Pt-Decorated Magnetic Nanozymes for Facile and Sensitive Point-of

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Pt-Decorated Magnetic Nanozymes for Facile and Sensitive Point-of-Care Bioassay Min Su Kim, Soon Ho Kweon, Seongyeon Cho, Seong Soo A An, Moon Il Kim, Junsang Doh, and Jinwoo Lee ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b12326 • Publication Date (Web): 25 Sep 2017 Downloaded from http://pubs.acs.org on September 25, 2017

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ACS Applied Materials & Interfaces

Pt-Decorated Magnetic Nanozymes for Facile and Sensitive Point-of-Care Bioassay Min Su Kim,†, # Soon Ho Kweon,‡, # Seongyeon Cho,§ Seong Soo A. An,§ Moon Il Kim,§, * Junsang Doh,‡,∥, * Jinwoo Lee†, *

†

Department of Chemical Engineering, Pohang University of Science and Technology

(POSTECH), Pohang, Gyeongbuk 37673, Republic of Korea ‡

School of Interdisciplinary Bioscience and Bioengineering (I-Bio), POSTECH, Pohang,

Gyeongbuk 37673, Republic of Korea §

Department of BioNano Technology, Gachon University, Seongnam, Gyeonggi 13120,

Republic of Korea

∥

Department of Mechanical Engineering, Pohang University of Science and Technology

(POSTECH), Pohang, Gyeongbuk 37673, Republic of Korea #

These authors contributed equally to this work.

KEYWORDS: biosensors, immunoassays, nanozymes, enzyme-mimetics, diagnostics

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ABSTRACT

Development of facile and sensitive bioassay is important for many point-of-care applications. In this study, we fulfilled such demand by synthesizing Pt-decorated magnetic nanozymes and developing a bioassay based on unique properties of the newly synthesized nanozymes. Fe3O4-Pt/Core-Shell nanoparticles (MPt/CS NPs) with various compositions were synthesized and characterized. Fe3O4 NP itself is a good nanozyme with catalytic activity superior to natural enzymes, but catalytic activity can be further improved by incorporating Pt to the outer layers of the Fe3O4 NPs and building heterogeneous nanostructures. Magnetic properties of MPt/CS NPs enable magnetic enrichment of liquid samples, whereas catalytic properties of MPt/CS NPs allow signal amplification by enzymelike reactions. By integrating MPt/CS NPs in lateral flow immunoassay strips, one of the widely used point-of-care bioassay devices, and harnessing magnetic and enzyme-like properties of MPt/CS NPs, two orders of magnitude increase in sensitivity was achieved compared to conventional lateral flow immunoassay based on Au nanoparticles.


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Introduction Developing a reliable, accurate, and user-friendly diagnostic device for point-of-care (PoC) applications is one of the most important objectives in clinical medicine. Lateral flow immunoassay (LFIA) strips are among the most widely-used PoC bioassay devices for many field applications, including the diagnosis of blood infections, contamination, drug abuse, and pregnancy.1-5 It is extremely simple to perform assay using LFIA strips: 10~15 min after applying a drop of liquid sample on one end of the strip, a single line or double lines are visible on the other side of the strips. Typically, double lines indicate presence of target analytes above certain concentrations, thus we can easily obtain qualitative information about sample analytes with our bare eyes. Dark-colored nanoparticles (NPs) conjugated with antibodies capable of capturing target analytes have been used for the visualization of lines on the LFIA strips. In particular, Au NPs with ~30 nm are widely used for two reasons:6 their unique plasmon absorption generates dark purple color, readily detectable by bare eyes. In addition, their size is ideal for the transportation through pores of nitrocellulose membranes by capillary forces. Although colorimetric assay based on Au NPs allow analyte detection without using any sophisticated detectors, applications of Au NP-based LFIA have been limited due to its relatively low sensitivity.6,

7

To overcome this limitation, various signal-amplification

methods have been introduced such as fluorescence,8,

9

chemiluminescence,10 or heat

generation by plasmonic NPs11. However, these methods require specialized devices to detect the corresponding signals, and therefore have not been widely used in PoC applications. LFIA based on colorimetric method with increased sensitivity would be more desirable. To achieve this goal, natural enzymes that further generate colors by converting substrates were loaded on Au NPs.12 However, natural enzymes are fragile biomolecules that can lose catalytic activity by denaturation even under normal assay conditions, thus are not suitable 3 ACS Paragon Plus Environment


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for PoC applications.13 Furthermore, the purification and storage of natural enzymes is usually laborious and expensive.14 Recently developed nanozymes based on inorganic nanomaterials can be an attractive alternative for Au NP-loaded with natural enzymes because they exhibit enzyme-like catalytic activities without denaturation, thus can be stored and used under wide ranges of pHs and temperatures.15-21 In addition, nanozymes can be easily produced in large scales and exhibit increased catalytic activities due to their large surface area and specific catalytic potentials.16, 22 Our research group harnessed these advantages of nanozymes and developed nanozymebased LFIA strips.23 We first designed and synthesized hierarchically structured Pt NPs (H-Pt NPs) optimal for LFIA applications. Then, we devised a new method that enabled us to perform H-Pt NP-mediated peroxidase reaction for signal amplification on LFIA strips. Of note, H-Pt NPs-based LFIA exhibited one order of magnitude enhancement in sensitivity compared with the conventional Au NP-based LFIA strips. Based on this achievement, in this study, we have developed Pt-decorated magnetic nanozymes that enables nanozyme-based LFIA after magnetic enrichment, thus can further increase sensitivity. Enrichment of target analytes using magnetic NPs is well established techniques from which sample analytes could be concentrated to several dozens of times.24 Importantly, this method only require magnets, thus can be readily applied for PoC applications. Fe3O4 NPs are promising candidate that have both magnetic properties and peroxidase-like properties.16 However, catalytic activity of bare Fe3O4 NPs can be further improved by introducing noble metals on Fe3O4 NPs. For example, PtPd-Fe3O4 dumbbell-like heterogeneous NPs have superior peroxidase-like activity; the increased catalytic activity is a result of a synergy between PtPd and Fe3O4 NPs.25 Also Fe3O4-Pt composite NPs have higher 4 ACS Paragon Plus Environment

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peroxidase-like catalytic activity than do Fe3O4 NPs.26 Although these works revealed that noble metal-Fe3O4 heterogeneous composites can significantly increase catalytic activity of Fe3O4 NPs, reported heterostructured NPs are synthesized in organic solvent, and surface of the NPs were subsequently further functionalized with hydrophilic polymer surfactant such as poly(ethylene glycol) to disperse NPs in aqueous environments for bio-related applications. Considering polymeric surfactant may significantly hinder an access route of substrate,17 small molecule chain surfactant-stabilized NPs are more desirable for high catalytic activity. In short, to increase the sensitivity of nanozyme-based LFIA, four requirements must be met: (i) nanozymes with magnetic properties for magnetic enrichment (ii) improvement of peroxidase-like activity by decorating a small amounts of noble metals (iii) optimization of noble metal-metal oxide heterogeneous composite, and (iv) short surfactant layers. To satisfy these requirements, we synthesized various compositions of Fe3O4-Pt/Core-Shell (MPt/CS) NPs in aqueous phase using citrate as a reducing and surface-stabilizing agent. By following procedures in Scheme 1, magnetic enrichment, LFIA-based detection, and nanozymemediated signal amplification, two orders of magnitude sensitivity improvement were achieved compared with conventional Au NP-based LFIA.



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Results and Discussion Synthesis and characterization of MPt/CS NPs To construct the MPt/CS NPs, we first synthesized Fe3O4 NPs using a co-precipitation method.27 Then Pt was decorated onto Fe3O4 NPs by a seed-growth method using sodium citrate to reduce chloroplatinic acid. The synthesized MPt/CS NPs were characterized using transmission electron microscopy (TEM), X-ray diffraction (XRD), and high-resolution transmission electron microscopy (HR-TEM). The compositions of MPt/CS NPs were controlled by the mass ratios of Pt precursor/Fe3O4 NPs and were measured by inductively coupled plasma atomic emission spectroscopy (ICP-AES). The nomenclatural representation we have devised to describe the composition of MPt/CS NPs is, MPt/CS-X NPs, where X is the weight percentage of Pt. Figure 1 shows typical TEM images of MPt/CS-7, MPt/CS-15 and MPt/CS-30 NPs. The average size and size distributions of the Fe3O4 and MPt/CS NPs are shown in Figure S1 in supporting information (SI). Most of the Fe3O4 and MPt/CS NPs were ~15 nm in diameter, and separate Pt NPs were not observed. However, when the loading amount of Pt was increased to 60 wt%, large Pt aggregates were generated, because the amount of Pt exceeded the amount required to decorate the Fe3O4 NPs surfaces (Figure S2 in SI). Elemental mapping of MPt/CS NPs (Figure 1) and HR-TEM images (Figure 2) confirmed successful decoration of Pt on Fe3O4 NPs surfaces. MPt/CS-7 NPs have few layers of Pt on Fe3O4 NPs, whereas MPt/CS-30 NPs have ~1 nm Pt islands on the surface of Fe3O4 NPs. These distinct morphologies of MPt/CS-7 and 30 NPs can be explained by a heterogeneous nucleation and growth model.28 In particular, lattice mismatch between Pt and Fe3O4 determines the growth mode and final morphology of nanostructures. Excess energy ∆γ during the growth of Pt on Fe3O4 NPs is: ∆γ = γa + γi – γb 6 ACS Paragon Plus Environment

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(γa: surface energy of shell material, γb: surface energy of core material, γi: interfacial energy) Depending on the sign of ∆γ, three different types of growth modes can be observed (Figure S3 in SI). First, if the lattice mismatch between core and shell is small, the sum of surface energy of depositing metal and interfacial energy is smaller than the surface energy of substrate material (∆γ < 0), so growth follows Frank-van der Merwe (FM, layer-by-layer) mode.29, 30 If the mismatch between core and shell is relatively large, or ∆γ > 0, growth follows Volmer-Weber (VW, island growth) mode to minimize interfaces during the growth.30 Lastly, if ∆γ is initially negative, but changes to positive during growth, it follows Stranski-Krastanov (SK, island-on-wetting-layer growth) mode.28 Similar to FM mode, SK mode begins with layer-by-layer growth of metal on substrate material because ∆γ < 0 initially; however, the strain energy increases gradually with increase in the number of layers of depositing metal. If the number of layers reaches a threshold value, ∆γ becomes positive and metal growth switches to VW mode. The growth mode of MPt/CS NPs fits well with the SK mode: with low Pt loading (MPt/CS-7), multiple atomic layers of Pt were formed, whereas with increased Pt loading (MPt/CS-30), nanoscale Pt islands formed. XRD patterns of the MPt/CS NPs clearly show peaks for the cubic crystal structure of magnetite (JCPDS, #89-0691) (Figure 3a). No appreciable peaks of Pt were observed for MPt/CS-7 and 15 NPs, but as the weight percentage of Pt increased, clear peaks for cubic structure of Pt (JCPDS, #88-2342) appeared for MPt/CS-30 NPs. The crystallite sizes calculated by the Scherrer equation were 13.21 nm for Fe3O4 NPs, which is similar to the average size measured from the TEM images. Both TEM images and XRD patterns of MPt/CS NPs showed that the crystallite size of magnetite from MPt/CS-7, 15 and 30 NPs were comparable, suggesting that introduction of Pt only affected the loading amount of Pt on 7 ACS Paragon Plus Environment


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the surface of Fe3O4 NPs, not the structure of Fe3O4 NPs. In addition to these structural characterizations, we measured magnetic properties of MPt/CS NPs (Figure 3b), which is a critical property for magnetic separation. The magnetic curves of Fe3O4 NPs showed typical curves for superparamagnetic nanomaterials without any hysteresis. The saturated magnetization value of Fe3O4 NPs was 58.5 emu/g, and it gradually decreased as Pt amounts increased: 54.9 emu/g (6.15% decrease) for MPt/CS-7 NPs, 47.4 emu/g (19.0% decrease) for MPt/CS-15 NPs, and 37.2 emu/g (36.4% decrease) for MPt/CS-30 NPs.

Catalytic activity of MPt/CS NPs We next examined the catalytic activities of the MPt/CS NPs by performing the representative peroxidase-mediated 3,3’,5,5’-tetramethylbenzidine (TMB) oxidation reaction in the presence of H2O2. The peroxidase activity of MPt/CS-30 NPs was compared with natural horseradish peroxidase (HRP), an enzyme that is widely used in immunoassays. The blue color of the solution deepened more after reaction with MPt/CS-30 NPs than with HRP (Figure 4a) when the applied concentrations of MPt/CS-30 NPs and HRP were the same (2.9 × 10-10 M). Therefore, we concluded that MPt/CS-30 NPs has higher catalytic activity than HRP. Also, MPt/CS-30 NPs can catalyze the oxidation of several other colorimetric substrates such as 3,3’-diaminobenzidine (DAB, producing reddish product) and ophenylendiamine (OPD, producing yellow/orange product) as well as TMB (Figure 4c, d). To further demonstrate the catalytic mechanism, steady-state kinetic parameters of Fe3O4, MPt/CS-7, MPt/CS-15, and MPt/CS-30 NPs were determined. Under our experimental conditions, the optimal pH was 3.5 and optimal H2O2 concentration was 100 mM (Figure S4 in SI). Thus, subsequent experiments were performed under these experimental conditions. 8 ACS Paragon Plus Environment

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Because Pt NPs exhibit considerable catalytic activity in oxidizing TMB even without H2O2, kinetic parameters of only TMB were calculated by performing a series of experiments with various concentrations of TMB while keeping the concentration of H2O2 at 100 mM. Kinetic parameters were extracted from the experimental data using Michaelis-Menten model. Michaelis-Menten model has been successfully applied to describe the kinetic behavior of nanozymes, even though it does not completely recapitulate enzyme-like reaction mechanisms of nanozymes.16, 21, 25 Similar to the previous study, our data fitted well with the Michaelis-Menten model when the initial reaction rate vs. TMB concentration was plotted for each type of NP (Figure S5 in SI). The curves were then fitted to Lineweaver-Burk plots (Figure S6 in SI), and the kinetic parameters for Fe3O4 and MPt/CS NPs were calculated (Table 1). The Km value of Fe3O4 NPs was the smallest, and as the loading amount of Pt increased, the Km values gradually increased, indicating the affinity of TMB-Pt is lower than that of TMB-Fe3O4. Although MPt/CS NPs exhibited lower affinity for TMB substrate than Fe3O4 NPs, kcat, which means turnover number, was significantly higher than that of Fe3O4 NPs due to super-activity in catalyzing redox reactions of Pt. Furthermore, keff = kcat/Km, which measures the efficiency of catalyst, was an order of magnitude higher for all MPt/CS NPs than for Fe3O4 NPs. Taken together, the synthesized MPt/CS NPs exhibited superior catalytic activities than Fe3O4 NPs as well as conventional HRP.

Facile and sensitive detection of sample analytes by MPt/CS NP-based LFIA Based on these unique properties of MPt/CS NPs, we developed an ultra-sensitive immunoassay (Scheme 1). MPt/CS NPs conjugated with antibodies (Ab-MPt/CS NPs), which can selectively bind to target analytes, were used. First, target analytes in solution were captured and enriched using Ab-MPt/CS NPs by magnetic enrichment (Step 1). Second, Ab9 ACS Paragon Plus Environment


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MPt/CS NPs capturing target analytes were applied to the sample pad of a LFIA strip to perform LFIA (Step 2). Ab-MPt/CS NPs containing sufficient amounts of target analytes were captured on the T-line whereas the remaining Ab-MPt/CS NPs were captured on the Cline.23 Lastly, solution containing substrates was applied to the test pad to further amplify signals in T- and C-lines by enzyme-like reaction mediated by MPt/CS NPs (Step 3). Human chorionic gonadotropin (hCG), a biomarker for pregnancy, was used as a model biomarker, and LFIA strips were prepared by modifying commercially-available pregnancy test strips. Anti-hCG antibodies were attached on the surfaces of MPt/CS NPs for selective capturing of hCG. Various concentrations of hCG (0.01~1 ng/mL) in 15 mL PBS were prepared. Then, 15 µL of Ab-MPt/CS NPs in PBS (7 mg/mL) was added to each sample and mixed with gentle shaking. After 2 h of mixing, sample solution containing Ab-MPt/CS NPs were placed close to a magnet to separate hCG/Ab-MPt/CS NPs. Optimal separation time for Ab-MPt/CS NPs depended on the saturation magnetization values of MPt/CS NPs (Figure S7 in SI); more than 90% of Fe3O4, MPt/CS-7, and MPt/CS-15 NPs were recovered within 10 min of magnetic separation, whereas ~20% of NPs were still in solution for MPt/CS-30 NPs even after 20 min of magnetic separation. For all NPs, magnetic separation was performed for 20 min. After carefully removing the supernatant, the concentrated hCG/Ab-MPt/CS NPs were resuspended in 50 µL of conjugation buffer (PBS with 0.1% Tween 20, 3% BSA, 10% sucrose), and directly applied to the sample pad of the modified LFIA strip. In this process, hCG/Ab-MPt/CS NP complexes are immobilized in the T-line by antibodies that capture hCG, whereas Ab-MPt/CS NPs will be immobilized in the C-line by antibodies that bind to anti-hCG antibodies. After 5 min, 60 µL of sodium acetate buffer (0.1 mM, pH 4.5) was added to the sample pad to wash away unbound NPs and to provide acidic environments in 10 ACS Paragon Plus Environment

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the strips for the peroxidase-like reaction. Then, small droplet containing H2O2 and 3.8 mM 3-amino-9-ethylcarbazole (AEC), which generates red precipitate when oxidized by peroxidases, was applied to the test pad for 5 min to amplify signals in T- and C-line. Digital images of the test pads for various concentrations of hCG samples were acquired (Figure 5a) and further processed to quantify signal intensities of T-lines.23 Absolute intensity values of T-lines may vary depending on image acquisition conditions, including light illumination and camera settings. To compensate for such variations, the intensity ratio = (T-line intensity)/(Cline intensity) was calculated after background subtraction.23 Average intensity ratio for various [hCG]s were analyzed by linear fitting (Figure 5b). For all MPt/CS-based LFIAs, average intensity ratio increased linearly with [hCG]. Overall, MPt/CS-15-based LFIAs exhibited the highest intensity ratio for most [hCG]s. Using the linear equation obtained by linear fitting for each MPt/CS-based LFIA, [hCG] corresponds to the (average background + 3 (standard deviation of background)) was calculated as limit of detection (LOD; Table 2).31, 32

MPt/CS-15-based LFIAs exhibited the lowest LOD, or the most sensitive detection. Taken

together, MPt/CS-15 NPs was the best material for the detection method developed in this study (Scheme 1) among three MPt/CS NPs tested in our study. The amounts of antibodies attached to MPt/CS NPs were not significantly different from each other (Figure S8 in SI), so these results are mostly due to compositions of MPt/CS NPs. Increase in Pt content in MPt/CS NPs increases the catalytic activity (Table 1), which is critical for signal amplification (Step 3 in Scheme 1), but reduces maximum magnetization (Figure 3b), which is important for magnetic separation (Step 1 in Scheme 1). Therefore, even though MPt/CS30 NPs had the best catalytic activity, poor recovery after magnetic separation (Figure S8 in SI) limited its overall efficiency. Importantly, LODs of all MPt/CS-based methods were approximately two orders of magnitude lower than conventional Au NP-based LFIAs, and



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approximately one order of magnitude lower than our previously-developed enzyme-mimetic NP (H-Pt NP)-based LFIAs (Table 2).

Conclusions We synthesized MPt/CS NPs and incorporated them into LFIA strips as highly-active and magnetically-separable probes. We proved that MPt/CS NPs have high catalytic efficiency owing to high catalytic properties and high affinity for the colorimetric substrate. Moreover, we demonstrated that this new bioassay platform has two orders of higher sensitivity than conventional Au NP-based LFIA. These results suggest that MPt/CS NPs combined LFIA strips enable efficient PoC detection in clinical diagnostics that require ultra-low detection limits.


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SCHEME

Scheme 1. Schematic illustration of analyte detection using magnetic nanozyme-based LFIA strips.



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FIGURES AND TABLES

Figure 1. TEM and elemental mappings of (a) MPt/CS-7, (b) MPt/CS-15 and (c) MPt/CS-30 NPs.


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Figure 2. HR-TEM images of (a, b) MPt/CS-7, and (c, d) MPt/CS-30 NPs.



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Figure 3. (a) XRD patterns of Fe3O4, MPt/CS-7, MPt/CS-15 and MPt/CS-30 NPs. (b) magnetization curves for Fe3O4, MPt/CS-7, MPt/CS-15 and MPt/CS-30 NPs.


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Figure 4. (a) Absorption spectra of color generation by TMB oxidation mediated by identical concentration of MPt/CS-30 NPs and HRP (2.9 × 10-10 M) and representative images (inset). (b) Kinetics of TMB oxidation mediated by various concentrations of MPt/CS-30 NPs accessed by absorbance at 652 nm. (Molar concentration of MPt/CS-30 NPs was calculated based on the assumption that MPt/CS-30 NP is a sphere with a diameter of 15 nm) (c, d) Photographs of the color-generation reaction of TMB, DAB and OPD in the presence of H2O2 for identical concentration of MPt/CS-30 and HRP. (In a typical reaction, a solution containing the colorimetric substrate (0.5 mM TMB, 5.5 mM DAB, or 6 mM OPD), 2.9 × 1010

M MPt/CS-30 NPs, and 100 mM H2O2 in a reaction buffer (sodium acetate, 0.1 M, pH 3.5)

was incubated at room temperature for 5 min. For the HRP-mediated colorimetric reaction, 2.9 × 10-10 M HRP was added into a reaction buffer (sodium acetate, 0.1 M, pH 4.0) containing 10 mM H2O2 and the same amount of the colorimetric substrate (0.5 mM TMB, 5.5 mM DAB, or 6 mM OPD)) 17 ACS Paragon Plus Environment


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Table 1. Kinetic parameters of the Fe3O4, MPt/CS-7, MPt/CS-15 and MPt/CS-30 NPs (TMB as a substrate). Km: Michaelis-Menten constant; kcat = Vmax/[E], and keff = kcat/Km where Vmax is the maximal reaction velocity and [E] is the concentration of the nanozymes.

MPt/CS-30 MPt/CS-15 MPt/CS-7 Fe3O4

Used (µg/mL)

[E] (M)

Km (mM)

Vmax (nM/s)

kcat (s-1)

keff (106)

3

2.89×10-10

0.055

634.6

2196

39.94

3

3.78×10

-10

0.029

255.9

677

23.34

4.60×10

-10

0.028

257.1

559

19.95

-9

0.026

329.4

58

2.23

3 30

5.64×10


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Figure 5. Quantitative detection of [hCG] using MPt/CS NP-based LFIA. (a) Representative digital images of MPt/CS-15 NP-based LFIA strips. (b) Mean intensity ratio (T-line/C-line) versus [hCG] of MPt/CS NP-based LFIA. (error bar: standard deviation, n=3)



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Table 2. LOD of MPt/CS, our previously developed H-Pt and conventional Au NP-based LFIAs.

LOD (ng/mL)

MPt/CS-7

MPt/CS-15

MPt/CS-30

H-Pta)

Aua)

0.039

0.025

0.044

0.20

3.7

a)

H-Pt NP-based LFIA and commercially available Au NP-based LFIA data are from our previous study23


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Experimental Section Chemicals: Iron (II) chloride tetrahydrate, iron (III) chloride hexahydrate, ammonium hydroxide solution (28%), hydroxylamine hydrochloride, tetramethylammonium hydroxide pentahydrate (TMAOH), sodium citrate tribasic dehydrate, chloroplatinic acid hexahydrate, horseradish peroxidase (HRP), 3,3´,5,5´-tetramethylbenzidine (TMB), 3,3´-diaminobenzidine (DAB), o-phenylenediamine dihydrochloride (OPD), sodium acetate, and phosphate buffered saline (0.01M, pH 7.4) were all purchased from Sigma-Aldrich (Milwaukee,Wi), and 35% H2O2 was purchased from Junsei Chemical Co. (Japan). All other chemicals were of analytical grade or higher. All solutions were prepared with DI-water purified by a Milli-Q Purification System (Millipore, USA).

Characterization: Structures of synthetic materials were examined using TEM with a JEOL JEM-2010 microscope, and HR-TEM with a JEOL JEM-2200FS (with Image Cs-corrector). Powder X-ray diffraction patterns were obtained using the PANalytical X’Pert diffractometer with Cu Kα radiation (=1.5306 Å). Magnetic property measurement was obtained using the MPMS XL-7 (Quantum Design Co.). Spectrophotometry was measured using a DU-800 UVVisible spectrophotometer (Beckman). Digital images of the LFIA strips were acquired using a smart cellular phone (GALAXY S4 SHV-E300S, Samsung).

Synthesis of Fe3O4 NPs by chemical co-precipitation: The Fe3O4 NPs were prepared by chemical co-precipitation:27 0.63 g of Iron (II) chloride tetrahydrate and 1.83 g of iron chloride hexahydrate were added to 20 mL of deionized water (DI-water) and heated to 80 °C under Ar. Then, ammonium hydroxide solution (5 mL; 28%) was injected into the solution 21 ACS Paragon Plus Environment


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and heated for 1 h at 80 °C. Synthesized Fe3O4 NPs were magnetically decanted and washed several times with deionized water (DI-water), then resuspended in DI-water.

Synthesis of MPt/CS NPs: Various compositions of MPt/CS NPs were synthesized using a seed-growth method: 50 mg of Fe3O4 NPs, 50 mg of hydroxylamine hydrochloride and 136 mg of TMAOH were diluted with 75 mL DI-water and heated to 80 °C in a three-neck roundbottom flask under vigorous stirring and Ar gas protection. Then, a solution of 40 mL chloroplatinic acid hexahydrate (MPt/CS-7: 11.2 mg, MPt/CS-15: 37.29 mg and MPt/CS-30: 111.86 mg in 40 mL DI-water) was added dropwise into the mixed solution and 100 mL of 15 mM sodium citrate was added incrementally within 2 h, and the mixture was stirred for 3 h after the addition. Finally, the MPt/CS NPs were purified by washing them several times with DI-water.

Evaluation of the peroxidase-like activities of the MPt/CS-30 NPs: Peroxidase-like activity of the MPt/CS-30 NPs were determined by the catalytic oxidation of colorimetric substrates TMB, DAB, and OPD in the presence of pre-determined concentration of H2O2. In a typical reaction, a solution containing the colorimetric substrate (0.5 mM TMB, 5.5 mM DAB, or 6 mM OPD), 3 µg/mL MPt/CS-30 NPs (equal to 2.9 × 10-10 M of MPt/CS-30 NPs), and 100 mM H2O2 in a reaction buffer (sodium acetate, 0.1 M, pH 3.5) was incubated at room temperature for 5 min. For the HRP-mediated colorimetric reaction, 13 ng/mL HRP was added into a reaction buffer (sodium acetate, 0.1 M, pH 4.0) containing 10 mM H2O2 and the same amount of the colorimetric substrate (0.5 mM TMB, 5.5 mM DAB, or 6 mM OPD). The absorbance of the solution was measured using a microplate reader in a scanning mode (Synergy H1, BioTek, VT). 22 ACS Paragon Plus Environment

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Characterization of peroxidase-like activities of MPt/CS NPs: Peroxidase-like activities of MPt/CS NPs were assessed by using standard colorimetric assays to measure oxidation of TMB. To measure kinetics of TMB oxidation, 100 µL of H2O2 (1 M), 100 µL of various [TMB] (31.23 µM ~ 1000 µM) and 30 µL of MPt/CS NPs (0.1 mg/mL) were added to 770 µL reaction buffer (sodium acetate, 0.1 M, pH 3.5) in a cuvette mounted on the spectrophotometer. Then, the reactions were monitored by measuring absorbance changed in kinetic mode at 652 nm for 1 min. Using early reaction data, the kinetic parameters for Michaelis-Menten equation (v = Vmax × [S]/(Km+[S])) were calculated, where v is the initial rate, Vmax is the maximal rate, [S] is the concentration of the substrate, and Km is the Michaelis-Menten constant.

Preparation of Ab-MPt/CS NPs conjugates: Antibody targeting β-hCG (Anti-β-hCG, clone: M705159, Fitzgerlad) was conjugated to MPt/CS NPs by physical adsorption. Briefly, 400 µL of MPt/CS NPs (7.0 mg/mL) in DI-water and 200 µL of anti-β-hCG (408 µg/mL) in Phosphate buffered saline (PBS, 10 mM sodium phosphate, 140 mM sodium chloride, pH 7.4) were mixed and incubated for 24 h at 4 °C with gentle shaking. Then 400 µL of blocking buffer (0.1% Tween-20 and 3% Bovine Serum Albumin (BSA) in PBS) was added to block the residual surface of the MPt/CS NPs for additional 24 h at 4 °C. The obtained solution was centrifuged three times at 14,000 rpm for 30 min at 4 °C and supernatant was discarded. Then 400 µL of blocking buffer was added to the antibody-conjugated MPt/CS NPs (Ab-MPt/CS NPs) for resuspension. Ab-MPt/CS NPs solution was stored at 4 °C.



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Magnetic separation of Ab-MPt/CS NPs: Ab-MPt/CS NP solution (15 µL; 7.0 mg/mL) was added to 15 mL of PBS in 20 mL glass vial (Wheaton) and vigorously mixed. Then, the vial was placed onto a permanent magnet, and 1 mL of supernatant was removed every 5 min for 20 min. Absorbance of supernatant at 220 nm was measured using DU-800 UV-Visible spectrophotometer (Beckman), and converted to normalized absorbance to assess the fraction of Ab-MPt/CS NPs remaining in the supernatant.

Quantification of antibodies conjugated to Ab-MPt/CS NPs: To determine the amount of antibody attached to the Ab-MPt/CS NPs, the amount of antibody remaining in the supernatant after conjugation was measured by the Bradford assay using a commercial kit (Pierce), and subtracted from the amount of antibody used for the conjugation.

Analyte Capturing and magnetic enrichment: Various concentrations of hCG diluted in 15 mL PBS (0.01 ≤ [hCG] ≤ 1 ng/mL) were prepared. Then, 15 µL of Ab-MPt/CS NP solution (7 mg/mL) was added to each samples and mixed with gentle shaking for 2 h. The samples were located in close contact with magnet for 20 min for magnetic separation. After carefully removing the supernatant, hCG/Ab-MPt/CS NP aggregates were resuspended with 50 µL of conjugation buffer (PBS containing 0.1% Tween 20, 3% BSA, 10% sucrose).

Preparation of LFIA: Commercially available LFIA strips for hCG detection (BioCards, IlYang Bio) were modified and used. First, conjugation pads were gently detached from the strips, rinsed three times in DI-water to remove Au NPs, dried overnight in vacuo, and


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reloaded in the strips. Second, half of sample pads were removed to minimize retention of hCG/Ab-MPt/CS complexes in the sample pads.

Analyte detection using MPt/CS NP-based LFIA: First, 50 µL of MPt/CS aggregates in conjugation buffer (PBS containing 0.1% Tween 20, 3% BSA, 10% sucrose) was gently dispensed onto a sample pad and allowed to migrate across a test pad for 5 min. Then 20 µL of sodium acetate buffer (0.1 mM, pH 4.5) was dispensed three times onto a sample pad to clear away unbound MPt/CS NPs in test pad and generate an acidic environment for enzymatic reaction. For peroxidase-like reaction, 50 µL of 3-amino-9-ethylcarbazole (AEC, Santa Cruz) in sodium acetate buffer (final concentration 3.8 mM) mixed with H2O2 (final concentration: 0.3%) was dropped to the test pad for 3 min. Then digital images of the LFIA strips were acquired using a smart cellular phone.

Image analysis: Image analysis was performed as previously described.23 Briefly the acquired LFI strip images were converted to 8-bit grayscale images and inverted by in ImageJ software (NIH). Then, the average intensity of background was subtracted from those of the T-line and C-line, and Intensity ratio (T-line/C-line) was calculated and plotted against [hCG]. Limit of detection (LOD) was defined as [hCG] corresponds to background intensity plus three times of its standard deviation.31, 32



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AUTHOR INFORMATION Corresponding Author *E-mail: (M. I. Kim) [email protected], (J. Doh) [email protected], (J. Lee) [email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. # These authors contributed equally to this work. Notes The authors declare no competing financial interests. ACKNOWLEDGMENT This work was supported by the Basic Science Research Program through the National Research Foundation (NRF) of Korea (J.L., grant number: NRF-2017R1A2B3004648; M.I.K., grant number: NRF-2017R1C1B2009460), a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare (J.D., grant number: HI17C0574), Republic of Korea, and J.L. and J.D. acknowledge the support by POSCO Green Science Project.

ASSOCIATED CONTENT Supporting Information: More information of samples, TEM, and elemental mapping images, schematic illustration of growth mode, optimal pH and H2O2 concentration, Michaelis-Menten plot, Lineweaver-Burk plot, magnetic separation images and quantification 26 ACS Paragon Plus Environment

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of antibodies. The following files are available free of charge via the Internet at http://pubs.acs.org.



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TABLE OF CONTENTS


Pt-Decorated Magnetic Nanozymes for Facile and Sensitive Point-of

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