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Biological and Medical Applications of Materials and Interfaces

An in situ Pt Staining Method for Simple, Stable, and Sensitive Pressure-based Bioassays Jiuxing Li, Fang Liu, Zhi Zhu, Dan Liu, Xiaofeng Chen, Yanling Song, Leiji Zhou, and Chaoyong James Yang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b03567 • Publication Date (Web): 27 Mar 2018 Downloaded from http://pubs.acs.org on March 27, 2018

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ACS Applied Materials & Interfaces

An in situ Pt Staining Method for Simple, Stable, and Sensitive Pressure-based Bioassays Jiuxing Li1, Fang Liu1, Zhi Zhu1, Dan Liu1, Xiaofeng Chen1,Yanling Song1, Leiji Zhou1, Chaoyong Yang1,2*

1: MOE Key Laboratory of Spectrochemical Analysis & Instrumentation, Key Laboratory of Chemical Biology of Fujian Province, Key Laboratory of Chronic Liver Disease and Hepatocellular Carcinoma of Fujian Province, State Key Laboratory of Physical Chemistry of Solid Surfaces, Collaborative Innovation Center of Chemistry for Energy Materials, Department of Chemical Engineering, Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, China

2: Institute of Molecular Medicine, Renji Hospital, Shanghai Jiao Tong University, School of Medicine, Shanghai, 200127, China

* To whom correspondence should be addressed. Tel: (+86) 592-218-7601, E-mail: [email protected] KEYWORDS: ELISA, AuNPs, Pt staining, pressure-based bioassays, point-of-care testing

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ABSTRACT:

Pressure-based bioassays (PASS) integrate a molecular recognition process with a catalyzed gas generation reaction, enabling sensitive and portable quantitation of biomarkers in clinical samples. Using platinum nanoparticles (PtNPs) as catalyst has significantly improved the sensitivity of PASS compared with protein enzyme-based detection. However, PtNPs are easily deactivated during storage or after being decorated with antibodies. Moreover, non-specific adsorption of PtNPs on substrate has been a problem, resulting in significant background. To solve these problems of PtNP-based detection, we report a robust, simple, stable, and sensitive Pt staining method for PASS. Detection antibody-decorated gold nanoparticles (AuNPs) are used to perform enzyme-linked immunosorbent assay (ELISA), followed by Pt staining to stain AuNPs with Ag and Pt bimetallic shells (Au@AgPtNPs), which endow AuNPs with catalytic activity. The concentration of target can be quantitatively determined by measuring the pressure due to O2 gas (g) formed by the decomposition of H2O2 catalyzed by Au@AgPtNPs. C-reactive protein (CRP) and avian influenza hemagglutinin 5 neuraminidase 1 (H5N1) can be quantitatively detected with detection limits of 0.015 and 0.065 ng/mL, respectively. The simple, stable, and sensitive properties of the Pt staining-based method will largely broaden the applications of PASS in clinical diagnosis and biomedicine.


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Introduction In the past decade, many detection platforms have been developed for disease diagnosis, including fluorescence,1-2 Raman,3-4 electrochemical,5 and magnetic methods.6 Compared with traditional assays, these methods showed significant advantages in sensitivity, selectivity, and practicality. However, due to the requirement of complex optical and electronic systems, their use in point-of-care testing (POCT) is limited. Recently reported PASS integrate a molecularrecognition process with a catalyzed gas-generation reaction,7-8 transducing the molecularrecognition event into an increase of gas pressure in a confined space. The concentration of targets can be accurately determined by measuring the increased pressure using a portable pressuremeter. Because the pressuremeter is simple, inexpensive, and portable, PASS adequately meets the affordable, equipment-free, user-friendly, and deliverable criteria of POCT9-10 and can quantitatively determine the concentrations of targets with high sensitivity and selectivity. Currently, nanomaterials with optical,11 electronic,12 magnetic,13 and catalytic14 activities are widely applied in clinical diagnosis and biomedicine. To improve the sensitivity of PASS, nanoparticles with high catalytic activity for the decomposition of H2O2 to produce O2 (g) are needed. As highly efficient catalysts, platinum nanoparticles (PtNPs) have been widely applied in chemical engineering, environmental monitoring, and medical diagnoses.15-18 It has been shown that PtNPs can efficiently catalyze the decomposition of H2O2 to generate O2 (g).7 Moreover, in bimetallic nanoparticles or alloys with other kinds of metal, the catalytic efficiency of Pt can be increased further.19-20 Using PtNPs as catalyst, the sensitivity of PASS is enhanced by 4 orders of magnitude compared to that of protein enzyme-based detection.7 However, nanomaterials with high catalytic activities are easily poisoned, and the catalytic activity decreases noticeably after decoration or adsorption of molecules.21-22 Furthermore, we found that


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PtNPs suffer from non-specific adsorption during the detection process, resulting in high backgrounds. Due to their unique electrical and optical properties, facile synthesis, easy functionalization, low cost, and excellent stability,23-24 AuNPs have been widely combined with biomolecules to develop commercially available immunochromatographic assays for the detection of human chorionic gonadotropin (HCG), hepatitis, influenza virus, pesticides, mycotoxins, veterinary drugs, malaria parasites, syphilis, and tuberculosis,25-30 guaranteeing human health and food security and improving the living environment.31-32 Based on the wide commercial application of AuNPs and in order to avoid decreased catalytic activity due to decoration or surface poisoning of pure PtNP catalyst and non-specific adsorption, we have developed a Pt staining method to stain AuNPs with a Pt shell and perform PASS detection. First, AuNP-labelled detection antibodies are used to carry out ELISA detection. Then, metal precursor and reductant are added to stain AuNPs with Ag and Pt bimetallic shells (Au@AgPtNPs) with catalase activity. Consequently, H2O2 can be decomposed by Au@AgPtNPs to generate O2 (g), increasing the gas pressure in a confined space (a sealed microtiter well in this application). Eventually, the concentration of target can be accurately determined by measuring the gas pressure using a portable pressuremeter. Due to the mature technology of synthesis and functionalization of AuNPs, well-defined spherical structures of AuNPs, and strong interaction between AuNPs and protein, non-specific adsorption of molecules on AuNPs can be prevented by coating with bovine serum albumin (BSA).33-34 Moreover, by freshly staining AuNPs with Ag and Pt bimetallic shells after ELISA detection, the poisoning of active sites and the decrease of enzyme activity after decoration are avoided. The simple, stable,


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portable, and high sensitivity of the Pt staining-based method will significantly broaden the use of PASS in clinical diagnoses and medical applications. Results and discussion Working principle of Pt staining-based ELISA detection. In order to develop a robust, simple, stable, and sensitive method for PASS, we propose a Pt staining-based ELISA method. The working principle is depicted in Figure 1, 16-nm AuNPs were synthesized according to the literature35 and then conjugated with detection antibodies by physical adsorption. Afterwards, the detection antibody-capped AuNPs are used for the immunosorbent assay in the wells of a microtiter plate. Due to the strong binding affinity between protein and AuNPs, non-specific adsorption of AuNPs on the microtiter plate is effectively prevented. Subsequently, AgNO3 solution and hydroquinone solution are added to the wells and used as Ag precursor and reducing agent to coat AuNPs with silver shells (Au@AgNPs), which can be used as a sacrificial layer for Pt staining. Subsequently, H2PtCl6 solution and ascorbic acid solution are added and used as Pt precursor and reductant to coat Au@AgNPs with a Pt layer, resulting in the formation of Ag and Pt bimetallic-coated AuNPs. Compared with AuNPs, catalytic activity of Au@AgPtNPs is significantly enhanced due to the intrinsic high catalytic activity of Pt, as well as the synergistic and electronic effects between Au, Ag, and Pt. Finally, H2O2 is introduced to initiate the gas generation reaction and produce a large amount of O2 (g), resulting in increased gas pressure for highly sensitive readout by a portable pressuremeter (Figure S1). Due to the strong and specific interaction between antigen and antibody, the number of AuNPs fixed on the substrate is proportional to the target concentration. Thus, the concentration of target can be quantitatively measured by the increased pressure.


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In comparison with immunoassays based on decoration of catalytic active nanomaterials with antibodies, the Pt staining-based immunoassay shows several advantages. First, it avoids the decoration of catalysts with detection antibodies and prevents the decrease in catalytic activity due to blocking of catalytic active sites. Second, because the catalytic active sites of the Pt staining-based method are freshly formed each time immediately after immunoassay, loss of catalytic activity during storage is avoided. Moreover, by inheriting the excellent properties of AuNPs, including low-cost, easy synthesis and functionalization, and high stability, 36-37 36-37 36-37 36-37 36-3734-35

Pt staining-based detection solves the problems of instability, complexity, and poor

reproducibility of other methods.

Figure 1. Working principle of Pt staining-based ELISA for PASS. Limitations of PtNPs for PASS. PASS is a simple, low cost, and portable method for disease diagnosis and adequately meets the criteria of POCT. Use of PtNPs as catalyst significantly increases the detection sensitivity of PASS.7-8 However, different kinds of molecules are easily adsorbed onto the surfaces of PtNPs, resulting in decreased catalytic activity. As shown in Figure 2A, after incubating with DNA, HS-DNA, BSA or 2-mercaptoethanol (ME), catalytic activity of PtNPs decreased significantly. The decrease of catalytic activity of PtNPs is closely related to the binding affinity and loading density of the ligands. HS-DNA bound to the surfaces


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of PtNPs more strongly than DNA or BSA, leading to more severe inhibition of catalytic activity of PtNPs. Similarly, the density of 2-mercaptoethanol on the surfaces of PtNPs was higher than that of HS-DNA. Hence, the inhibition of catalytic activity of PtNPs by 2-mercaptoethanol was worse than inhibition by HS-DNA. Moreover, the catalytic activity of PtNPs is not stable during storage. As shown in Figure 2B, after being stored at room temperature for two months, catalytic activity of PtNPs decreased severely with time. For example, after being stored at room temperature for a month, catalytic activity of PtNPs dropped to 20%. Particle aggregation and adsorption of other species onto the surfaces of nanoparticles are two possible reasons for the loss of activity over time.21-22 Furthermore, it should be pointed out that physical adsorption of PtNPs on the microtiter wells, leading to high detection background, was more problematic than adsorption of AuNPs, even after coating with bovine serum albumin (Figure S2). This is probably due to the weaker binding affinity of protein to the PtNP surface compared to the AuNP surface. Hence, it was necessary to develop a new coating method in order to avoid poisoning of catalyst, decrease of catalytic activity after decoration, and non-specific adsorption to the microtiter plate surface.

Figure 2. (A) Relative catalytic activities of PtNPs after being capped with different ligands. (B) Stability of catalytic activity of PtNPs after being stored at room temperature for different times.


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Activity and stability of in situ synthesized Au@AgPtNPs. Due to their excellent properties, including low-cost, easy synthesis and functionalization, and good storage stability, AuNPs have been widely applied in bioanalysis and diagnosis.36-37 In order to develop a stable and sensitive method to meet the commercial demands of PASS, we designed an in situ Pt staining-based method to coat AuNPs with Pt shells after immunosorbent assay to increase the catalytic activity of AuNPs. However, we found it difficult to stain protein-capped AuNPs with Pt shells. As an alternative, Ag staining has been widely used to stain protein-coated AuNPs with Ag shells to increase the detection sensitivity.38-39 Inspired by these reports, we introduced Ag staining as a medium to stain protein-functionalized AuNPs with Pt shells and optimized the staining conditions. First, we investigated the concentrations of different reagents to carry out Pt staining. Catalytic activity of Au@AgPtNPs and background were compared to determine the optimum concentrations of different reagents used for Pt staining. AuNPs were adsorbed on a microtiter plate and then capped with BSA by physical adsorption, using BSA-coated microtiter plates as control. As shown in Figures 3A-B and S3, the optimum concentrations for AgNO3, H2PtCl6, hydroquinone, and ascorbic acid to perform Pt staining were 0.25, 0.5, 1, and 10 mM, respectively. Second, using the same criteria, we studied the time needed for Ag and Pt staining and found that the best staining time for Ag and Pt were 5 and 20 min, respectively (Figure 3CD). Thereafter, in order to verify the successful coating of Ag and Pt shells, we characterized nanoparticles after each coating step in solution by UV-vis absorption spectroscopy, scanning electron microscopy (SEM), transmission electron microscopy (TEM), and high resolution transmission electron microscopy (HRTEM). As illustrated in Figures 4A-B and S4, AuNPs with a diameter of 16 nm show a light red appearance and have a distinct UV-vis absorption


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peak at 520 nm. After coating with Ag shells, the solution appearance changed to yellow-orange, while the UV-vis absorption peak blue-shifted to 490 nm with a new peak appearing at 400 nm. Moreover, TEM and SEM analyses showed that a 3 nm shell was formed outside AuNPs, demonstrating the successful coating of AuNPs with silver shells. Thereafter, Pt staining was achieved using H2PtCl6 and ascorbic acid as metal precursor and reducing reagent. The solution appearance changed from yellow-orange to black and the UV-vis absorption peaks at 400 and 490 nm both disappeared. TEM and SEM images showed that the nanoparticles grew from 20 nm to 27 nm with the nanoparticle surface becoming rough, proving the successful formation of Pt shells. Finally, to characterize the elemental distribution of Au@AgPtNPs, we analyzed the structure of Au@AgPtNPs using HRTEM. In Figure 4C, Au, Ag, and Pt elements are marked with fake green, blue, and red, respectively, to show the distributions of different elements. It is obvious that Ag and Pt formed an amorphous bimetallic shell around the surface of AuNPs. Subsequently, we compared the catalytic activity of Au@AgPtNPs to PtNPs having the same size and concentration. Au has been reported to improve the catalytic activity of Pt by synergistic and electronic effects,40-41 while Ag was found to have a toxic effect on the catalytic activity of Pt.42-44 However, it was found that Au@AgPtNPs showed catalytic activity similar to that of PtNPs (Figure 4D), which may be attributed to the opposing effects of Au and Ag. Finally, we investigated the stability of the Pt staining method to stain protein-capped AuNPs with Pt shells and catalyze the decomposition of H2O2. As shown in Figure 4E, the Pt staining-based method endows AuNPs with catalytic activity stable for two months, demonstrating the good reproducibility and stability of the Pt staining method.


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Figure 3. Optimization of (A) AgNO3 and (B) H2PtCl6 concentrations for Pt staining. Optimization of incubation time for (C) Ag staining and (D) Pt staining. Au@BSA and BSA represent 96-well microtiter plate coated with Au@BSA or BSA.


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Figure 4. (A) UV-vis absorption spectra (inset: photographs) and (B) TEM images of AuNPs, Au@AgNPs, and Au@AgPtNPs. (C) Elemental imaging analysis of Au@AgPtNPs. (D) Comparison of the catalytic activities of PtNPs and Au@AgPtNPs with diameters of 30 nm. (E) Stability of Pt staining-based method over two months. Pt staining-based ELISA for clinical diagnosis. Pt staining is simple, and stable, which can be used to develop immunoassay methods for clinical diagnosis. In the present study, Pt staining was combined with ELISA for quantitative detection of CRP. A pentameric protein produced by the liver with a molecular weight of 114 kDa, CRP, is a crucial marker for coronary heart disease, inflammation, and acute coronary syndromes.45 To carry out CRP detection by Pt staining-based ELISA, polyclonal and monoclonal antibodies specific for CRP were coated on a microtiter plate and on AuNPs, respectively. In the presence of CRP targets, antibody-capped AuNPs were captured on the microtiter plate and were stained with AgPt bimetallic shells to increase the catalytic activity. Thereafter, H2O2 was utilized as substrate and was catalyzed by the Pt outer shell to generate O2 (g). The gas increased the pressure inside the sealed microtiter well, and was measured by a portable pressuremeter. As shown in Figure S5A, the increased pressure was proportional to the concentration of CRP with a limit of detection of 0.015 ng/mL, which was about one order of magnitude lower than that of standard ELISA (0.16 ng/mL, Figure S5B) and five orders of magnitude lower than the limit of cardiovascular events (1 µg/mL).46 Furthermore, we studied the specificity of Pt staining-based ELISA using thrombin, human serum albumin (HSA), prostate specific antigen (PSA), and avian influenza H5N1 as control (Figure S5C). It was found that only CRP showed significant response, demonstrating the excellent specificity of this method.


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Moreover, to test the versatility of Pt staining-based ELISA, H5N1 was chosen as the target. As shown in Figure 5A, the increased pressure showed a linear response to the concentration of H5N1 with a limit of detection of 0.065 ng/mL, which was about 3 times lower than that of standard ELISA (0.21 ng/mL, Figure S5D). Moreover, when severe acute respiratory syndrome (SARS) specific antigen, hemagglutinin 7 neuraminidase 9 (H7N9), and hemagglutinin 3 neuraminidase 2 (H3N2) were present, only H5N1 led to a significant increase of pressure (Figure 5B), again verifying the good selectivity of Pt staining-based ELISA. Furthermore, to prove the practical utility of this method for clinical diagnosis, we spiked H5N1 in real serum samples. As shown in Figure 5C, the increased pressure demonstrated good linear response to the concentration of H5N1, showing that Pt staining-based ELISA can work well in a complex system. Finally, to verify the accuracy of Pt staining-based ELISA, we compared the detection results with standard ELISA for the detection of H5N1. As shown in Figure 5D, the detection results of Pt staining-based ELISA agreed commendably with those of standard ELISA, demonstrating the good accuracy of Pt staining-based ELISA.


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Figure 5. (A) Sensitivity and (B) selectivity of Pt staining-based ELISA for the detection of H5N1. The concentrations of H5N1, SARS, H7H9, and H3N2 were 1 ng/mL. (C) The application of Pt staining-based ELISA for the detection of H5N1 in real samples. (D) Detection of H5N1 by Pt staining-based method and standard ELISA, showing the consistency between two different strategies.

Conclusions In conclusion, we have demonstrated a robust, simple, and stable in situ Pt staining method for pressure based bioassays. There are several advantages of in situ Pt staining for the pressurebased bioassays. First of all, in situ Pt staining prevents the blocking of active sites of the catalytic surface during antibody coating to maintain their high catalytic activity. Second, because the catalytic active sites of the Pt staining-based method are freshly formed each time immediately after immunoassay, loss of catalytic activity during storage is avoided. Moreover, Pt


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staining is based on the staining of widely commercial used protein-capped AuNPs, which inherits the advantages of AuNPs such as low cost, easy synthesis, excellent storage stability, and low non-specific adsorption. Finally, Pt staining tremendously increases the detection sensitivity of pressure-based assay by transforming low-activity catalysts (AuNPs) to highactivity catalysts (Au@AgPtNPs). Due to the robust, simple, stable, and sensitive properties of in situ Pt staining for the pressure based bioassays, this method will significantly broaden the applications of PASS in clinical diagnosis and biomedicine.

Experimental Section Instruments and Reagents. The increased pressure in a sealed microtiter well was determined by a portable pressuremeter with a needle (Passtech, Xiamen, China). UV-vis absorption spectra were obtained by a NanoDrop spectrometer (Thermo Scientific, MA, U.S.A.). TEM images were acquired by a JEM 1400 microscope operating at 100 keV. Elemental imaging analysis of nanoparticles was performed on a high resolution transmission electron microscope (Tecnai F30, FEI, Netherlands). SEM analysis was carried out on a SEM-4800 microscope at 10 keV. DNA (AAA AAA AAA AAA) and HS-DNA (HS-AAA AAA AAA AAA) were synthesized on a PolyGen 12-Column DNA synthesizer (PolyGen GmbH, Germany). HAuCl4·4H2O, ascorbic acid, AgNO3, H2PtCl6, sodium citrate, and H2O2 were purchased from Sinopharm Chemical Reagent Co., Ltd.. Hydroquinone was obtained from J&K Chemical Technology (Guangdong, China). 2-Mercaptoethanol was purchased from Xiya Reagent Co., Ltd. (Chengdu, China). Thrombin, human serum albumin (HSA), prostate specific antigen (PSA), severe acute respiratory syndromes (SARS) specific antigen, sheep anti-human CRP polyclonal antibody (capture antibody), human C-reactive protein, and mouse anti-human C-


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reactive protein monoclonal biotinylated antibody (detection antibody) were acquired from R&D Systems. Hemagglutinin 7 neuraminidase 9 (H7N9), hemagglutinin 3 neuraminidase 2 (H3N2), and ELISA pair set (SEK002) for H5N1 (including anti-H5N1 capture antibody, recombinant H5N1, and anti-H5N1 biotinylated detection antibody) were obtained from Sino Biological Inc. (Beijing, China). BSA, rubber plugs, and 96-well microtiter plates were supplied by Taijing Biological Inc. (Xiamen, China). Synthesis of PtNPs. PtNPs were synthesized directly by the reduction of H2PtCl6 using ascorbic acid. Briefly, H2PtCl6 (10 µL, 100 mM) was diluted with water (900 µL) in a centrifuge tube and incubated at 80 °C for 20 min. Then, ascorbic acid (100 µL, 400 mM) was quickly added into the mixture and then incubated at 80 °C for 30 min. During this period, the solution gradually changed from light yellow to brown. Finally, PtNPs were cooled to room temperature and stored at 4 °C before use. Determination of enzymatic activity of PtNPs. PtNPs can effectively catalyze the decomposition of H2O2 to produce O2 (g). To determine the catalytic efficiency of PtNPs, we measured the production of O2 (g) by the increased pressure in a sealed microtiter well over time. Briefly, a 2 µL aliquot of freshly synthesized PtNPs was added to the wells of a 96-well microtiter plate. Then, H2O2 (100 µL, 1 M) solution was added to mix with PtNPs, followed by sealing the wells using a rubber plug. Subsequently, the mixture was reacted at 37 °C for 20 min, during which H2O2 was decomposed to generate O2 (g) and increase the pressure inside the well. Finally, the increased pressure was measured by a portable pressuremeter and used to indicate the catalytic activity of PtNPs. To verify that the adsorption of molecules on PtNPs will result in decreasing enzyme activity, PtNPs (2 µL) were mixed and incubated with water, DNA, HS-DNA, BSA, and 2-


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mercaptoethanol (10 µL, 1 mg/mL) at room temperature for 20 min. Then, H2O2 (100 µL, 1 M) was added to each PtNP sample, followed by sealing the reaction chamber using rubber plugs. Thereafter, H2O2 was decomposed to produce O2 (g) by incubating at 37 °C for 20 min, leading to an increase of pressure in the sealed well. The catalytic activities of different PtNP samples could be compared by measuring the increase of pressure with a portable pressuremeter. Synthesis of AuNPs. 16-nm AuNPs were synthesized based on the literature.35 First, HAuCl4 (100 mL, 0.01 wt%) was boiled in a round-bottom flask (250 mL) with stirring. Then, sodium citrate (1 mL, 3 wt%) was added immediately to the solution, and AuNPs could be synthesized after 30 min. Thereafter, the AuNP solution was cooled to room temperature and stored in the dark before use. After washing three times with water, the morphology and size of AuNPs were characterized by TEM. According to the amount of metal precursor and the size of AuNPs, the concentration of AuNPs synthesized by this method was determined to be 2.5 nM (extinction coefficient of 16 nm AuNPs: 2.4×108 M−1cm−1). Prevention of physical adsorption of nanoparticles on microtiter plates by BSA. Due to their high specific surface area, nanoparticles are easily adsorbed on microtiter plates by physical adsorption. BSA is a globulin in bovine serum that can bind strongly with both nanomaterials and the microtiter plate, leading to the prevention of physical adsorption of nanoparticles on the plate. To evaluate the efficiency of BSA on preventing physical adsorption between nanoparticles and the microtiter plate, we carried out adsorption experiments of nanoparticles on microtiter plates in the presence of different concentrations of BSA. First, PtNPs and AuNPs (100 µL) were synthesized as described above and added to the wells of a 96-well microtiter plate. Then, different concentrations of BSA solution (10 µL) were added and mixed with nanoparticles by tapping on the 96-well microtiter plate. The UV-vis absorption of different


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samples at 405 nm was measured by a microplate reader, referred to it as original absorbance (Ao). Thereafter, the 96-well microtiter plate was incubated at 37 °C for 1 h to facilitate the adsorption of nanoparticles, followed by washing three times using water to remove free nanoparticles. Finally, each well was measured by UV-vis absorption spectroscopy at 405 nm, denoted as final absorbance (Af). The relative amount of nanoparticles adsorbed on microtiter plate could be compared by dividing Ao by Af. Optimization of Pt staining conditions. The conditions to stain AuNPs with Pt shells were optimized to obtain the best signal-to-background ratio. First, BSA was dissolved in phosphate buffered saline (PBS) to prepare a 2 wt% solution. Then, the BSA solution (100 µL, 2 wt%) was added to the wells of a 96-well microtiter plate and incubated at 37 °C for 1 h to coat the microtiter plate with BSA. After washing with washing buffer (PBS, containing 0.05 wt% Tween 20) three times, the BSA-coated microtiter plate was obtained. Afterwards, in an effort to prepare an AuNP-coated microtiter plate, AuNP solution (100 µL, 2.5 nM, 16 nm) was added to the wells of the BSA-coated 96-well microtiter plate and incubated at 37 °C for 1 h. Thereafter, AuNP solution was siphoned off, followed by the addition of BSA/PBS solution (200 µL, 2 wt%) to block AuNPs with BSA. After incubating at 37 °C for 1 h, the BSA/PBS solution was removed. Finally, a 96-well microtiter plate coated with Au@BSA was obtained and stored in the dark before use. Pt staining was performed on the Au@BSA-coated microtiter plate using a BSA-coated microtiter plate as control. Briefly, AgNO3 (50 µL, 0.25 mM) and hydroquinone (50 µL, 2 mM) were added to a 96-well microtiter plate coated with Au@BSA or BSA. After incubating at 37 °C for 20 min, each well was washed once with water. Then, H2PtCl6 (50 µL, 1 mM) and ascorbic acid (50 µL, 10 mM) were mixed in the wells and kept at 37 °C for 20 min, followed by


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washing with water. Subsequently, H2O2 (100 µL, 1 M) was added to initiate the catalytic gas generation reaction, followed by sealing the wells of the microtiter plate using rubber plugs. Finally, after incubating at 37 °C for 20 min, the pressure inside the wells was measured by a portable pressuremeter. The catalytic activities of AuNPs stained with Pt under different conditions were compared by the magnitude of the increased pressure. In this manner, the concentrations of AgNO3, hydroquinone, H2PtCl6, and ascorbic acid were optimized successively to obtain the best Pt staining conditions. Characterization of AuNPs stained with Ag and Pt bimetallic shells. In order to characterize the structure of AuNPs after being stained with Ag and Pt bimetallic shells (Au@AgPtNPs), we synthesized Au@AgPtNPs directly in solution. First, 16-nm AuNPs were synthesized as described above. Then, AgNO3 (50 µL, 2.5 mM) and hydroquinone (50 µL, 20 mM) were mixed with AuNP solution (1 mL, 2.5 nM) in a centrifuge tube, followed by incubation at 37 °C for 20 min to coat AuNPs with Ag shells. Afterwards, H2PtCl6 (50 µL, 10 mM) and ascorbic acid (50 µL, 100 mM) were added into the mixture and kept at 37 °C for 20 min. Finally, Au@AgPtNPs were obtained and analysized by UV-vis spectroscopy, SEM, TEM, and HRTEM. Comparison of stability of PtNPs and Pt staining. Because Pt staining is performed each time after the binding of AuNPs, storage of catalytically active nanoparticles is avoided, thus preventing the poisoning of active sites during storage. Hence, the Pt staining-based detection method was thought to have excellent stability. To compare the stabilities of PtNPs and Pt staining, we monitored their catalytic activities over two months. PtNPs were synthesized as described above and kept at room temperature for different times. The catalytic activities of PtNPs were monitored by their abilities to catalyze the decomposition of H2O2. First, PtNPs (2


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µL) and H2O2 solution (100 µL, 1 M) were mixed in the wells of a 96-well microtiter plate, followed by sealing the wells immediately using rubber plugs. Subsequently, the reaction was kept at 37 °C for 20 min, during which O2 (g) produced by the decomposition of H2O2 increased the pressure inside the wells. Finally, the pressure, indicating the catalytic activity of PtNPs, was measured by a portable pressuremeter. Afterwards, to determine the stability of the Pt staining-based method, an Au@BSA-coated microtiter plate was prepared as described above and stored at room temperature for different times. During this period, Au@BSA on the wells of microtiter plate was stained with Ag and Pt bimetallic shells by the Pt staining method. First, AgNO3 (50 µL, 0.25 mM) and hydroquinone (50 µL, 2 mM) were added to the wells of the Au@BSA-loaded microtiter plate. After incubating at 37 °C for 20 min, each well was washed once with pure water. Then, H2PtCl6 (50 µL, 1 mM) and ascorbic acid (50 µL, 10 mM) were mixed in the wells and kept at 37 °C for 20 min, followed by washing with water. Finally, H2O2 (100 µL, 1 M) was added to each well, followed by sealing the wells using rubber plugs. After incubating at 37 °C for 20 min, the pressure in each well was measured by a portable pressuremeter. Pt staining-based ELISA detection. In order to perform ELISA detection by the Pt stainingbased method, streptavidin (SA)-conjugated AuNPs were prepared. First, 16-nm AuNPs were synthesized as described above. Then, SA/PBS solution (5 µL, 1 mg/mL) and Na2HPO4 solution (50 µL, 0.2 M) were added to the AuNP solution (1 mL, 2.5 nM, 16 nm) and incubated at 37 °C for 20 min to attach SA to the surfaces of AuNPs by physical adsorption. Afterwards, BSA/PBS solution (50 µL, 2 wt%) was added to the mixture and incubated at 37 °C for 20 min to block the residual space on the AuNP surfaces. Finally, SA-conjugated AuNPs were obtained by


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centrifuging the AuNP solution at 14000 rpm for 10 min and then washing twice using BSA/PBS solution (1 mL, 0.1 wt%). The process of Pt staining-based ELISA detection for C-reactive protein (CRP) was similar to that of standard ELISA. Capture antibody (100 µL, 4 µg/mL) for CRP was added to the wells of a 96-well microtiter plate and incubated at 4 °C for 12 h, followed by washing each well with washing buffer (300 µL, 20 mM Tris, 0.05 wt% Tween 20, 150 mM NaCl, pH 7.4) three times. Thereafter, blocking buffer (300 µL, 20 mM Tris, 0.05 wt% Tween 20, 150 mM NaCl, 2 wt% BSA, pH 7.4) was added to the wells, followed by and incubating at 37 °C for 1 h and washing three times. Then, CRP samples were added into the wells. After incubating at room temperature 2 h, each well was washed with washing buffer for three times. Subsequently, biotinylated detection antibody (100 µL, 0.2 µg/mL) for CRP was added into the wells. After incubating at room temperature for 1 h and washing the wells for three times, SA-capped AuNPs (100 µL, 10 nM) were added, followed by incubating at room temperature for 1 h. Each well was washed with washing buffer three times, followed by washing once with water. Thereafter, AgNO3 (50 µL, 0.25 mM) and hydroquinone (50 µL, 2 mM) were added to the wells to stain AuNPs with Ag shells. After incubating at 37 °C for 20 min, the reagents in the wells were removed. Then, H2PtCl6 (50 µL, 1 mM) and ascorbic acid (50 µL, 10 mM) were added and incubated at 37 °C for 20 min. Extra reagents were removed by washing the wells using ultrapure water (100 µL) three times. Finally, H2O2 (100 µL, 1 M) was added to each well, followed by sealing the wells using rubber plugs. After incubating at 37 °C for 20 min, H2O2 was decomposed to form O2 (g), which increased the pressure inside the wells. The increased pressure was measured by a portable pressuremeter. The concentration of targets could be determined by comparing the


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increased pressure with a standard working curve. The process to detect H5N1 by Pt stainingbased PASS was similar to that for CRP except that the antibodies were specific for H5N1.


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ASSOCIATED CONTENT Supporting Information. Device structures for pressure-based bioassay, physical adsorption properties of PtNPs and AuNPs, optimization of conditions for Pt staining, size and morphology analyses of different nanoparticles, and detection of CRPs using different methods. This material is available free of charge on the ACS Publications website at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author Phone: +86 592-218-7601; E-mail: [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. Notes The authors declare no competing financial interests. ACKNOWLEDGMENT We thank the National Natural Science Foundation of China (21775128, 21735004, 21435004, 21521004, 21325522, 21422506), and Program for Changjiang Scholars and Innovative Research Teams in University (IRT13036) for financial support. REFERENCES (1) Fujita, H.; Kataoka, Y.; Tobita, S.; Kuwahara, M.; Sugimoto, N., Novel one-tube-one-step real-time methodology for rapid transcriptomic biomarker detection: Signal amplification by ternary initiation complexes. Anal. Chem. 2016, 88, 7137-7144.


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Table of Contents (TOC)


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In Situ Pt Staining Method for Simple, Stable, and ... - ACS Publications

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