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Tobacco mosaic virus with peroxidase-like activity for cancer cells detection through colorimetric assay Jiawang Guo, Xia Zhao, Jun Hu, Yuan Lin, and Qian Wang Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b00921 • Publication Date (Web): 04 Jan 2018 Downloaded from http://pubs.acs.org on January 5, 2018

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Molecular Pharmaceutics

Tobacco mosaic virus with peroxidase-like activity for cancer cells detection through colorimetric assay Jiawang Guo,†,‡ Xia Zhao,† Jun Hu,† Yuan Lin,*,† Qian Wang†,§ †

The State Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of Applied

Chemistry, Chinese Academy of Sciences, Changchun 130022, P.R. China. ‡

University of Science and Technology of China, Hefei 230026, P.R. China.

§

Department of Chemistry and Biochemistry, University of South Carolina, Columbia, South

Carolina 29208, USA.

*To whom correspondence should be addressed: Yuan Lin, Ph.D. The State Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, P.R. China. Phone: +86-0431-85262658. E-mail: [email protected]

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ABSTRACT Cell-based ELISA (CELLISA) has been widely used in disease diagnoses due to its simplicity and low cost. Recently, peroxidase-like nanomaterials have emerged as promising systems for CELLISA applications. In this work, Tobacco mosaic virus (TMV) was simultaneously tailored with peroxidase-like inorganic nanoparticles (platinum nanoparticles) and cancer cell target groups (folic acid) to obtain TMV-FA-Pt nanoparticles for cancer cell detection. Induced by the uniformly distributed reactive groups and well-defined structure of the TMV particle, platinum nanoparticles could be grown in situ on the exterior surface of TMV with excellent monodispersity and uniform spatial distribution. Meanwhile, folic acid (FA) with a PEG1000 linker was successfully conjugated to the coat proteins of TMV through the Cu(I)-catalyzed alkyne-azide cycloaddition reaction, an efficient “click” chemistry. Our study demonstrated that the resultant TMV-FA-Pt had specific affinity to cancer cells and were successfully used to detect cancer cells through CELLISA. Less than 1.0 × 104 cells/mL of cancer cells could be readily detected. KEYWORDS: Tobacco mosaic virus, platinum nanoparticles, peroxidase-like activity, cancer detection, CELLISA


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Table of Content



1. INTRODUCTION Facile and economic diagnostic methods for the detection of disease biomarkers are essential to improve clinical practice, particularly in the resource limited countries and areas. Enzyme-based colorimetric assay, such as enzyme linked immunosorbent assay (ELISA), is the most commonly used disease detecting technology.1,2 Cell-based enzyme linked immunosorbent assay (CELLISA) is a test that can directly detect the biomarkers expressed by cells,3,4 and has been used to detect the target cells and determine the relative protein expression levels.5,6 Horseradish peroxidase (HRP), which catalyzes the oxidization of several substrates in the presence of H2O2 producing color change of 3,3',5,5'-tetramethylbenzidine (TMB), 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS), and o-phenylenediamine (OPD), is mostly used in CELLISA. However, HRP has some intrinsic drawbacks, including limited activity, susceptibility of its activity to environment, hard for long term storage, which greatly limit its application potentials.7,8 Recently, various nanomaterials such as Fe4O3 nanoparticles,9,10 gold nanoparticles,11,12 platinum nanoparticles,13,14 cerium oxide nanoparticles,6 and oxide graphene,15 have been proven to possess peroxidase-like activity, and can catalyze the oxidization of several substrates for sensing applications. Due to their unexpected catalytic activity, stability and low cost, a number of peroxidase-like activity nanomaterials have been applied in CELLISA.16-18 Furthermore, their detection sensitivity can be further improved by depositing nanoparticles with peroxidase-like activity on silicon oxide or gold carriers.19,20 Alternatively, the peroxidase-like activity of nanomaterials can be tuned by controlling the topography, chemical composition of the carrier as well as the coating density on the carrier surface.21,22 Herein, it is extremely beneficial to create the carrier with identical size and to distribute peroxidase-like nanoparticles uniformly on the carrier surface for the augmentation of the detection sensitivity of CELLISA. Native Tobacco mosaic virus (TMV) consists 2130 identical coat protein subunits helically assembled around single RNA to form a rod-like structure. It is 300 nm in length and 18 nm in diameter,23 and has a well-defined uniform chemical property and regular topography which has


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been resolved at a resolution of 2.9 Å by X-ray crystallography.24 It was demonstrated that TMV particles could be applied as supporting scaffolds to regulate cell differentiation25,26 and to construct conductive nanowire.23 Furthermore, a number of amino acid residues on the exterior surface of TMV could be used to assemble metal nanoparticles27 and induce various metal precursors to form uniform nanoparticles,28 including gold nanoparticles,29 palladium nanoparticles30 and platinum nanoparticles.31 In the last decade, it has been demonstrated that organic functional groups and biological functional molecules could be conjugated to the exterior and interior of TMV, which expanded the applications of TMVs in the field of drug delivery,32 immunology33, imaging34 and sensing.35 Genetic engineering is a general method to insert functional peptides on the coat proteins of TMV,36 while chemical modification is a popular strategy to conjugate functional groups to TMV using the exposed lysine,37 aspartic/glutamic,38 cysteine39 and tyrosine40 residues. “Click” chemistry was demonstrated as a quick and efficient protocol for the bioconjugation of viral nanoparticles.41 For example, the tyrosine residues on TMV exterior surface could be easily coupled with alkyne groups and subsequently conjugated via Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction.32-34,40 With this strategy, our group have incorporated many functional groups on TMV, for instance, phosphate group for regulating osteogenic differentiation,26

estriol

(E3)

for

enhancing

antibody

response33

and

Tn

antigen

(GalNAc-α-O-Ser/Thr) for cancer recognizing.42 In this study, we chose TMV as the nano-platform to simultaneously incorporate cancer target moieties and peroxidase-like inorganic nanoparticles for cancer cells detection (Scheme 1). Folic acid (FA), whose receptors are overexpressed in many types of cancer cells,43 was selected as a model target unit, and platinum nanoparticles, which could be generated in situ, served as the peroxidase surrogate. The resulted nano-system, namely, TMV-FA-Pt NPs, could specifically interact with the cancer cells and result in an efficient colorimetric detection.



Scheme 1. The construction of TMV-FA-Pt NPs and the assay of cancer cells detection with TMV-FA-Pt NPs. Briefly, TMV-alkyne was conjugated with N3-PEG1000-FA by CuAAC reaction and subsequently platinum was deposited on the exterior surface of TMV-FA to form TMV-FA-Pt NPs. After co-incubated with folate receptor overexpressed cancer cells, TMV-FA-Pt NPs specially adsorbed on the cell membranes, and then transduced and amplified signal by catalyzing the oxidation of TMB by H2O2, which can be monitored by spectrophotometer.

2. EXPERIMENTAL SECTION 2.1 Chemicals and Materials. TMV was extracted and purified according to protocol previously reported.44 Chloroplatinic acid hexahydrate (H2PtCl6∙H2O, AR) was purchased from Shanghai Chemical Reagents Corporation (Shanghai, China). Sodium borohydride (NaBH4, 98%), sodium 2-mercaptoethanesulfonicacid (MPS, 98%), 3,3',5,5'-tetramethylbenzidine (TMB, 97%), L-ascorbic acid sodium salt, cupric sulfate (CuSO4, AR), p-tosyl chloride (PTSC), 3-aminophenylacetylene (98%), triphenylphosphine, folic acid (FA, 97%), dimethyl sulfoxide (DMSO, GC) were purchased from Aladdin Chemistry (Shanghai, China). Polyethylene glycol


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(PEG1000, Mw= 1000 Da) was purchased from J&K Chemical Technology (Beijing, China). p-Toluenesulfonic acid was purchased from Sinopharm (Shanghai, China). Sodium azide (NaN3) was purchased from Tianjin Fuchen Chemical Reagents Factory (Tianjin, China). Polyethylene glycol (PEG8000, Mw=8000 Da) were purchased from Beijing Dingguo Changsheng Biotechnology Co.Ltd. (Beijing, China). Dichloromethane (CH2Cl2), pyridine, methylbenzene, ether, ammonium chloride, sodium bicarbonate and potassium hydroxide were purchased from Beijing Chemical Reagents Corporation (Beijing, China). N3-PEG1000-FA was synthesized in our lab (see SI). 2.2 Measurements. Dynamic light scattering (DLS) was carried out by Nano ZS90 (Malvern Instruments, UK). Transmission electron microscopy (TEM) analyses were performed on a FEI TECNAI F20 EM with an accelerating voltage of 200 kV. The absorbance of solution was measured through Nano Drop 2000 and microplate reader (Tecan, Switzerland). Centrifugation were carried out by centrifugal machines from Beckman Coulter Corporation. 1H NMR analysis was characterized with Bruker AVANCE DRX 400M. FT-IR data was measured by Nicolet 6700. MALDI-TOF MS was carried out by the established protocol previously reported.40 2.3 Synthesis of TMV-Pt NPs. 1.50 mL of 2.00 mg/mL TMV was added to 8.00 mL of 6.30 mM H2PtCl6 solution (pH=7.00, adjusted with 1.00 M NaOH). Then 0.50 mL of 5.00 mM MPS was added to above solution before 10.00 mL of 5.00 mM cold NaBH4 solution was injected, and further stirring for 1 h. The TMV-Pt NPs were purified by centrifugation at 8000 rpm for 20 min to remove excess reactants and platinum nanoparticles, and washed three times with water. 2.4 Synthesis of TMV-FA-Pt NPs. N3-PEG1000-FA molecules were conjugated to the exterior surface of TMV by CuAAC reaction to obtain TMV-FA using established protocols.40 Firstly, alkyne groups were introduced at the exterior tyrosine group (Tyr 139) with diazonium salts. Briefly, diazonium salts were synthesized by mixing 0.80 mL of 0.30 M p-toluenesulfonic acid, 0.15 mL of 0.67 M 3-aminophenylacetylene and 0.05 mL of 3.00 M sodium nitrite. After 1 h, 30.00 mg TMV were treated with 0.40 mL of above diazonium solution in a pH 9.0 buffer solution



for 2 h to get the TMV grafted alkyne groups (TMV-alkyne). Next, 24.00 mg (50 eq) of N3-PEG-FA were dissolved in 10.00 mM pH 7.8 phosphate buffer of 4.70 mL containing 5.00 mg (1 eq) of TMV-alkyne and 0.30 mL of the mixture including equal volume of 0.10 M CuSO4 and 0.20 M ascorbic acid sodium salt were added, then stirring for 24 h to form TMV-FA. The sample was purified by sucrose gradient, and MALDI-TOF were carried out to confirm the conjugation between N3-PEG-FA and TMV-alkyne. Finally, TMV-FA-Pt NPs were prepared through the above protocol of synthesis of TMV-Pt NPs. 2.5 Peroxidase-like Activity of TMV-Pt NPs and TMV-FA-Pt NPs. The platinum concentrations of Pt NPs and TMV-Pt NPs and TMV-FA-Pt NPs were quantified by Inductively Coupled Plasma-Atomic Emission Spectroscopy (ICP-AES, Thermo Scientific iCAP6300). Time-dependent absorbance at 652 nm were measured from the reaction solutions containing water, 9.26 µg/mL TMV NPs, 25 nM Pt NPs, 25 nM TMV-Pt NPs or 25 nM TMV-FA-Pt NPs in the present of 0.80 mM fresh TMB and 1.00 M H2O2, in 0.20 M HAc/NaAc buffer (pH 4.50) at room temperature. The peroxidase-like activity of TMV-Pt NPs was evaluated by measuring the absorbance at 652 nm of reaction solution including fresh TMB and H2O2 in 0.20 M HAc/NaAc buffer at different conditions (temperature, pH and the concentrations of H2O2). The peroxidase-like activity of TMV-FA-Pt NPs was estimated at the different pH and the concentrations of H2O2 following the above protocol. The relative activity was expressed relative to the maximal absorbance after 5 min. The TMV-Pt NPs were recycled by ultrafiltration and washed with water for 3 times after the oxidization of TMB by H2O2 in 0.20 M HAc/NaAc buffer (pH 4.50) for 5 min. TMV-Pt NPs in water were ultra-filtrated and washed as control for excluding the influence of the loss of TMV-Pt NPs. The relative activity was expressed relative to the absorbance before recycled. 2.6 Kinetic Assay. All steady-state kinetic assays were carried out at 30 °C in 4.5 mL cuvettes with a path length () of 1.00 cm. The absorbance (A652nm) of the solution containing 25 nM TMV-Pt NPs or 25 nM TMV-FA-Pt NPs and corresponding substrates (TMB and H2O2) in 0.20 M


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NaOAc/HOAc (pH 4.50) were monitored every 10 s for 3 min. The concentrations of oxTMB were calculated by C = A652 nm / εl, where ε was the extinction coefficient of oxTMB (ε = 3.90 × 104 M-1cm-1).45 These “oxTMB vs time” plots were used to obtain the initial velocities which were the slope at initial point of each reaction. The plots of against substrates concentrations were fitted using Michaelis-Menten equation: ν = νmax× [S] / (Km + [S]). The Lineweavere-Burk plots were transformed from Michaelis-Menten to get the parameters Km and νmax. 2.7 Colorimetric Detection of Cancer Cells. L929, Hela and MCF-7 cells were grown in DMEM medium containing 10 % fetal bovine serum (FBS) in a 37 °C incubator with 5 % CO2. The cells were collected by digestion and centrifugation. Then, the cells suspending in 4 % paraformaldehyde PBS solution (pH 7.4) were fixed after mildly rotating for 15 min. In order to guarantee that the pH was at 4.5 where the activity peak of TMV-FA-Pt located, water was used to wash the cells. The number of cells was determined with cell counter under optical microscope. For the detection of cancer cell, 5.00 × 105 cells/mL were blocked by treated with 10% FBS for 1.5 h and collected through being centrifuged for 5 min at 1000 rpm. The precipitate of cells was co-incubated with 0.10 mL of 70.00 µg/mL different nanoparticles for 1.5 h and washed with 1 mL water for 3 times. Then, 0.10 mL of 0.80 mM TMB solution (pH 4.5) were added to tubes to collect and transfer the cells to 96-well plate and subsequently 0.10 mL of 1.00 M H2O2 solution (pH 4.5) was added. After 30 min, the absorbance at 652 nm was measured by microplate reader. As for the detection of cancer cell number, the varying numbers of Hela and L929 cells were incubated with 0.10 mL of 70.00 µg/mL TMV-FA-Pt NPs and treated following similar protocol above. For the cancer cell detection in mixed cell population, the Hela and L929 cells were mixed in different ratios while the total cell numbers were maintained the same level of 1.0×105 cells/mL and the similar analytical protocol was executed.

3. RESULTS



3.1 Synthesis and Characterization of TMV-Pt NPs. TMV-Pt NPs were fabricated by incubating with chloroplatinic acid hexahydrate (H2PtCl6), and then injecting the equal volume of cold NaBH4 solution containing sodium 2-mercaptoethanesulfonate (MPS), further stirring for 1 h. The TMV-Pt NPs were purified by centrifugation. TMV-Pt NPs maintained the rod-like structure (Figure 1A, B), and the platinum nanoparticles uniformly distributed on the outer surface of TMV (Figure 1C, D). The size of platinum nanoparticles on TMV exterior, i.e. 2.0 ± 0.5 nm (Figure 1F), were measured from TEM images, which were smaller than Pt NPs in solution (4.8 ± 1.6 nm, Figure S7). High-resolution transmission electron microscopy (HRTEM) indicated that the continuous lattice spacing of 0.22 nm (Figure 1E) that nicely corresponded to the (111) facet of the face-centered (fcc) platinum crystal, which was the same as Pt NPs in solution (Figure S7).

Figure 1. (A) DLS analysis of TMV NPs (black) and TMV-Pt NPs (red). (B-D) TEM images of TMV-Pt NPs at different magnification. (E) HRTEM of Pt NPs on TMV exterior. The lattice spacing of Pt NPs was ~0.22 nm. Scale bars: 2 µm for (B), 100 nm for (C), 10 nm for (D) and 3 nm for (E). (F) The histogram illustration of the size distribution of Pt NPs on TMV exterior surface.


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3.2 Peroxidase-like Activity of TMV-Pt NPs. The chromogenic reaction of 3,3′,5,5′-tetramethylbenzidine (TMB) and hydrogen peroxide (H2O2) was employed to investigate the peroxidase-like activity of TMV-Pt NPs. The color of the substrate solution containing TMB and H2O2 changed from colorless to blue due to the oxidation of TMB by H2O2, and the UV-vis absorbance at 652 nm of the solution was measured to monitor the conversation of TMB.46,47 TMV had a very weak peroxidase-like activity because the negative charges on the exterior surface of TMV caused the increasing of local concentration of TMB (Figure 2A).11 As comparison, the ability of TMV-Pt NPs to catalyze the oxidation of TMB was remarkably increased. The active sites of Pt NPs on TMV-Pt NPs were partially capped by TMV which caused the activity decreasing of Pt NPs. However, the smaller size of Pt NPs on TMV exterior would compensate their activity. Consequently, the activities were not significantly different between Pt NPs on TMV exterior and Pt NPs in solution (Figure 2A). More importantly, the TMV-Pt NPs could be reused while the peroxidase-like activity of TMV-Pt NPs remained almost constant after several recycles (Figure S8). Furthermore, like other peroxidase-like activity nanomaterials,9 the catalytic activity of TMV-Pt NPs largely depended on temperature, pH and the concentration of H2O2. It showed high activity at the conditions of 15~35 °C (Figure 2B) and pH 3~5 (Figure 2C). With the concentration of H2O2 increasing, the relative activity of TMV-Pt NPs was raising and started to stabilize (Figure 2D) when the concentration of H2O2 was up to 3 M.



Figure 2. (A) Time-dependent UV-vis absorbance at 652 nm from the reaction solutions containing water (black), TMV NPs (red), Pt NPs (pink), or TMV-Pt NPs (blue). The peroxidase-like activity of TMV-Pt depended on temperature (B), pH (C) and the concentrations of H2O2 (D). The relative activities were expressed relative to the maximal UV-Vis absorbance after 5 min. In order to study the peroxidase-like activity of TMV-Pt NPs, we investigated the steady-state kinetics of the oxidation of TMB catalyzed by TMV-Pt NPs. The initial reaction rates vs substrate concentrations were obtained by varying the concentrations of TMB (Figure 3A) or H2O2 (Figure 3B), which could be fitted to Michaelis-Menten curves. Michaelis constant (Km) could be obtained through transforming the plots to Lineweaver-Burk plots (Figure 3C, D). For nature enzyme, the value of Km is generally used to demonstrate the enzyme affinity to substrate, i.e. the smaller the value of Km is, the higher affinity the enzyme has towards the substrate.48 Table 1 suggests that the Km value of TMV-Pt NPs towards H2O2 (1.4 × 10-1 M) was higher than the one of HRP (3.7 × 10-3 M),9 implying that the affinity of TMV-Pt NPs to H2O2 was weaker than HRP. The Km value of TMV-Pt NPs (1.5 × 10-4 M) to TMB was in the same magnitude of HRP (2.3 × 10-4 M), which


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revealed the affinity of TMV-Pt NPs to TMB was approximate to HRP. The values of catalytic constants (kcat) of Pt NPs on TMV suggests its activity was slightly lower than that of HRP.

Figure 3. Steady-state kinetics of TMV-Pt NPs toward TMB (A) and H2O2 (B). Lineweaver-Burk plots of TMV-Pt NPs for TMB (C) or H2O2 (D) were calculated from (A) and (B), respectively. Table 1. Comparison of the kinetic parameters of different nanoparticles. [E]: nanoparticle concentration, Km: Michaelis constant, and kcat: catalytic constant. Catalyst

[E] (M)

Substrate

Km(M)

kcat (s-1)

TMV-Pt NPs

8.1 × 10-11

H2O2

1.4 × 10-1

6.4 × 102

8.1 × 10-11

TMB

1.5 × 10-4

3.0 × 102

2.5 × 10-11

H2O2

3.7 × 10-3

3.5 × 103

2.5 × 10-11

TMB

4.3 × 10-4

4.0 × 103

HRP9

3.3 Colorimetric Detection of Cancer Cells. It was reported that folate receptors were overexpressed in certain cancer cells (e.g. Hela cell, MCF-7 cell).22 Therefore, we conjugated



N3-PEG1000-FA to TMV through “click” chemistry to get TMV-FA (Figure 4A). Initially, alkyne groups were linked to the exterior surface of TMV by the reaction between tyrosine residues and diazonium salt using established protocols.40 Subsequently, N3-PEG1000-FA molecules were conjugated to TMV through CuAAC reaction. MALDI-TOF-MS spectrogram (Figure 4B) shows the appearance of the ion intensity peak of TMV-alkyne coat proteins (17654.9 Da) and the one of the coat proteins coupled with N3-PEG1000-FA (19039.7 Da), which suggests that coat proteins were partially linked with N3-PEG1000-FA. Subsequently, TMV-FA-Pt NPs were prepared following the aforementioned protocol the same as TMV-Pt NPs. Since the Pt NPs deposition was done after N3-PEG-FA has been conjugated, the size and the peroxidase-like activity of Pt NPs on TMV-FA was not significantly influenced by the conjugation (Figure S9-S13, Table S1).

Figure 4. (A) The reaction scheme between tyrosine residues of TMV on the exterior surface and N3-PEG1000-FA via diazonium coupling reaction and the subsequent CuAAC reaction. (B) MALDI-TOF-MS spectrogram of TMV-FA. The ion intensity peak at 17654.9 Da represents the molecular weight of alkyne-modified TMV coat proteins, and the one at 19039.7 Da is the molecular weight of TMV coat proteins conjugated with N3-PEG1000-FA. To verify the ability of TMV-FA-Pt NPs to detect folate receptors overexpressed cancer cells, we selected MCF-7 and Hela cells as target cells while L929 cells that lack of folate receptors for control groups. In order to eliminate endocytosis of cells to TMV-FA-Pt NPs, cells were fixed with 4 % paraformaldehyde before detecting. Therefore, 5.0 × 105 cells/mL various fixed cells were


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collected by centrifugation and co-incubated with the solution of Pt NPs, TMV-Pt NPs and TMV-FA-Pt NPs for 1.5 h, whose concentrations of platinum were 70 μg/mL. Then, unbound nanoparticles were removed by centrifugation and washing with water for 3 times. In the presence of TMB and H2O2, the nanoparticles bounding cells would promote the oxidization of TMB to color change that could be quantitatively monitored through measuring the absorbance at 652 nm. Compared with the cells co-incubated with Pt NPs and TMV-Pt NPs, MCF-7 and Hela cells treated by TMV-FA-Pt NPs had higher absorbance because of their recognizing interaction with folate receptors (Figure 5A). Furthermore, the different extent of color changes can be obviously differentiated with naked eyes (Figure 5B). To determinate the sensitivity of this assay, different numbers of Hela and L929 cells were screened. Figure 6 shows that, with the increase number of cells, the absorbance of both L929 cells and Hela cells was increasing while the absorbance of Hela cells went up more rapidly and reached plateau when the cell number was higher than 5.0 × 105 cells/mL. Especially, the absorbance was still significantly different between normal cells (L929 cells) and folate receptor overexpressing cancer cells (Hela cells) even if the cell concentrations were as low as 1.0 × 104 cells/mL. In order to further test the sensitivity and detection of this assay, different ratios of a mixed cells population of Hela and L929 cells were prepared. As shown in Figure S14, the absorbance was increased with the increase number of Hela cells.

Figure 5. (A) Cancer cell detection with TMV-FA-Pt NPs. The UV-Vis absorbance of the solutions respectively containing TMB, H2O2, various cells at 5 × 105 cells/mL treated by different



nanoparticles were measured at 652 nm. (B) Photographs of cancer cell detection at 5 × 105 cells/mL using TMV-FA-Pt NPs (1: water, 2: L929, 3: MCF-7, 4: Hela).

Figure 6. Detection of folate receptor overexpressed Hela cells with TMV-FA-Pt NPs. The UV-Vis absorbance of the solutions respectively containing TMB, H2O2, various cells (L929 and Hela) treated by TMV-FA-Pt NPs were measured at 652 nm.

4. DISCUSSION CELLISA is a low cost and practical diagnostic method. As the most used signal transducer and amplifier in the assay, HRP has obvious drawbacks which limit its application scopes. Recently, various peroxidase-like nanomaterials, such as Fe3O4 nanoparticles, platinum nanoparticles and oxide graphene, were developed and applied in disease diagnostic applications on account of their excellent activity, stability and low cost.9,13,16 To improve the detection sensitivity of CELLISA, an ideal peroxidase-like nanomaterial is expected to possess high catalytic activity, robust strategy to conjugate target motifs and good monodispersity in aqueous solution. TMV had a highly uniform rod-like nanostructure consisted of 2130 coat proteins helically assembled around single RNA. The resolved X-ray diffraction demonstrated that the helix formed


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by TMV coat protein subunits have identical pitch of 23 Å.24 Due to the uniform chemical properties and identical topography, inorganic particles and organic groups have been readily displayed on the TMV exterior surface. It was stated that regularly distributed ligands of metal ions, like glutamate and aspartate residues on the exterior surface of TMV, were able to modulate the deposition of various metal nanoparticles.49-51 Based on our results, platinum precursor preferentially deposited to generate narrow-dispersed Pt NPs evenly distributed on the outer surface of TMV (Figure 1). It is well known that the distance between the closest coat proteins TMV along the length direction is ~ 2.5 nm, the distance between the nearest coat proteins along circle direction is ~ 3.5 nm, and the distance between neighbor pitches of TMV is ~ 2.3 nm.24 The size of Pt NPs (2.0 ± 0.5 nm) on the TMV exterior was appropriate to the distance between neighbor pitches, while the size of Pt NPs (4.8 ± 1.6 nm) in solution was larger. Hence, we deduced that the subtle structure of TMV led to the uniform and smaller sizes of Pt NPs on TMV exterior. However, the TMV coated metal nanoparticles were unavoidable to aggregate due to the high interface energy of metal nanoparticles.52 Sodium 3-mercapto-1-propanesulfonate (MPS) has been previously used to stabilize metal nanoparticles because of their sulfonate groups with negative charge and sulfhydryl which has strong chelate affinity to metal nanoparticles.53,54 In this work, MPS was used to protect TMV-Pt NPs through generating strong electrostatic repulsion between Pt NPs. Consequently, TMV-Pt NPs were able to disperse in water and remained the good monodispersity as TMV (Figure 1A). Otherwise, TMV instinctively possessed high surface area and groups which could be efficiently incorporated functionalities through “Click Chemistry”. In this work, tyrosine residues of TMV outer surface were completely coupled with alkyne via diazonium reaction. Subsequently, 7 % of alkyne modified TMV coat proteins were conjugated with N3-PEG1000-FA via CuAAC reaction (Figure 4). The low modifying ratio of TMV coat proteins was mainly attributed to the high binding affinity of copper to PEG backbone lowered the efficient of catalyst.40



Platinum nanoparticles were demonstrated to equip with peroxidase-like activity and have been utilized in biomolecule monitoring and pollution detection.14,46 In general, the smaller size of nanoparticles is, the higher catalytic activity is in the equal amount of catalyst.6,9,30 The size of Pt NPs in the solution was controlled by the concentration and type of precursors, reductant and protectant.55 In our study, the size and density of Pt NPs on TMV hit a plateau when the concentration of [PtCl6]2- was 2.5 mM on account of well-defined topography of TMV exterior (Figure S16, Table S2). The size of Pt NPs on TMV was smaller than Pt NPs in solution, while their activity was comparable. We speculate that Pt NPs were partly embedded in the grooves of TMV which caused the decreasing of active sites of Pt NPs.56 It was demonstrated that the oxidization of TMB by H2O2 in the present of platinum metals was ascribed to the charge transfer between TMB and HO• radical generated through decompositions of H2O2 on platinum surface.57 Hence, the affinity to substrates would significantly affect the activity of catalyst. Since the affinity to TMB of TMV-Pt NPs was approximate to HRP, the lower affinity to H2O2 was the major factor that the catalytic activity of Pt NPs on TMV exterior was lower than HRP (Table 1). Folate receptors were overexpressed in certain cancer cells and have commonly been used as biomarkers for drug delivery and cancer detection. Here, N3-PEG1000-FA, with a length of > 2.3 nm,58 which was larger than the diameter of Pt NPs on the TMV exterior, was employed to construct TMV-FA-Pt NPs. The specific affinity to cancer cells of TMV-FA-Pt NPs (Figure 5) indicated that some of folic acid moieties were available for cell binding. Cancer cells could be detected as low as 1.0 × 104 cells/mL (Figure 6) by quantifying the color changes. Here, TMV not only served as a template for Pt nanoparticle growth, but also provided the reactive sites for the conjugation of functional units. In addition, due to the large surface area of TMV for interaction with cells, the incorporation of Pt nanoparticles on TMV increased the numbers of Pt NPs on cell surface, thus improving the CELLISA sensitivity. The achieved cancer cell detection in mixed cell population suggests the application potential of TMV-FA-Pt NPs for cells detection in histology section and tissue homogenate. In consideration of the favorable performance of these TMV-based


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nanoparticles, we can expect to introduce other recognition ligands to the outer surface of TMV, such as single domain antibodies for antigen test,59 target molecules for endotoxin check,60 aptamers for pesticides detection,61 either by chemical reaction or genetic engineering methods, which may expand this strategy for a broad scope of applications in bio-detection, food security and pollutants detection. We believe that this virus-based nanoparticles equipped with both recognition ligands and stimuli-responsiveness will contribute to its extensive future applications in biomedicine fields and environment monitoring.

ACKNOWLEDGEMENT: This study was partially supported by the National Natural Science Foundation of China (Programs21374119, 21429401). YL thanks the financial support from the Youth Innovation Promotion Association of the Chinese Academy of Sciences (2014200) and Youth Science Foundation of Jilin Province (20160520004JH, 20170101189JC) as well as State Key Laboratory of Precision Measuring Technology and Instruments (Tianjin University). QW would like to acknowledge the partial financial support from the NSF OIA-1655740.

ASSOCIATED CONTENT The supporting information is supplied. This material is available free of charge via the Internet at http:// pubs.acs.org. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected].

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