Pt peroxidase mimic and its bioanalytical applications

Accepted Manuscript Bimetallic Bi/Pt peroxidase mimic and its bioanalytical applications Gang-Wei Wu, Yi-Min Shen, Xiao-Qiong Shi, Hao-Hua Deng, Xiao-Qing Zheng, HuaPing Peng, Ai-Lin Liu, Xing-Hua Xia, Wei Chen PII:

S0003-2670(17)30323-9

DOI:

10.1016/j.aca.2017.03.028

Reference:

ACA 235129

To appear in:

Analytica Chimica Acta

Received Date: 20 January 2017 Revised Date:

4 March 2017

Accepted Date: 13 March 2017

Please cite this article as: G.-W. Wu, Y.-M. Shen, X.-Q. Shi, H.-H. Deng, X.-Q. Zheng, H.-P. Peng, A.L. Liu, X.-H. Xia, W. Chen, Bimetallic Bi/Pt peroxidase mimic and its bioanalytical applications, Analytica Chimica Acta (2017), doi: 10.1016/j.aca.2017.03.028. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Graphical Abstract

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Bimetallic Bi/Pt nanoparticles in bovine serum albumin biomolecular scaffold (BSA-Bi/PtNPs) were synthesized through a facile and green method. The resulting BSA-Bi/PtNPs exhibit enhanced peroxidase-like activity as compared with BSA-PtNPs. Moreover, when protected by the bovine serum albumin, the

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BSA-Bi/PtNPs are more stable than other material-capped PtNPs in harsh environment such as high temperature, extreme pH environments, and high ionic

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strength, as well as in common biological matrixes. Bioassays, such as serum glucose detection, extracellular hydrogen peroxide monitor, and cancer cells labeling, have been realized with satisfying results.

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Bimetallic Bi/Pt peroxidase mimic and its bioanalytical applications

Gang-Wei Wu,a,c Yi-Min Shen,a,b Xiao-Qiong Shi,a,b Hao-Hua Deng,a,b Xiao-Qing

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Zheng,a,b Hua-Ping Peng,a,b Ai-Lin Liu,a,b Xing-Hua Xia,d Wei Chen* a,b

Department of Pharmaceutical Analysis, Fujian Medical University, Fuzhou 350004,

China

Higher Educational Key Laboratory for Nano Biomedical Technology of Fujian

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Province, Fujian Medical University, Fuzhou 350004, China

Department of Pharmacy, Fujian Provincial Hospital, Fuzhou 350001, China

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State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry

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and Chemical Engineering, Nanjing University, Nanjing 210093, China. * Corresponding author. Tel./fax: +86 591 22862016.

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E-mail address: [email protected] (W. Chen).

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Gang-Wei Wu and Yi-Min Shen contributed equally to this work.

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Abstract In this work, bimetallic Bi/Pt nanoparticles in bovine serum albumin biomolecular scaffold (BSA-Bi/PtNPs) were synthesized through a facile and green method. As

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compared with BSA-PtNPs, the BSA-Bi/PtNPs possess enhanced peroxidase-like catalytic activity. Moreover, the BSA-Bi/PtNPs are stable in harsh conditions such as high temperature, extreme pH environments, and high ionic strength, as well as in

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common biological matrixes. These prominent advantages enable the BSA-Bi/PtNPs to be applied to a wide range of fields. Bioassays, such as serum glucose detection,

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extracellular hydrogen peroxide (H2O2) monitor, and cancer cells labeling, have been realized with satisfying results. The linear range of glucose determination was from 1 to 100 µM and the limit of detection (LOD) was 0.2 µM. The H2O2 released from each MCF-7 cell after stimulation was calculated to be 2.66×10-16 mol/s. By utilizing folic acid

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as a recognition element, tumor cell could be readily distinguished by BSA-Bi/PtNPs and the LOD for MCF-7 cell detection was 90 cells. Keywords: Bovine serum albumin, platinum, bismuth, nanoparticle, peroxidase mimic,

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bioassay

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1. Introduction

Because of their remarkable merits such as high catalytic activity and excellent substrate specificity, Natural enzymes have been widely applied in medicine, chemical industry, food processing, and agriculture. Peroxidase, which can effectively catalyze the reaction between hydrogen peroxide and electron donors, is a widely-used detection tool in enzymatic analysis and popular labeling probe for enzyme-linked immunosorbent

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assay (ELISA) and DNA detection system [1]. Nevertheless, natural enzymes also have some serious disadvantages, which limit their wide applications. For example, they are highly sensitive to environmental conditions and have low stability in operation because

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of denaturation and digestion. Besides, preparation and purification processes of natural enzymes are time-consuming and expensive. What’s more, false-negative results are more-likely to be caused by the instable horseradish peroxidase (HRP). Therefore, it is

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highly attractive to discover or construct stable enzyme mimetics.

Manufactured nanostructures have recently received considerable interest in the field

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of enzyme mimics [2]. Nanomaterials, which can be facilely obtained by large-scale and low-cost preparation, are much more stable over a wide pH and temperature range than their natural counterparts. Moreover, their catalytic activities can be tuned by flexibly structure and composition design [3-9].

In virtue of these advantages, various

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nanomaterials have been applied as peroxidase mimics in the biomedicine and environmental chemistry fields [10-27].

Although the sensing systems based on catalytic nanomaterials are selective and

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sensitive for analytes, applications in the real biological samples are still limited. The major problem is the aggregation of the nanomaterials and adsorption of proteins or other

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biomolecules on the nanomaterials. The catalytic activity is correspondingly reduced because less active site is available for the substrate. Recently, we have reported the biomineralization synthesis of Pt nanoparticles in bovine serum albumin (BSA) template [28]. It has been proven that the consequent BSA-PtNPs can catalyze the reaction between hydrogen peroxide and various peroxidase substrates. Owning to the anti-fouling and anti-aggregation effects of the albumin shell, BSA-PtNPs can preserve their

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peroxidase-like catalytic activity even in serum or the environment with high ionic strength, making them suitable for widespread applications in biological analysis [29]. The catalytic activity of nanomaterials is closely related to their surface or interfacial

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properties [30]. Recently, it is discovered by Huang and his co-workers that the catalytic activity of citrate-capped AuNPs can be dramatically stimulated by Hg2+ [31]. Apart from Hg2+, other metal ions, such as Ag+, Pb2+, and Bi3+, have also been found to have the

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similar promoting effect [32-34].

In this work, we developed a facile approach to prepare BSA-encapsulated bimetallic

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Bi/Pt nanoparticles (BSA-Bi/PtNPs). It is a green synthetic method, where only three reactants of H2PtCl6, Bi(NO3)3, and BSA are involved without extra stabilizers or reducers. The resulting BSA-Bi/PtNPs exhibit enhanced peroxidase-like activity as compared with BSA-PtNPs. Moreover, when protected by the bovine serum albumin, the

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BSA-Bi/PtNPs are more stable than other material-capped PtNPs in harsh environment such as high temperature, extreme pH environments, and high ionic strength, as well as in common biological matrixes [14,24,35]. Sensitive detection of hydrogen peroxide at the

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nanomolar level can be achieved based on the BSA-Bi/PtNPs catalyzed colorimetric reaction. Further analytical applications for monitor serum glucose and extracellular

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release of H2O2 were realized with satisfying results. The BSA-Bi/PtNPs can be facilely functionalized on the protein shell. Folic acid (FA) modified BSA-Bi/PtNPs were prepared for monitoring tumor cells through folic acid-folate receptor interaction (Scheme 1).

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Bi/PtNPs.

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Scheme 1. Schematic diagram of synthesis and bioanalytical applications of BSA-

2.1. Apparatus

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2. Materials and methods

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Transmission electron microscope (TEM) and high-resolution transmission electron microscope (HRTEM) images were collected using a JEM-2100 microscope (JEOL, Japan). X-ray photoelectron spectroscopic analysis of the product was carried out with an ESCALAB 250XI spectrometer (Thermo Scientific, USA). The Fourier transform infrared spectroscopy (FTIR) was measured using an Avatar 360 FTIR spectrophotometer (Nicolet, USA). Zeta-potential experiments were carried out on a Zetasizer Nano-ZS. The absorption spectra were obtained with a UV-2450 UV-visible spectrophotometer 5

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(Shimadzu, Japan). 2.2. Chemicals and reagents 3, 3’, 5, 5’-tetramethyl benzidine (TMB), Folic acid (FA), glucose, glucose oxidase, N-

hydroxy-3-sulfopropyl)-3-methylaniline

sodium

salt

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(3-Dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDC), N-ethyl-N-(2(TOOS),

and

3-methyl-2-

benzothiazole hydrazone (MBTH) were purchased from Aladdin Reagent Company.

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H2PtCl6·(H2O)6, EDTA, Bi(NO3)3·5H2O, Na2HPO4, NaH2PO4·2H2O, NaOH, bovine serum albumin and 30% H2O2 were all from Sinopharm Chemical Reagent Co. Ltd.

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Dimethyl sulfoxide (DMSO) was purchased from Xilong Chemical Reagent Co. Ltd. 3(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazoliumbromide (MTT) and Phorbol 12myristate-13-acetate (PMA) were purchased from Sigma-Aldrich Co. Ltd. All chemicals were of analytical reagent grade. Water used in the experiments was purified with a Milli-

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Q purification system (Millipore, USA). 2.3. Synthesis of BSA-Bi/PtNPs

In a typical process, BSA-PtNPs aqueous solution [28] (23.8 mg/mL, 0.6 mL) was

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mixed with Bi(NO3)3 (0.5 mM, 0.6 mL) and PB (pH=7, 10 mM, 1.8 mL). After incubated at 80 oC for 2.5 h, the obtained BSA-Bi/PtNPs were purified by ultrafiltration (Millipore,

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30 kDa). The as-prepared BSA-Bi/PtNPs could be stored at 4 °C for at least six months with negligible changes in their catalytic activities. 2.4. Synthesis of FA-BSA-Bi/PtNPs 1 mL solution of 0.04 M EDC was uniformly mixed with the 1 mL solution of 0.0068 M folic acid at room temperature for 15 min. Then 5 mL BSA-Bi/PtNPs (4.76 mg/mL) was added into the mixture. After incubated at room temperature for 2 h, the FA-BSA-

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Bi/PtNPs were purified by ultrafiltration (Millipore, 30 kDa). 2.5. Cell culture The human breast cancer cells (MCF-7) and human vascular endothelial cells

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(EAhy926) were cultured in routine medium which was made up of RPMI-1640 and 6% fetal calf serum. 2.6. MTT assay

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MCF-7 and EAhy926 cells were placed in 96-well plates at a density of 5000 cells/well for adherence and then allowed to incubate with BSA-Bi/PtNPs, respectively. The control

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well was treated without BSA-Bi/PtNPs. After cultured for 24 h, MTT solution was added into each well and incubated for 4 h. A dark blue formazan product was formed in the live cells. After then, DMSO was added into the wells to dissolve the dark blue crystals in the cells. The absorbance was measured by Thermo Multiskan MK3

2.7. Detection of H2O2

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microplate reader at 570 nm for each well.

100 µL of H2O2 was added into 200 µL PB (100 mM, pH 5.0) containing 0.238 mg/mL

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BSA-Bi/PtNPs and 0.4 mM TMB. After incubated at 37 oC for 15 min, 50 µL H2SO4 (0.5 M) was added to terminate the reaction. The yellow product was quantitatively monitored

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at 450 nm.

2.8. Detection of extracellular release of H2O2 In a typical process, human breast cancer cells (MCF-7) and human renal epithelial cells (HEK 293) were adhered in 96-well plate with 1×10-5 cells each circular hole. Then, 100 µL of 250 ng/mL phorbol 12-myristate-13-acetate solution was added into the wells. After 10 min, the released flux of H2O2 was added into 200 µL PB (100 mM, pH 5.0)

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containing 0.238 mg/mL BSA-Bi/PtNPs and 0.4 mM TMB. After incubated at 37 oC for 15 min, 50 µL H2SO4 (0.5 M) was added to terminate the reaction. The yellow product was quantitatively monitored at 450 nm.

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2.9. Glucose detection

80 µL of 1 mg/mL GOx and 640 µL glucose of different concentrations were added into 80 µL PB (10 mM, pH=7.0). After incubated at 37 oC for 30 min, 1.3 mL PB (10 mM,

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pH=7.0), 800 µL of 0.5 mM MBTH, 1 mL of 3 mM TOOS, and 100 µL of 4.76 mg/mL

product was measured at 590 nm.

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BSA-Bi/PtNPs were added and incubated at 37 oC for 30 min. The absorbance of the

2.10. Endpoint method for glucose detection

80 µL of 1 mg/mL GOx and 640 µL glucose of different concentrations were added into 80 µL PB (10 mM, pH=7.0). After incubated at 37 oC for 30 min, 2.9 mL PB (100

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mM, pH=7.0), 200 µL of 16 mM TMB, and 100 µL of 0.5 U/mL HRP were added and incubated at 37 oC for 10 min. The absorbance of the product was measured at 652 nm. 2.11. Cell detection

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MCF-7 and EAhy926 cells adhered in 96-well plate were incubate with RPMI 1640 medium containing 47.6 µg of FA-BSA-Bi/PtNPs at 37 °C for 1.5 h. In one of the control

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group, cells were incubated with RPMI 1640 medium containing 47.6 µg of BSABi/PtNPs. In another control group, cells were pretreated with 30 µg of FA for 30 min and then incubate with RPMI 1640 medium containing 47.6 µg of FA-BSA-Bi/PtNPs. Unattached FA-BSA-Bi/PtNPs were removed by rinsing each cell well with PB (10 mM, pH 7.4) for three times. Then, 200 µL PB (100 mM, pH 5.0) containing 1 M H2O2 and 0.8 mM TMB was added into each well. After 15 minutes of reaction at room temperature, 50

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µL H2SO4 (0.5 M) was added to terminate the colorimetric reaction. The absorbance of the colorimetric product was quantitatively monitored at the wavelength of 450 nm by

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Thermo Multiskan MK3 microplate reader.

3. Results and discussion 3.1. Characterization of BSA-Bi/PtNPs

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BSA-Bi/PtNPs were prepared by incubating BSA-PtNPs with Bi(NO3)3 at 80 oC for 2.5 h and purified by ultrafiltration. TEM image shown in Fig. 1A revealed an average

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diameter of 4.0±0.4 nm for the BSA-Bi/PtNPs. The size distribution was not statistically different between the BSA-Bi/PtNPs and BSA-PtNPs [28], suggesting that only a monolayer or submonolayer of Bi formed on the surface of PtNPs. High-resolution TEM image showed 0.227 nm of the interplanar spacing corresponding to the Pt (111) facet,

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reflecting the same platinum core structure of BSA-Bi/PtNPs and BSA-PtNPs [36]. The conjugation of bismuth on PtNPs surface was further ascertained by X-ray photoelectron spectroscopy (Fig. S1). The atom ratio of Bi:Pt in the formed BSA-Bi/PtNPs was found

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to be 1:10. As is shown in Fig. 1B, the Pt 4f electron spectrum of BSA-Bi/PtNPs could be well resolved with two doublets. The line at 72.5 eV is due to Pt0 species, whereas that at

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74.0 eV is due to Pt4+ species [37]. The relative composition of Pt0 and Pt4+ species was calculated to be 87% and 13%, respectively, which were almost the same as those of BSA-PtNPs. The XPS spectrum of bismuth reveals high ratio of Bi3+ (87%) and low ratio of Bi0 (13%) on the PtNPs surface, which greatly affected the surface properties (Fig. 1C). The small number of Bi0 existed on the particle surface may be ascribed to the reduction capability of residues (e.g. tyrosine) in BSA. After reaction with Bi3+, negative-charged

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BSA-PtNPs with zeta potential of -3.48 mV converted to positive-charged BSA-Bi/PtNPs with zeta potential of +21.29 mV. As shown in Fig. 1D, there is no distinct difference between the IR spectra of BSA-Bi/PtNPs and BSA-PtNPs. These results indicate that

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BSA can still preserve its conformation after bismuth combination.

Fig. 1. (A) TEM image of BSA-Bi/PtNPs. (B) XPS spectrum of BSA-Bi/PtNPs in the Pt 4f region. (C) XPS spectrum of BSA–PtNPs in the Bi 4f region. (D) FT-IR spectra of BSA-PtNPs and BSA-Bi/PtNPs.

3.2. Enhanced peroxidase-like activity of BSA-Bi/PtNPs The peroxidase-like catalytic activity of BSA-Bi/PtNPs was assessed by using H2O210

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TMB chromogenic reaction and compared with that of BSA-PtNPs. Peroxidase mimics could catalyze the oxidation of TMB by H2O2 to yield a blue color and the blue-colored product could further oxidized to a yellow diimine by the introduction of H2SO4. As

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shown in Fig. 2A, the BSA-PtNPs exhibited relatively low catalytic activity for the H2O2mediated TMB oxidation. The catalytic activity of BSA-Bi/PtNPs, which increased with the concentration of Bi3+ used in the fabrication process, is significantly enhanced relative

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to that of the BSA-PtNPs (Fig. 2B). This can be mainly ascribed to the formation of a larger fraction of bimetallic Bi-Pt complex. The enhanced activity of BSA-Bi/PtNPs was

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further proved by other types of chromogenic peroxidase substrate such as ABTS, pyrogallol, and 4-aminoantipyrine/TOPS. As shown in Fig. S2, the BSA-Bi/PtNPs could catalyze the oxidation of these chromogenic substrates of peroxidase by H2O2, which give

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higher peroxidase-like activity than BSA-PtNPs.

Fig. 2. (A) UV-vis spectra of (a) TMB + H2O2, (b) TMB + H2O2 + BSA-PtNPs, and (c) TMB + H2O2 + BSA-Bi/PtNPs. (B) Catalytic activity of the BSA-Bi/PtNPs prepared by uisng different concentrations of Bi3+.

Steady-state kinetics of the TMB-H2O2 reaction system was further investigated to

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better understand the effect of bismuth modification on properties of BSA-PtNPs. Michaelis-Menten kinetics equation, v=vmax×[S]/(Km+[S]), is used to describe the rate of enzymatic reactions. As a special constant for enzymes, the Michaelis constant Km means

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the substrate concentration at which the reaction rate is half of vmax which can reflect the affinity to the substrates for a given enzyme. Fig. 3 shows the substrates concentrationdependent reaction rates and corresponding fitting curves of Michaelis-Menten equation.

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The Km values of BSA-Bi/PtNPs and BSA-PtNPs with TMB as the substrate are 0.054 and 0.083 mM, and with H2O2 as the substrate the corresponding Km values are 13.24 and

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17.38 mM. The Km(TMB) and Km(H2O2) values of BSA-Bi/PtNPs are smaller than those of BSA-PtNPs, demonstrating that the enhanced peroxidase-like activity of BSABi/PtNPs derived from their stronger affinity towards both substrates. It is worth to mention that the Km(TMB) value of BSA-Bi/PtNPs is much lower than HRP (0.23 mM)

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toward TMB.

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[21], suggesting that the BSA-Bi/PtNPs have a considerably higher binding affinity

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Fig. 3. Steady state kinetic assay of (A, C) BSA-Bi/PtNPs and (B, D) BSA-PtNPs.

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Experiments were performed in PB (100 mM, pH 5.0) at 37 °C. (A, B) The concentration

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of H2O2 was varied and that of TMB was fixed at 0.8 mM. (C, D) The concentration of TMB was varied and that of H2O2 was fixed at 500 mM.

3.3. Stability and biocompatibility of BSA-Bi/PtNPs.

Stability is an important property to reflect whether the nanomaterials could be used in

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practical applications. The stability of BSA-Bi/PtNPs in harsh conditions was validated by measuring their catalytic activity for TMB-H2O2 system after incubation at various pH values (3-11), temperatures (30-80 oC), and sodium chloride solutions with different

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concentration (0-7.5 M) for 2 h (Fig. 4A-C). Typically, nature enzymes are unable to

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tolerate harsh conditions and tend to be inactivated. In contrast, BSA-Bi/PtNPs primely maintained their peroxidase-like catalytic activity in harsh environments such as extreme pH values, high temperature, and high ionic strength. Exposed in common external environment at room temperature, the catalytic activity of HRP is significantly decreased in a very short time. After 24 h, most of its activity is lost [28]. In contrast, the BSABi/PtNPs could preserve 98.9% of their original catalytic activity even storage at room temperature for 1 month.

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Stability of BSA-Bi/PtNPs in common biological matrixes was further investigated. As shown in Fig. 4D, BSA-Bi/PtNPs were quite stable and preserved their catalytic activity (> 90%) in the presence of proteins (human serum albumin and bovine serum albumin),

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serum (fatal calf serum), and cell culture medium (RPMI-1640), which makes them an

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ideal candidate for biological applications.

Fig. 4. (A-C) Catalytic activity of the BSA-Bi/PtNPs after pretreated at various (A) pH values, (B) temperatures, and (C) concentrations of NaCl for 2 h. (D) Stability of BSABi/PtNPs in common biological matrixes.

For further biomedical applications, cytotoxicity assessment of BSA-Bi/PtNPs on cells is essential to be evaluated. MCF-7 is a human breast cancer cell line, while EAhy926 is 14

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a normal human vascular endothelial cell line. Cell viability of MCF-7 and EAhy926 incubated with different concentrations of BSA-Bi/PtNPs was investigated by MTT assay. As shown in Fig. 5A and 5B, physiological state change and cell death of MCF-7 and

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EAhy926 were negligible, even in the presence of the highest dosage (960 µg/mL) of BSA-Bi/PtNPs. From the results of MTT assay (Fig. 5C and 5D), no significant cytotoxicity of BSA-Bi/PtNPs has been found. Cell viability is more than 80% in

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comparison with control group in the concentration range from 0 to 960 µg/mL. These results indicated that BSA-Bi/PtNPs possessed good biocompatibility and low

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cytotoxicity.

Fig. 5. (A, B) Morphology of (A) MCF-7 cells and (B) EAhy926 cells after treated with 960 µg/mL of BSA-Bi/PtNPs. (C, D) Dark toxicity of BSA-Bi/PtNPs to (C) MCF-7 cells

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and (D) EAhy926 cells. Cell viability was detected by MTT assay after incubated with

3.4. Bioanalytical applications of BSA-Bi/PtNPs

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BSA-Bi/PtNPs at 37 °C for 24 h (n=5).

Hydrogen peroxide is widely present in a variety of cellular biochemical reaction mediated by reactive oxygen, participating in numerous physiological and pathological

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processes. Therefore, the sensitive, reliable and accurate detection of H2O2 at physiological levels is of great significance for studying physiological processes. On the

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basis of enhanced peroxidase-like activity and well biocompatibility of BSA-Bi/PtNPs, an effective colorimetric method was established for extracellular H2O2 determination. Under the optimized conditions, the absorbance of TMB was gradually increased with the increase H2O2 concentration. The absorbance had linear relationship with the

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concentration of hydrogen peroxide in the range of 500 nM to 30 µM, r=0.997 (Fig. S3). The limit of detection (LOD) was calculated to be 68 nM at a signal-to-noise ratio of 3σ. The BSA-Bi/PtNPs-catalyzed colorimetric reaction was applied to perform the

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detection of H2O2 released from living cells. The MCF-7 and HEK 293 were chosen as model cells. When cancer cells (MCF-7) with the number of 1.0×105 was stimulated by

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250 ng/mL PMA for 10 min, a large amount of H2O2 was released from the cells. An absorbance of 0.802 (average value of three parallel test) was obtained by a microplate reader set at 450 nm, corresponding to 2.03×10-5 mol of H2O2. After further calculation, 2.66×10-16 mol of H2O2 releasing from each cell per second was obtained. This result agreed well with the value reported previously [38]. This measurement provided a practical and effective approach to evaluate the released H2O2 from living cells. As shown

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in Fig. 6A, the flux of H2O2 release from MCF-7 cells is positively corresponded to PMA dose. On the contrary, no detectable H2O2 releasing was observed for the normal cells (HEK 293), indicating a cell type-dependent manner. The time-dependent response was

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further studied in the time period of 0 to 20 min. A positive correlation between

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absorbance and time could be found for MCF-7 cells stimulated by PMA (Fig. 6B).

Fig. 6. (A) Dose-dependent H2O2 release curve. (B) Time-dependent H2O2 release curve.

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Error bars represent the standard deviations of three independent experiments.

Hydrogen peroxide is also considered to be an important final or intermediate product

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in various biochemical reactions. Hence these biochemical reactions can be monitored for medical diagnosis by H2O2 assay. As a demonstration, H2O2 produced in the process of

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glucose oxidase (GOx)-catalyzed glucose oxidation was detected by BSA-Bi/PtNPscatalyzed colorimetric reaction. Taken into consideration of the suitable pH environment for GOx, MBTH-TOOS was used as chromogenic substrate for glucose monitor. BSABi/PtNPs can catalyze TOOS with MBTH by the action of H2O2 to produce a blue-purple product. The linear range of glucose determination is from 1 to 100 µM (Fig. S4). The LOD for glucose detection (0.2 µM) was lower than that using BSA-PtNPs as the catalyst (3.5 µM). As shown in Fig. 7, the results demonstrated that the colorimetric response of 17

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different reaction systems containing maltose, lactose, or fructose was negligible, verifying the selectivity of this method. In order to explore the feasibility and applicability of this approach, glucose level in human serum samples was detected. As

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shown in Table1, the results obtained by this approach are consistent with those determined by glucose oxidase endpoint method. In addition, the recoveries of glucose added to serum samples were found to be in the range of 98.6-104.3%. These results

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show that our proposed approach is suitable for monitoring glucose level in real samples.

Fig. 7. Selectivity of the method toward glucose.

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Table 1. Determination of glucose level in human serum samples by the proposed method and glucose oxidase endpoint method. Proposed method (mM) Endpoint method (mM) 4.63±0.04 4.78±0.01 6.34±0.23 6.79±0.08 3.18±0.05 3.36±0.12 10.23±0.11 10.64±0.23 3.81±0.07 3.63±0.01

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Sample 1 2 3 4 5

Relative deviation (%) -3.1 -6.6 -5.3 -3.8 4.9

Rapid, economical, and sensitive methods are very important for the early diagnosis of cancer in biomedical and clinical applications. The peroxides-like property makes BSABi/PtNPs a signal transducer. Folic acid (FA), which can effectively combined with folate 18

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receptor overexpressed on the membrane of various tumor cells [39], was used as recognition element and conjugated with BSA using covalent crosslink method. The formed FA-BSA-Bi/PtNPs can recognize tumor cells and transduce the recognition

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process to detectable signals. In order to verify the feasibility of this proposal, MCF-7 with overexpressed folate receptors was used as target cell. As shown in Fig. 8A, no obvious signal can be observed for BSA-Bi/PtNPs without FA modification in the

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absence or presence of MCF-7 cells, indicating nonspecific adsorption of BSA-Bi/PtNPs on the microplate or cells could be neglected. When FA-BSA-Bi/PtNPs incubated with

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MCF-7 cells, the BSA-Bi/PtNPs conjugated on the cell membrane catalyzed TMB to form the naked-eye observable chromogenic product. The interaction between FA-BSABi/PtNPs and folate receptor was further proved by FA block experiment, in which no color change reaction occurred.

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In order to verify whether the proposed method can be used to distinguish between tumor cells and normal cells, MCF-7 and EAhy926 were detected. As shown in Fig. 8B, all the samples with different amount of normal cells (EAhy926) remain almost colorless.

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However, darker color could be observed when more MCF-7 cells presented. Spectrophotometric detection results show that the absorbance values at 450 nm

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increased with the MCF-7 cell numbers in the range from 500 to 2000, consistent with the naked-eye observation. As for normal cells, the UV-vis signals were similar to the blank regardless of different cell numbers. The standard deviations of MCF-7 cell detection ranged from 4 to 13%. The LOD was calculated to be as low as 90 cells, which was superior to that of previously reported methods employing the peroxidase-like activity of nanomaterials [18, 40-42]. In view of above results, this approach has good

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sensitivity and selectivity for direct monitoring of tumor cells.

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Fig. 8. (A) The chromogenic reaction results for (a) 47.6 µg BSA-Bi/PtNPs without cells, (b) 47.6 µg BSA-Bi/PtNPs with 2000 MCF-7 cells, (c) 47.6 µg FA-BSA-Bi/PtNPs with 2000 MCF-7 cells, and (d) 47.6 µg FA-BSA-Bi/PtNPs with 2000 MCF-7 cells pretreated with 30 µg FA. (B) A450 vs. the cell number of EAhy926 (red column) and MCF-7 (green

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column).

4. Conclusion

In summary, a green and facile method was established for the synthesis of bimetallic

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Bi/Pt nanoparticles in bovine serum albumin scaffold. The developed BSA-Bi/PtNPs possess enhanced substrate affinity and peroxidase-like activity due to the bismuth

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modification. Moreover, the BSA-Bi/PtNPs are stable in harsh chemical and thermal conditions, as well as in common biological matrixes. These advantages, coupled with the easy of chemically or biologically modification, make BSA-Bi/PtNPs suitable candidates for widely biomedical applications. As demonstrations, serum glucose detection, extracellular hydrogen peroxide monitor, and cancer cells labeling have been realized with satisfying results.

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Acknowledgment We sincerely acknowledge the financial support of the National Natural Science

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Foundation of China (21175023), the Program for New Century Excellent Talents in University (NCET-12-0618), the Medical Elite Cultivation Program of Fujian Province (2013-ZQN-ZD-25), the Natural Science Foundation of Fujian Province (2016J01427),

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ACCEPTED MANUSCRIPT Highlights

● Synthesis of bimetallic Bi/Pt nanoparticles in bovine serum albumin scaffold. ●BSA-Bi/PtNPs possess enhanced peroxidase-like catalytic activity.

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●BSA-Bi/PtNPs are stable in biological matrixes. ● Analytical applications for monitor serum glucose and extracellular release of H2O2.

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● Synthesis of folic acid modified BSA-Bi/PtNPs for monitoring tumor cells.