognizer. This suggested probe is accordingly capable of recognition, separation, and visualization of influenza virus with the colorimetric analysis in one-step. Molecular dynamics (MD) simulation is conjointly implemented for catalyticity analysis depending on their surface properties. It resulted the LOD of MagLISA was significantly improved up to 10-14 gmL-1 with the assistance of conventional commercial microreader equipment, which is the lowest record to monitor influenza virus as far as we know. Analytical performances using clinical samples of influenza patients additionally demonstrated its prominent specificity, sensitivity, and reliability. It is plausible that the advanced sensing system acquires resulting feasibility for early diagnosis of influenza virus due to its sound constitution, accessible manipulation, and simple storage of inorganic nanozymes.

Experimental Section Materials Hydrogen tetrachloroaurate (III) trihydrate (HAuCl4·3H2O), cysteamine, sodium 7



borohydride (NaBH4), trisodium citrate dihydrate, ammonium hydroxide (NH4OH, 28-30 wt%), tetraethyl orthosilicate (TEOS, 99%), (3-aminopropyl) triethoxysilane (APTES, 99%), bovine serum albumin (BSA), 3,3′,5,5′-tetramethylbenzidine (TMB, T4444), and stop reagent for TMB substrate were purchased from Sigma-Aldrich (St. Louis, MO, USA). The ECL™ anti-mouse IgG, horseradish peroxidase (HRP)-conjugated whole antibody (Ab) was obtained from GE Healthcare Ltd. (Buckinghamshire, UK). Goat anti-rabbit IgG-HRP was purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Anti-Influenza A virus HA antibody (Ab66189), which is a mouse monoclonal antibody (mAb), anti-swine influenza A HA antibody (Ab91530), which is polyclonal antibody (pAb) for the influenza A virus HA H1 and A/New Caledonia/20/99 (H1N1) was purchased from Abcam Inc. (Cambridge, UK). Recombinant influenza virus A (New Caledonia/20/1999) (H1N1) (Cat No.: 11683-V08H) and anti-H3 (H3N2) antibody HA mAb (Lot: HB04N0160) were purchased from Sino Biological Inc. (Beijing, China). A clinically-isolated influenza virus A/Yokohama/110/2009 (H3N2) was kindly provided by Dr. C. Kawakami, Yokohama City Institute of Health, Japan and was used to monitor the versatility of the assay system. All experiments were carried out using high purity deionized (DI) water (>18 MΩ) and phosphate-buffered saline (PBS). PBS (1 M, pH 7.4) was obtained from BD Biosciences (San Jose, CA, USA) and diluted to 0.01 M prior to use.

Preparation of silica-coated magnetic nanobeads (MagNBs) Monodispersed FNCs were synthesized by the solvothermal process, which is currently tech-transferred and can be purchased from AMO Life Science Inc. (Amo-MagTM, 8


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Kimpo, Korea).45 The FNCs were coated with silica shells only to serve as capture probes, not as nanozyme. Since ELISA binds target materials using two inorganic particles, only one part requires sufficient enzymatic activity to discriminate other moieties, and it is known that Fe3O4 possesses a weak enzyme-like activity as well. The purchased FNCs were silica-coated as follows: a 0.6 mL aqueous solution of the FNCs (concentration of 25 mgmL-1) was diluted with 4 mL ethanol followed by the addition of 200 µL of ammonia (28%). The mixture was sonicated for 10 min. 250 µL TEOS was injected into the mixture under mechanical stirring. After 4 h, APTES (125 µL) was added to be amine-functionalized, and the reaction was allowed to proceed under mechanical stirring for further four h at room temperature. After that, the solution was refluxed at 90oC for one h. The final product, amine-functionalized silica-coated FNC (i.e., MagNB) was washed several times with ethanol and water, followed by drying under vacuum at 60oC for 6 h.

Preparation of the antibody conjugated MagNBs (MagNB-Abs) and AuNZs (AuNZ-Abs) The MagNBs were functionalized with anti-Influenza A virus HA antibody (ab66189, Ab1) as follows: the MagNBs were rinsed 3 times with water and re-dispersed in 0.01 M PBS (1 mgmL-1). 4 µg of Ab1 was mixed into 1 mL of the MagNB suspension followed by incubation at 15°C for 4 h with gentle shaking at 150 rpm. In this step, the Ab1 was adsorbed onto the MagNBs through the combination of ionic and hydrophobic interactions. The remaining active sites on MagNBs were blocked by 100 µL of 0.01% BSA in 0.01M PBS. After separation of Ab1 conjugated MagNBs (MagNB-Ab1) by a magnet, the supernatant was discarded, and the sediment was rinsed with PBS. The MagNB-Ab1 were finally reconstituted in PBS and stored at 4°C for further use. Anti-H3 (H3N2) antibody HA mAb 9



antibody (Ab3) were also conjugated to the surface of the MagNBs by using the same procedure (MagNB-Ab3). The positively-charged AuNZs were prepared by chemical reduction of HAuCl4 in the presence of cysteamine. Then, the preparation and confirmation of Ab2-conjugated AuNZs (AuNZ-Ab2) were referred to our previous work. 31

Peroxidase-like catalytic activity of nanoprobes The peroxidase-like activity of the MagNBs and AuNZs probes was investigated from the oxidation of TMB by H2O2. A typical experiment was carried out at room temperature with 5.0 × 10-11 M of AuNZs and MagNBs using working solution which prepared by mixing TMB reagent and 500 mM of H2O2 (3:7 volume ratio of TMB and H2O2) in deionized water. After the working solution was added to the probe solution and incubated for 5 min, color reactions were observed at 653 nm using UV-vis spectroscopy. To verify time and concentration-dependent catalytic activity, the assays were performed under standard reaction conditions as described above with varying concentration of Au NPs (2 × 10-12 to 2 × 10-9 M) and MagNBs (5.2 × 10-12 to 5.2 × 10-9 M).

Molecular dynamic simulations between substrate and nanoprobes The computational simulation was performed to determine the interactions energy between the substrate and nanoprobe surface by using the Material Studio 4.2 (Accelrys, San Diego, CA).46

First, the substrate models were built with the amorphous cell module in the material

studio, i.e., H2O2 model, and •OH model. For AuNZ surface model, space group of Au is Fm3m, and the (111) plane of the surface was constructed via crystallographic cleavage. 10


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Surface of Au (111) was modified with cysteamine, its lattice parameters are a = b = 11.53, c = 31.77 Å; α = β = 90°, γ = 120°. To produce a surface of MagNB, the SiO2 surface structure was imported from MS studio structure library, followed by adjustment with amine groups on their surface and its lattice parameters are a=b=28.51, c=40.00 Å; α = β = γ = 90°. All simulation models are shown in Figure S1. These models were based on the experimental condition for the enzymatic activity of nanoprobes. The adhesion of substrate on the surface of nanoprobes can be appraised by comparison of the interaction energy between them. The interaction energies were calculated using the following equation (1). Einteraction = Etotal − (Eadsorbate + Esurface )

-------------------------------------

(1)

Where Esurface is the energy of the nanoprobe surface, Eadsorbate is the energy of the adsorbate, H2O2, and OH radical, and Etotal is the energy of the adsorbate on the surface. High interaction energy values indicate high adhesion between H2O2, OH radical and nanoprobes surfaces.

Detection of Influenza Viruses A series of diluents of influenza virus A (H1N1) ranging from 5 × 10-15 to 5 × 10-6 gmL-1 were prepared using pH 7.4 PBS (0.01 M) to perform sensing experiments. The mAb against HA of the H1N1 virus were selected to conjugate on the MagNBs for selective recognition and targeting (MagNB-Ab1). 100 µL of MagNB-Ab1 (10-3 gmL-1) and 100 µL aliquot of viruses with various concentrations were mixed into an Eppendorf tube to detect the influenza virus. The MagNB-Ab1 can exclusively conjugate with the designated virus during incubation (37°C for 1h, shaking with 150 rpm) to be a MagNB-Ab1-virus complex. Other moieties were removed by washing three times with PBS and finally re-dispersed in 11



100 µL PBS. Afterward, 100 µL of AuNZ-Ab2 was injected into the MB-Ab1-virus complex solution with incubation at 37°C with shaking for 1h. A sandwich-structured immunocomplex, MagNB-Ab1-virus-Ab2-AuNZ was formed finally. The resulting immunocomplex was washed with PBS at least 3 times by magnetic separation. A working solution was added into immunocomplex solution and incubated at 25°C for 10 min, which resulted in the development of blue color. After 10 min, 50 µL of 10% H2SO4 solution was added to stop the reaction. Subsequently, the immunocomplex was collected with an external magnet, and 150 µL of supernatant was transferred to new 96 well-plate, followed by the absorbance at 450nm was measured using an ELISA reader. Furthermore, this sensing platform was directly utilized to clinical specimens isolated from patients (influenza virus A/Yokohama/110/2009, H3N2), but different Abs, with MagNB-Ab3 and AuNZ-Ab3. A stock solution of H3N2 viruses was serially diluted with the serum and used for detection from 6 × 10-1 to 6 × 106 PFUmL-1. The LOD was calculated in the usual way with little modification.47 To detect viruses in complex matrices, saliva, urine, and serum were mixed with 10-8 gmL-1 of H1N1 and 104 PFUmL-1 of H3N1 virus. The MagNB-Abs and AuNZ-Abs were added into these complex samples to capture and identify the target virus. The same procedure that excluded viruses was conducted for negative controls. This study was approved by the institutional review board (IRB Protocol No. 14-157) at Dong-A University.

Characterization The absorbance was measured using UV-vis spectroscopy (model 8453, Agilent Technologies Inc., Santa Clara, CA). The surface potentials and size distributions were determined using a Zeta-sizer (ZS Nano, Malvern Instruments, Malvern, U.K.). The shapes and sizes were 12


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characterized by high-resolution transmittance electron microscopy (HRTEM) (JEM-2100F, JEOL, Ltd., Tokyo, Japan) and field-emission scanning electron microscopy (FESEM, S4700, Hitachi, Tokyo, Japan). The chemical reactions were monitored by Fourier Transform Infrared (FT-IR) spectroscopy (JASCO, FT-IR 6300, Tokyo, Japan). Magnetic measurements were performed using a superconducting quantum interference device (SQUID) magnetometer (Quantum Design MPMS XL-7, USA) at room temperature. The absorption of the immunocomplex was measured using a filter-based multimode microplate ELISA reader (Infinite F500, TECAN, Ltd, Mannedorf, Switzerland).

Results and Discussion Working principle Scheme 1 illustrates fundamental concepts of the developed sensing platform for rapid and sensitive detection of influenza virus, which composes of two different probes that can specifically recognize the target virus. To prepare the detection probe, Ab2 was immobilized on positive charged AuNPs (i.e., AuNZs) via electrostatic attraction

31

. As a

capture probe, monodispersed Fe3O4 nanoclusters (FNCs) were modified with silica shell to prohibit enzyme activity from the surface of the iron oxide. Aminopropyl triethoxysilane (APTES) was coated on its surface to produce a positive charge for conjugation of antibodies. The monoclonal antibody (mAb) was used for capture probes, and polyclonal antibody (pAb) was used to generate the AuNZ probes (Scheme 1a). The MagNB-Abs serves as a capture probe to recognize target virus by specific antigen-antibody interaction while AuNZ-Abs have a role as signal amplifier to form a sandwich-like structure with MagNBs complex. 13



After the immunoreaction, the only sandwich-like structures are collected magnetically and others including AuNZ-Abs are dumped out. The unrecognized adsorption among nanomoieties is excluded by surface passivation using BSA as a non-specific blocker. The AuNZs on the sandwich-like immunocomplex as a catalyzer accelerate the oxidation reaction of TMB by H2O2, resulting in the generation of colorimetric signals. Meanwhile, the absence of virus cannot assemble the immunocomplex, leading to lack of oxidation reaction of TMB for colorimetric signaling. The concentration of influenza virus as directly correlating with TMB color changing can be detected by the catalytic activity of AuNZs with H2O2 as an electron acceptor (Scheme 1b).

Scheme 1. Schematic illustration of the magnetic nanobead−based nano(e)zyme linked 14


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immunosorbent assay (MagLISA). (a) Procedure for the preparation of the MagLISA nanoprobes. (b) Working principle for the quantification of influenza viruses using MagLISA -based colorimetric diagnostics kit.

Characterization of nanoprobes (MagNBs-Ab and AuNZs-Ab) TEM and SEM images demonstrate the solvothermally produced FNCs that are spherically shaped in the average size of 220 nm with 5.3 % of relative standard deviation (Figure 1a, S2a). The silanization step with amino group increased their sizes to 270 nm, indicating the thickness of the silica shell as 20−30 nm. No apparent change in their morphology was observed, still maintaining high monodispersity (relative std. = 8.25 %) (Figure 1b, and S1b). The surface charge was remarkably changed from -35.3 mV of FNCs to +29.9 mV after silanization with an amino group. It was changed to +18.1 mV and +22.1 mV with further bio-conjugation of the Ab1 and Ab3, respectively (Figure 1c). The analysis of FT-IR spectroscopy confirmed NH2 and SiO2 groups over the surface of MagNBs (Figure S3). The magnetite was verified by the wide strong absorption band between 560 and 630 cm−1 in both spectra

48

. The adsorption of silane polymer into the

surface of magnetite particles was figured out by the band at 1064 cm-1, corresponding to the Si-O-Si band of APTES to the surface of magnetic NPs. A band appeared 1231 cm-1 due to the formation of C-N bond between NH2 group in APTES and OH group. The C-H stretching bond appeared at 2911 cm-1 and 2980 cm-1 and C-H scissoring vibration present at 1395 cm-1 which confirmed the binding of APTES molecules at the surface of magnetite (MagNBs).49-50 The magnetic property of the MagNBs was evaluated using the SQUID at room temperature (Figure S4). Notice that the interaction of MagNBs with applied external magnetic field 15



makes these materials attractive for separation and concentration of attached entities. This result indicates that the prepared MagNBs behave like superparamagnetism with high saturation magnetization (Ms=60 emug-1). In UV-Vis spectra, broad absorbance peak from the MagNBs probes nearly dissipated to an optical density lower than 10-3 in the range of 350−900 nm (Figure S5a). The MagNB probes were readily separated by applying external magnet, showing a time-dependent variation of absorbance in which the solution color was transparent swiftly within less than 1 min (Figure S5b, inset). This data reflects the rapid separation of immunocomplex in this assay. Also, they can be readily re-dispersed in water by shaking, vortexing, or sonication after magnet removal from the solution. Thus, these MagNBs can be used as vehicles for magnetic separation and enrichment of target virus. Meanwhile, hexagonal AuNZs of the diameter of 30nm were synthesized with a high grade of monodispersity, which has an absorbance peak at 527 nm in UV-vis spectra (Figure 1d,e). Compared with bare AuNZs, the absorbance peak of AuNZs-Ab modestly shifted from 527 nm to 529 nm. The red-shift of the peak with a limited change in spectra shape reveals that the antibodies are conjugated stably on the surface of AuNZs without any aggregation.51 Moreover, the surface charge of the AuNZs was changed from +36.5 mV to +22.9 mV and +25.9 mV after modification with the Ab2 and Ab3, respectively (Figure 1f). The conjugation of antibody was double-checked using ELISA. Anti-mouse IgG-HRP was mixed with the probe solution. The Ab-conjugated nanoprobes can catch the anti-IgG-HRP, and enable substrate oxidation, followed by color formation while MagNBs and AuNZs without Ab-conjugation cannot oxidize the substrate (Figure 1g). The MagNB-Ab1 and MagNB-Ab3 displayed strong signals which indicate that antibodies were successfully conjugated, while bare MagNBs produced no absorbance signal. The successful conjugation of the Ab2 and 16


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Ab3 on AuNZs was also verified, where no distinguishable absorbance was observed in control AuNZs without Ab conjugation at 450 nm from the oxidized TMB. (Figure 1h).

Figure 1. Characterization of the MagLISA nanoprobes. (a, b) TEM images of FNCs and MagNBs (inset shows a histogram of size distribution) and (c) Zeta-potential results during surface modification process of MagNB probes. (d) TEM image of AuNZs, (e) UV-vis spectrum, and (f) Zeta-potential results of AuNZs modification with antibodies. (g,h) schematic illustration and results of ELISA confirmation for antibody conjugation.

Peroxidase-like activity of nanoprobes 17



The peroxidase-like activity of the MagNB and AuNZs probes was evaluated by catalyzing the oxidation of peroxidase substrates, TMB with H2O2. Similar to peroxidase, the highly charged AuNZs can oxidize TMB by H2O2 to develop a blue charge-transfer complex with a maximum absorbance at 655 nm (Figure 2a). The H2O2 might be broken into OH radical with adsorbing on the surface of the AuNZ. The generated OH radicals may contribute to the catalytic ability of AuNZs with the partial electron exchange interaction. As shown in Figure 2b, TMB solution in the presence of only H2O2 was nearly colorless with very low absorbance, indicating that the oxidation of TMB does not happen in the absence of the catalyst in the experimental condition. The AuNZs become catalyzer to oxidize TMB by H2O2 and develop a blue color. Meanwhile, it was noticeable that the absorbance change by MagNBs was not significant to be used as nanozyme (Figure 2c). The peroxidase-like activity of AuNZs was dependent on the physical reaction conditions, in particular, pH and temperature. Figure 2f shows the effects of pH (pH 2-12) and temperature (10-60 ℃), indicating that the catalytic activity is fairly stable over 90% of activity in the range of pH 210 and 10-60 ℃. Furthermore, the oxidation kinetics of TMB in the presence of H2O2 and different concentrations of AuNZs and MagNB was recorded by UV-vis spectroscopy. The absorbance of oxidized TMB was directly proportional to the particle concentration in the reaction solution. The representative absorbance of TMB in the presence of MagNBs shows no apparent absorbance intensity compared with the lowest concentration of AuNZs (Figure 2d, e). The catalytic reaction of MagNBs was nearly inhibited with silica shell coating. To compare the catalytic activity with negative charged Au NPs, trisodium citrate (TSC) coated Au NPs were prepared. Au NPs were well-synthesized with 25 nm size and had an absorbance peak at 525 nm (Figure S6). The TSC-Au NPs exhibited different enzyme-like

18


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behavior. As shown in Figure 2g, the TSC-Au NPs may not be efficient nanozyme to catalyze the TMB-H2O2 reaction, which shows lower catalytic activity than that of AuNZs. A kinetic of TMB oxidation was investigated by measuring the absorbance at 652 nm as a function of time after adding AuNZs and TSC-AuNPs solution to the working solution. The absorbance data at 652 nm were calculated to concentration by Beer-Lambert Law with a molar absorption coefficient of 36000 M-1 · cm-1. Apparent steady-state reaction rates were obtained by calculating the slopes of initial absorbance with time for each particle concentration. The dots are the experimental data, and the solid lines are the fittings to the Michaelis-Menten model for the enzyme kinetics. Typical Michaelis-Menten curves were obtained by plotting the initial reaction velocities against substrate concentrations for both H2O2 (Figure 2h) and TMB (Figure 2 i). The curves were then fitted to the double-reciprocal or Lineweaver-Burk plots (inset of Figure 2 h,i), from which the kinetic parameters were determined. The reaction rate has a drastic increase at low H2O2 concentration and reaches the maximum value (Vmax) when H2O2 exceeds 5 M. The Km measures the concentration of substrate at which the reaction reaches ½ Vmax. It is an important parameter to measure the binding affinity of the enzyme to the substrates and can be applied similarly here to study NP-TMB interaction, and the results are shown in Table S1. The magnitude of Km becomes smaller with increasing affinity between the enzyme and substrate. Km from the AuNZs is a lower value than TSC-AuNP and HRP, indicating that the AuNZs have a much higher binding affinity to TMB. On the other hand, Km(H2O2) is much greater than Km(TMB) for both kinds of AuNPs since H2O2 lacks this sort of interaction. This result indicated that like HRP, the AuNZs bind and react with one substrate (either TMB or H2O2) and then release a. product before reacting with the other substrate. These results indicated that AuNZs have higher 19



affinity to the substrates and stronger catalytic activity. It is concluded that AuNZs shows promise as a signal amplifier for applications requiring rapid and highly sensitive detection and analysis.

Figure 2. Peroxidase-like activity of nanoprobes catalyzing the oxidations of peroxidase substrate by H2O2 that produce colored products. (a) schematic illustration, (b) photograph and (c) UV-vis spectrum of the TMB- H2O2 reaction system catalyzed by AuNZs and MagNBs (t=10 min). Time- and particle concentration-dependent absorbance changes at 653 nm of TMB-H2O2 reaction by (d) AuNZs, and (e) MagNBs. (f) Effects of pH (left), temperature (right) on the catalytic oxidation of TMB with AuNZs. (g) UV-vis absorption spectra and image (inset) of the TMB-H2O2-mixed solution in the presence of AuNZs and TSC-AuNPs. (h,i) The Michaelis-Menten kinetic curves of AuNZ (red circle), and TSC20


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AuNPs (black square) in the presence of H2O2 and TMB with different concentrations at room temperature and double-reciprocal plots of activities (inset).

Computational simulation of interaction energy between substrate and nanoprobes The experimental results of peroxidase-like activity indicate that the silica shell is an inhibitor of the enzymatic activity in MagNBs while AuNZs exhibit higher enzyme-like activity than TSC-AuNPs. To unravel the mechanism for the unique enzyme activity of AuNZs, a computational study was carried out to observe H2O2 and OH radical interaction on the surface of MagNB and AuNZ. The thermodynamic energy states between substrate and surface of enzymatic materials, such as TSC-AuNP, bare AuNP, and the active site of HRP, were calculated to compare their adsorbing activity. Here, the adsorbing energy of H2O2 originates from the reactions occurring on the metal surfaces, which produces OH radicals.52 This reaction energy can be used as a descriptor of the surface of each model; more negative energy means greater H2O2 and OH radical adsorbing reaction. The molecular adsorption was investigated as being the first step of the decomposition reaction of H2O2 on the nanoprobe surface. Energy minimal geometries were found corresponding to the molecular adsorption of H2O2 and OH radical onto the surface of the nanoprobes for the two materials studied. Following the MD simulation, the structural alteration of molecules and the adsorption was observed on the surface of nanoprobes and other materials. The corresponding structures are depicted in Figure S7, for MagNB and AuNZ, in Figure S8, S9 for TSC-AuNP, bare AuNP, and HRP, respectively. The calculated adsorption energies between substrate and nanoprobes are given in Table 1. The interaction energies of MagNB surface model demonstrated positive values, showing that the substrates are difficult to bind to the surface of the MagNB.

21



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On the other hands, the AuNZ model has a negative value and the highest absolute compared with TSC-AuNP, bare AuNP and HRP model (Table S2). Therefore, it indicates that strong interaction occurs between the H2O2, OH radical and the AuNZ surface. The MD simulation data provide reliable evidence to demonstrate the related experimental results of the peroxidase-like activity of the nanoprobes.

Table 1. Interaction energy of the surface of AuNZ and MagNB with substrate molecule (H2O2 and OH radical). Substrate

Surface

H2 O2 H2 O2

Etotal

Eabsorbate -1

Esurface -1

Einteraction

(kcal·mol )

-1

(kcal·mol )

(kcal·mol )

(kcal·mol-1)

MagNB

-768.507

-790.828

21.573008

0.748234

AuNZ

-61.0016

-36.0653

21.54344

-46.4798

•

OH

MagNB

-44.4319

-43.911

-0.00309

-0.51784

•

OH

AuNZ

-65.9145

-51.7442

-0.08616

-14.0842

Sensitivity and reproducibility of detection for influenza virus To ensure efficiency of the sensing system, mAbs were conjugated with MagNBs for capture, and AuNZs were modified with pAbs. The high specificity of mAbs decreases background noise and cross-reactivity and helps provide reproducible results. pAbs could help to increase the signal produced by the target protein as the antibody can bind to more than one epitope. In our strategy, pAb conjugated AuNZ as signal amplifier shows higher color signal than mAb modified AuNZ. (Figure S10) The sensitivity of the proposed method was demonstrated by measuring H1N1 influenza virus with different concentrations in the range of 5 × 10-15 to 5 × 10-6 gmL-1. Figure 3a presents the photographs of the analyzed 22


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solution in various concentrations of virus. A distinct color change from pale to dark yellow at the absorbance wavelength of 450 nm was observed as the H1N1 virus concentration increases. The linear range was 5 × 10-15 to 5 × 10-6 gmL-1 with the correlation coefficient of 0.9811. The method was able to detect the H1N1 virus as low as 4.42 × 10-14 gmL-1 as the limit of detection (LOD) at the 3-fold signal against the noise.47 Remarkably, the color signal contrast could be distinguished at the sample concentration of 0.5 × 10-12 gmL-1 without any assistance of instrument. The intra-assay and inter-assay reproducibility were evaluated (Table S3), resulting in 3.26% and 4.76%, respectively, which confirms high reproducibility and stability.53 In order to evaluate the quantitative detection of the marker in various volumes, recovery experiments were carried out by the standard spiked method. 1 pg of the H1N1 virus was added into 1, 10, and 100 mL of PBS and analyzed by the proposed detection system. The recoveries were calculated with the corresponding relative standard deviation (RSD) in Table S5. The results showed that the recovery ranged from 90.6 % to 101.7 %, and the corresponding RSD ranged from 3.62 to 6.17 %, which revealed that the quantitative detection of our sensing system could be applied to the detection of ultra-trace biomarker in a large volume of specimen, and attributed to magnetic concentration strategy. A series of quantitative analyses with clinically isolated influenza virus A/Yokohama/110/2009 (H3N2) in human serum was also investigated (Figure 3b). Indeed, as the spiked test, the absorbance at 450nm was dramatically changed depending on the virus concentration. The calibration curve was obtained in the range of 6.0 × 10-1 to 6.0 × 106 PFUmL-1 and the LOD were calculated as 2.5 PFUmL-1 and the color could be identified by the human eye until 6.0 PFUmL-1. The intra-assay coefficients of variation (CV%) were calculated to be 3.47, and inter-assay CV was 4.30 %. (Table S4). The present sensing system was 2,000 times and 400

23



times more sensitive than the commercial kit and ELISA, moreover, 4-fold more sensitive than our previous work, which is combined conventional ELISA with AuNZs (Table 2). 31, 54

Selectivity of detection for H1N1 and H3N2 influenza virus In the clinical diagnosis, robust specificity is essential to avoid false positive interference and to ensure the accuracy of the results at an early stage. The specificity of the suggested method is evaluated by utilizing different subtype of viral samples including H1N1, H3N2, and BSA (Figure 3c). The entire diagnostic processes including the target virus separation and the conjugation of AuNZs were performed, followed by analyzing absorbance from the separated immunocomplexes with TMB oxidation. Owing to the highly efficient interaction of the antibody-binding MagNBs, they could capture target virus exclusively in complex samples. The data show that the average optical density (OD) of MagNB-Ab1 to H1N1 virus and MagNB-Ab3 to the H3N2 virus are 0.97 and 0.89, respectively. Meanwhile, the absorbance of the MagNB-Ab1 to H3N2 or BSA and MagNB-Ab3 to H1N1 or BSA showed no signal with very low intensity in the range of 0.11−0.14. The overall results indicated that different subtype of influenza virus was clearly identified from any negative controls. To investigate anti-interference ability in the complex biological samples, saliva urine, and serum was mixed with the virus and processed assay. As shown in Figure 3d, the saliva, urine, and serum positive samples generate a significant absorbance intensity compared to their corresponding negative controls. The recoveries and corresponding RSD of the proposed system for H1N1 and H3N2 virus added in clinical specimens were calculated 24


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in Table S6, and 7. The results revealed that our assay has reliable accuracy and precision with the RSD rage of 3 – 6 %; and good recoveries over 90%. The excellent magnetic concentration property of the MagNB probes helps detect target viruses directly in the complex biological matrices without purification process. This result shows that our detection assay has the excellent anti-interference ability and may be applied to complex matrices.

Figure 3. Sensing performance. Detection linearity of (a) H1N1 influenza virus, (b) clinically isolated H3N2 virus. (c) Selectivity of the developed virus detection with H1N1, H3N2, and BSA by using MagNB-Ab1 and MagNB-Ab3 probes and (d) Histograms of the 25



Page 26 of 33

detected absorbance at 450 nm in different biological matrices, saliva, urine, and serum to analyze the anti-interference ability. All data are presented as mean ± SD (n=3).

Table 2. Comparison of influenza virus (H3N2) detection using different methods Detection method

Virus concentration (PFUmL-1) 5,000 1,000

100

50

10

5

2.5

1

Commercial kit ** 53

+

-

-

-

-

-

-

-

Conventional ELISA 31

+

+

-

-

-

-

-

-

Previous work + AuNZs 31

+

+

+

+

-

-

-

-

Developed assay

+

+

+

+

+

+

+

-

* Note: + and - denote the positive and negative diagnosis, respectively. ** ImmunoAce Flu® (TAUNS Laboratories, Inc.) was used for the commercial kit.

Conclusion An enhanced colorimetric immunoassay system called MagLISA was developed for ultrasensitive detection of influenza viruses using MagNBs as a separable identifier and positively charged AuNZs as a signal amplifier. To adapt nanoprobes to the MagLISA system, we demonstrate silica-shelled MagNBs could inhibit their peroxidase-like activity, and AuNZ shows most efficient activity compared with other candidates by molecular dynamic simulation. This approach employing magnetic separation of MagNBs and the peroxidaselike activity of AuNZs detected target influenza virus A (H1N1) and clinically isolated influenza A (H3N2) up to 4.42 × 10-14 gmL-1 and 2.5 PFUmL-1, respectively. As a whole, this proposed system shows simple operation, ultra-sensitivity, and selectivity against 26


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potentially interfering bio-molecules and reliability and further provided a great possibility to the analysis of influenza virus in clinical samples and demonstrates its applicability to in situ point-of-care (POC) diagnosis. Furthermore, it can be also used for the rapid, automatic diagnosis of other acute infectious diseases with modification of the protocol.

Author Information Corresponding Author * Jaebeom Lee, E-mail: [email protected] * E.Y. Park, E-mail: [email protected] Present Address 1

Department of Mechanical and Industrial Engineering, University of Massachusetts

Amherst, Amherst, MA 01003, United States 4

Research Institute of Green Science and Technology, Shizuoka University, 836 Ohya,

Suruga-ku, Shizuoka 422-8529, Japan Author Contributions The manuscript was written through contributions of all authors. All authors have approved the final version of the manuscript.

Acknowledgments We thank Dr. Chiharu Kawakami in the Yokohama City Institute of Health, Japan, for supporting about clinical samples. This work was supported by the National Research Foundation of Korea (NRF) Grant funded by the Korean Government (MSIP) (2016R1A2B4012072, 2018K2A9A2A08000120), a grant from the Korean Health Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (HI16C1553), 27



Page 28 of 33

and research funds from Dong-A University. No additional external funding was received for this study. Supporting Information Supporting Information is available free of charge via the ACS Publication website. Includes details of the experimental methods, supporting figures mentioned in the main text, and details on the calculation and simulation of nanomaterials as Supporting Information.

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