Macromolecular Materials and Engineering

Peptide-induced synthesis of graphene-supported Au/Pt bimetallic nanoparticles for

electrochemical biosensor application

--Manuscript Draft--

Manuscript Number:

mame.202100886R1

Article Type:

Research Article

Corresponding Author:

Gang Wei, PhD

University of Bremen

Bremen, Bremen GERMANY

Corresponding Author E-Mail:

wei@uni-bremen.de

Order of Authors:

Bin Liu

Peng He

Hao Kong

Danzhu Zhu

Gang Wei, PhD

Keywords:

peptide nanofibers; self-assembly; graphene oxide; bimetallic nanoparticles;

electrochemical biosensors

Section/Category:

By Invitation Only: submission invited for regular issue

Abstract:

Understanding the self-assembly behavior of peptides is crucial for the design and

synthesis of functional peptide-based nanomaterials for various applications. In this

study, we design a bifunctional peptide molecule with a sequence of KIIIIKYWYAF,

which reveals multiple functions for self-assembling formation of peptide nanofibers

(PNFs), noncovalent graphene oxide (GO) binding, and biomimetic metallization of

nanoparticles (NPs). Well-defined PNFs are obtained through the optamization of

expermental conditions, which are further utilized to bind with GO to form GO/PNF

nanohybrids via noncovalent interactions. Ascribed to the biomimetic function of

peptide molecules, bimetallic gold/platnium nanoparticles (Au-Pt NPs) are created

along the PNFs by metallic ion adsorption and subsequent chemical reduction. The

synthesized GO/PNF/Au-Pt nanohybrids reveal improved electrochemical activity

compared to Au, Pt, and Au-Pt NPs, indicating potential contributions of both GO and

PNFs to the final electrochemical sensing performance of GO/PNF/Au-Pt-based

electrodes. The fabricated electrochemical non-enzymatic biosensors exhibit a

detection limit of 0.379 μM and linear detection ranges of 1 μM-1 mM and 1 mM- 20

mM. The current study provides a facile strategy for the creation of peptide-based

superstructures with multiple functions and will inspire the design and synthesis of

graphene-peptide based nanomaterials for biomedicine, tissue engineering, and

bioanalysis applications.

Author Comments:

Additional Information:

Question

Response

Please submit a plain text version of your

cover letter here.

Dear Dr. Weng,

Thank you very much for your kind consideration to our manuscript for the Macromol

Mater Eng (Research Article, No. mame.202100886). Also, we thank the referee for

her/his valuable suggestions and comments, which give us much help to improve the

quality of our manuscript.

According to the comments, we have revised the manuscript carefully to make it

suitable for the high standards of this journal. All the changes can be found in the

revised manuscript with tracked editing. The point-to-point replies towards all the

Powered by Editorial Manager? and ProduXion Manager? from Aries Systems Corporationcomments of the referee can be found in the Reply file.

Thank you very much for your kind consideration again and we are looking forward to

your decision.

best regards,

Gang Wei, PhD,

On behalf of all other authors

Do you or any of your co-authors have a

conflict of interest to declare?

No. The authors declare no conflict of interest.

Response to Reviewers:

1.Reply to the comments of Reviewer #1

Q1: The manuscript discussed the preparation of graphene-peptide-noble complex and

their application as H2O2 electrochemical sensors. The report is interesting overall and

I suggest publication after the following necessary revision.

R1: Thank you very much for your valuable comments and positive recommendation

on our manuscript. In this revised version, we modified the contents according to the

comments. We believe in the quality of this manuscript has been improved greatly. We

are looking forward to your kind consideration again.

Q2: Please discuss the potential applicatio scenario of the sensor with such detection

range.

R2: Thank you very much for this good suggestion. To answer this question, we have

added corresponding discussion of the potential application scenarios of our sensors

with this detection range.

"The potential application scenarios for electrochemical sensors with this detection

range are discussed further. First, the selective and sensitive detection of H2O2 is

becoming increasingly important in several fields such as environmental and industrial

analysis. The electrochemical sensor with low detection limit and wide detection range

can adapt to the requirements of environmental detection for different H2O2

concentrations.[49] Secondly, H2O2 is widely chosen as a strong oxidant in food

additives. The electrochemical H2O2 sensors are highly resistant to interference,

selective, and can accurately detect the H2O2 content in food products.[50] In addition,

the sensitive and selective determination of H2O2 plays an important role in biological

systems. The presence of H2O2 promotes a series of life activities such as intracellular

signal transduction, cell proliferation and protein synthesis.[51, 52] Excessive H2O2

concentration can lead to cellular damage, which can cause a series of diseases. The

wide detection range and low detection limit of our prepared chemical sensor is of

great importance for the accurate and reliable detection of endogenous H2O2."

(Page 15, marked with tracked editing)

In addition, four references were added.

[49]M. P. O'Halloran, M. Pravda and G. G. Guilbault, Talanta 2001, 55, 605-611

[50]H. Liu, Y. N. Ding, B. C. Yang, Z. X. Liu, X. Zhang and Q. Y. Liu, ACS Sustain.

Chem. Eng. 2018, 6, 14383-14393

[51]P. Balasubramanian, M. Annalakshmi, S. M. Chen, T. Sathesh, T. K. Peng and T.

S. T. Balamurugan, ACS Appl. Mater. Interfaces 2018, 10, 43543-43551

[52]Y. Zhang, X. Bai, X. Wang, K. K. Shiu, Y. Zhu and H. Jiang, Anal. Chem. 2014, 86,

9459-9465

Q3: The figure caption of Fig. 4 mentioned panel i and h, which do not exist in the

figure.

R3: Thank you very much for your careful reading.

We have double checked the description of Figure 4, and changed the label of "h" to

"e" and "i" to "f".

Q4: The chemical formula for chloroauric acid and potassium chloroplatinate need to

swith position on page 4 in the brancket.

R4: Thanks a lot for pointig out this problem. The chemical formula of "chloroauric acid

(K2PtCl6)" was changed to "chloroauric acid (HAuCl4)" and "potassium chloroplatinate

(HAuCl4)" was changed to " potassium chloroplatinate (K2PtCl6)".

Powered by Editorial Manager? and ProduXion Manager? from Aries Systems CorporationQ5: The atom identification represented by the black, red, and blue color in the model

need to be specified in figure 5.

R5: We agree with your comment. To respond this question, we made corresponding

modifications on Figure 5.

We have added new legends in Figure 5 representing the elements C, O, N, and H,

respectively, so that the reader can more clearly understand the atomic identity

represented by the different colors in the model.

Powered by Editorial Manager? and ProduXion Manager? from Aries Systems CorporationProf. Dr. Gang Wei · Qingdao University · Ningxia Road 308, 266071, Qingdao, PRC

Dr. Bo Weng

Editor

Macromolecular Materials & Engineering

Cover Letter

Dear Dr. Weng,

Thank you very much for your kind consideration to our manuscript for the Macromol Mater Eng

(Research Article, No. mame.202100886). Also, we thank the referee for her/his valuable

suggestions and comments, which give us much help to improve the quality of our manuscript.

According to the comments, we have revised the manuscript carefully

to make it suitable for the

high standards of this journal. All the changes can be found in the revised manuscript with tracked

editing. The point-to-point replies towards all the comments of

the referee can be found in the

Reply file.

Thank you very much for your kind consideration again and we are looking forward to your

decision.

best regards,

Gang Wei, PhD,

On behalf of all other authors

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

1. Reply to the comments of Reviewer #1.........................................................................P1-3

Prof. Dr. Gang Wei

College of Chemistry and Chemical Engineering

Qingdao University

Ningxia Road 308, Qingdao, Shandong, China

Tel. +86-150 66242101

E-Mail: weigroup@qdu.edu.cn

or wei@uni-bremen.de

Google Scholar:

http://scholar.google.de/citations?user=0b2QSWsA

AAAJ&hl=en

Qingdao, 15/12/2021

Response Letter to Reviewers1. Reply to the comments of Reviewer #1

Q1: The manuscript discussed the preparation of graphene-peptide-noble complex and their

application as H2O2 electrochemical sensors. The report is interesting overall and I suggest

publication after the following necessary revision.

R1: Thank you very much for your valuable comments and positive recommendation on our

manuscript. In this revised version, we modified the contents according to the comments. We

believe in the quality of this manuscript has been improved greatly. We are looking forward to your

kind consideration again.

Q2: Please discuss the potential applicatio scenario of the sensor with such detection range.

R2: Thank you very much for this good suggestion. To answer this question, we have added

corresponding discussion of the potential application scenarios of our sensors with this

detection range.

"The potential application scenarios for electrochemical sensors with this detection range are

discussed further. First, the selective and sensitive detection of H2O2 is becoming increasingly

important in several fields such as environmental and industrial analysis. The electrochemical

sensor with low detection limit and wide detection range can adapt to the requirements of

environmental detection for different H2O2 concentrations.[49] Secondly, H2O2 is widely chosen

as a strong oxidant in food additives. The electrochemical H2O2 sensors are highly resistant to

interference, selective, and can accurately detect the H2O2 content in food products.[50] In

addition, the sensitive and selective determination of H2O2 plays an important role in biological

systems. The presence of H2O2 promotes a series of life activities such as intracellular signal

transduction, cell proliferation and protein synthesis.[51, 52] Excessive H2O2 concentration can

lead to cellular damage, which can cause a series of diseases. The wide detection range and low

detection limit of our prepared chemical sensor is of great importance for the accurate and reliable

detection of endogenous H2O2."

(Page 15, marked with tracked editing)

In addition, four references were added.

[49] M. P. O'Halloran, M. Pravda and G. G. Guilbault, Talanta 2001, 55, 605-611

1[50] H. Liu, Y. N. Ding, B. C. Yang, Z. X. Liu, X. Zhang and Q. Y. Liu, ACS Sustain. Chem.

Eng. 2018, 6, 14383-14393

[51] P. Balasubramanian, M. Annalakshmi, S. M. Chen, T. Sathesh, T. K. Peng and T. S. T.

Balamurugan, ACS Appl. Mater. Interfaces 2018, 10, 43543-43551

[52] Y. Zhang, X. Bai, X. Wang, K. K. Shiu, Y. Zhu and H. Jiang, Anal. Chem. 2014, 86, 9459-

9465

Q3: The figure caption of Fig. 4 mentioned panel i and h, which do not exist in the figure.

R3: Thank you very much for your careful reading.

We have double checked the description of Figure 4, and changed the label of "h" to "e" and "i" to

"f".

Q4: The chemical formula for chloroauric acid and potassium chloroplatinate need to swith

position on page 4 in the brancket.

R4: Thanks a lot for pointig out this problem. The chemical formula of "chloroauric acid (K2PtCl6)"

was changed to "chloroauric acid (HAuCl4)" and "potassium chloroplatinate (HAuCl4)" was

changed to " potassium chloroplatinate (K2PtCl6)".

Q5: The atom identification represented by the black, red, and blue color in the model need to be

specified in figure 5.

R5: We agree with your comment. To respond this question, we made corresponding modifications

on Figure 5.

We have added new legends in Figure 5 representing the elements C, O, N, and H, respectively, so

that the reader can more clearly understand the atomic identity represented by the different colors

in the model.

231

Peptide-induced synthesis of graphene-supported Au/Pt bimetallic nanoparticles for

electrochemical biosensor application

Bin Liu, Peng He, Hao Kong, Danzhu Zhu, and Gang Wei*

B. Liu, P. He, H. Kong, D. Zhu, Prof. Dr. G. Wei

College of Chemistry and Chemical Engineering, Qingdao University, 266071 Qingdao, PR

China

E-mail: wei@uni-bremen.de or weigroup@qdu.edu.cn

Abstract

Understanding the self-assembly behavior of peptides is crucial for the design and synthesis of

functional peptide-based nanomaterials for various applications. In this study, we design a

bifunctional peptide molecule with a sequence of KIIIIKYWYAF, which reveals multiple

functions for self-assembling formation of peptide nanofibers (PNFs), noncovalent graphene

oxide (GO) binding, and biomimetic metallization of nanoparticles (NPs). Well-defined PNFs

are obtained through the optamization of expermental conditions, which are further utilized to

bind with GO to form GO/PNF nanohybrids via noncovalent interactions. Ascribed to the

biomimetic function of peptide molecules, bimetallic gold/-platnium nanoparticles (Au-Pt NPs)

are created along the PNFs by metallic ion adsorption and subsequent chemical reduction. The

synthesized GO/PNF/Au-Pt nanohybrids reveal improved electrochemical activity compared to

Au, Pt, and Au-Pt NPs, indicating potential contributions of both GO and PNFs to the final

electrochemical sensing performance of the GO/PNF/Au-Pt-based electrodes. The fabricated

electrochemical non-enzymatic biosensors exhibit a detection limit of 0.379 μM and linear

detection ranges of 1 μM-1 mM and 1 mM- 20 mM. The current study provides a facile strategy

for the creation of peptide-based superstructures with multiple functions and will inspire the

design and synthesis of graphene-peptide based nanomaterials for biomedicine, tissue

engineering, and bioanalysis applications.

Revised Manuscript

 2

Keywords: peptide nanofibers; self-assembly; graphene oxide; bimetallic nanoparticles;

electrochemical biosensors

1. Introduction

With the development of nanobiotechnology and material science, more and more

attentions have been attracted on the structural/functional design and tailoring of

nanomaterials.[1-3] Peptide molecules have multiple physical, chemical, and biological

properties, including designable molecular structure, controllable self-assembly ability, high

biocompatibility, adjustable molecular and material recognition functions, and therefore

exhibited wide applications in biomedicine and tissue engineering.[4-8]

Thanks to the achievement of peptide design and synthesis in the last years, a lot of

functional peptide molecules with unique self-assembly ability have been created, which could

be tailored to form various nanostructures from zero-dimensional (0D) nanosphere,[9, 10] to one

dimensional (1D) nanofibers/nanotubes,[11, 12] two-dimensional (2D) nanobelts/nanosheets,[13-

15] and three-dimensional (3D) hydrogels/aerogels.[16, 17] Generally, the self-assembly of

peptides to various nanostructures could be carried out in solutions, on solid substrates, and at

the air/water or oil/water interfaces through applying internal or external simulations to promote

the conformation transition and self-assembly of peptide molecules.[18]

Peptide nanofibers (PNFs) are especially popular in the biomedical and tissue engineering

fields as the formation of 1D peptide aggregates is dominant in the formation of amyloids.[4, 19]

Although self-assembled PNFs are useful nanoscale building blocks for the fabrication of

various functional biomaterials, their extended mechanical strength, catalysis, optical, magnetic,

and other properties are highly needed to fit the requirements of special applications of peptide

based materials. One simple and potential strategy for this aim is to bind PNFs with other

functional nanomaterials such as nanoparticles, polymers, carbon nanotubes, 2D materials, and

others to form hybrid nanomaterials.[4, 8, 20, 21] For instance, Very recently, we reported the

synthesis of ZrO2 nanoparticles on GO-supported PNFs for the creation of functional hybrid

3

membrane materials, which exhibited high-performance for the adsorption and removal of F

ions.[21] Pazos et al. demonstrated the nucleation and growth of silver nanoparticles (AgNPs)

on PNFs for the construction of a novel biocompatible antimicrobial propertiesmaterial. [22] The

formed metallized organic nanofibrous hybrid materials provided the typical example for

integrating PNFs and metallic contents together for improved functions. In another case, Li and

co-workers reported the binding of PNFs with graphene quantum dots and graphene oxide (GO)

nanosheets for the preparation of a combined hybrid nanomaterials,[23] which revealed potential

applications for the fabrication of electrochemical hydrogen peroxide (H2O2) biosensors.

Due to the adjustability of peptide sequences, it is possible to tailor the structure of the

amino acid sequence to obtain peptides with specific recognition ability towards different

materials interfaces,[24-26] by which the self-assembled PNFs could have multiple functions of

self-assembly, biomimetic synthesis, and material recognition. Previously, Okur et al.

demonstrated the design and self-assembly of nerve growth factor (NGF)-β binding peptide for

the creation of bioactive PNFs, which exhibited promoted effects on the outgrowth of sensory

neurons.[27] Through the screening of phage peptide library, the functionalization of self

assembled peptide materials could be achieved by rationally inserting functional motifs into the

sequences of peptide molecules.[28, 29]

As one of the most important 2D materials in the past years, graphene have shown

promising applications in various research fields.[30] It is well known that peptide molecules

could be adsorbed onto graphene surface via both covalent and noncovalent interactions.[31, 32]

The motif design of peptide molecules provided a versatile way to form uniform GO-peptide

nanohybrids by noncovalent interactions between GO and peptide molecules. It was reported

that a lot of functional peptide motifs, such as YWYAF,[33] GAMHLPWHMGTL,[34]

EPLQLKM,[35] FF,[36] and many others, [31] have been utilized for the recognition with graphene

surface for the construction of graphene-peptide nanocomposites for different applications.

Therefore, it is desirable to design a multifunctional peptide molecule with the functions of PNF

 4

formation, GO binding, and biomimetic mineralization, which will achieve in the facile green

synthesis of GO-PNF hybrids in one way, and at the same time mediate the growth of active

metallic nanomaterials through biomimetic strategy for electrocatalysis and electrochemical

sensor applications.

Herein, in this work we design an active peptide molecule with bifunctional motifs,

KIIIIKYWYAF, in which the head motif (KIIIIK) is responsible for the self-assembly

formaiton of PNFs, [37] and the tail graphene-binding motif (YWYAF)[38] can promote the

noncovalent binding of peptide with graphene surface. It is hypothesized that the self-assembled

PNFs can bind with GO to form GO-PNF nanohybrids, which can be further metallized into

GO/PNF/Au-Pt hybrid materials through the chemical reduction of adsorbed metallic cations.

The synthesized GO/PNF/Au-Pt nanocomposites were further used to modify the glassy carbon

electrode (GCE) for the fabrication of electrochemical H2O2 biosensors. The obtained results

show that the prepared GCE has strong electrocatalytic activity to H2O2. The detection limit of

the electrochemical H2O2 sensor is as low as 0.379 μΜ with two fine linear detection ranges

from 1 μM-1 mM and 1 mM-20 mM.

2. Results and discussion

2.1. Preparation and characterizations of GO/PNF/Au-Pt nanocomposites

Figure 1 presents the synthesis process of GO/PNF/Au-Pt nanocomposites. By designing

a peptide molecule KIIIIKYWYAF with two functional motifs, PNFs are formed by tailoring

the self-assembly of peptide molecules in solution. After that, ultrasonically dispersed GO

solution is added to the PNF solution to form GO-PNFs nanohybrids via specific binding

interactions between PNFs and GO. Then chloroauric acid (HAuCl4K2PtCl6) and potassium

chloroplatinate (K2PtCl6HAuCl4) solution are added to the mixed solution for ionic adsorption.

After a period of reaction, sodium borohydride (NaBH4) is added to reduce the adsorbed

metallic ions to form bimetallic Au-Pt NPs. Finally, peptide-induced biomimetic GO/PNF/Au

Pt nanocomposites are created.

Figure 1. Schematic preparation of biomimetic GO/PNF/Au-Pt nanocomposites for

electrochemical biosensor application.

There are a few advantages for using this ternary peptide-induced ternary GO/PNF/Au-Pt

nanocomposite. First, the aromatic groups in PNFs can interact with GO to produce strong,

specific binding force, thus promoting the compounding with GO nanosheets. Second, PNFs

can serve as flexible bridges between GO nanosheets and biomimetic Au-Pt NPs. In addition,

PNFs form a strong spacer layer between GO nanosheets, which plays an important role in the

dispersion of GO nanosheets and the diffusion of reactants. Third, there are many active sites

in on PNFs, which can adsorb different metal ions and biomimetic NPs through electrostatic

and coordination interactions. PNFs are well dispersed on GO nanosheets, which makes the

self-assembled PNFs can adsorb more metallic ions and create more Au-Pt NPs, thus revealing

better electrochemical sensing and catalytic performances.

2.2. Design and synthesis of PNFs

It is well known that the self-assembly of peptides could be induced by intermolecular

interactions such as hydrogen bonding, hydrophobicity, electrostatic interaction, and van der

 6

Waals force. As shown in Figure 2a, the designed KIIIIKYWYAF peptide sequence consists

of two typical peptide motifs, KIIIIK and YWYAF, in which KIIIIK peptide motif is a typical

β-sheet amino acid sequence. KIIIIK peptide has an acetyl group at the N terminal and an amine

at the C terminal. In the study of Lu et al., it was found that KIIIIK peptides can self-assemble

into monolayer nanotubes, and other similar short peptides tend to form nanofibers in their

experiment. [39] Based on this study, we suggest that KIIIIK peptide motifs can form stable PNFs

by tailoring the self-assembly conditions of peptide molecules. YWYAF groups can behas been

identified by material-specific materials and can be combined with graphene materials to form

graphene/PNF nanohybrids without using any surface functionalization. The aromatic groups

in YWYAF can interact with GO sheets by π-π stacking to form strong binding between PNFs

and GO nanosheets.

Figure 2. Design and synthesis of PNFs: (a) motifs of KIIIIKYWYAF peptides, (b) AFM

height images of PNFs, (c) section analysis of PNFs, and (d) corresponding height distribution

of PNFs.

When the designed peptides are dissolved in the mixed solution of ethanol and

trifluoroethanol (TFE), the ethanol system is found to be beneficial to the formation of PNFs in

our experiments. Obvious peptide monomers are distributed in the reaction solution at the

 7

beginning. After 5 days of reaction and self-assembly, PNFs with good morphology were

formed in the solution, and the formed PNFs exhibit a long-term stability. PNFs still maintained

a good morphology during the continuous detection period of 30 days. From the atomic force

microscopy (AFM) characterizations, it can be clearly seen that the PNFs with uniform height

and length are formed through molecular self-assembly (Figure 2b). The height of PNFs is

measured through the AFM section analysis (Figure 2c), and the statistic analysis of the PNF

height indicates that PNFs have an average diameter (height) of about 6 nm, as shown in Figure

2d.

2.3. Characterizations of GO/PNF/Au-Pt nanocomposites

The prepared PNFs are mixed with GO nanosheets for the formation of GO/PNF

nanohybrids under gentle stirring for 2 hours at room temperature. Because of the π-π stacking

and specific binding interactions between PNFs and GO, GO/PNF nanohybrids can be formed

easily. Figure 3a shows the AFM height image of the GO nanosheets. The size of these

monolayer GO nanosheets is about a few μm and the height is measured to be 1.1 nm (Figure

3c), which agree with the height and size data shown in previous report. [40] After mixing GO

and PNF solution together for 2 hours, PNFs are bound onto the surface of GO nanosheets

specifically, as indicated in Figure 3b. From the AFM image, it can be found that PNFs are

uniformly distributed on the GO nanosheets. In the area without GO, it is clear that no PNFs

are existed, which shows that PNFs have been adsorbed onto the GO nanosheet with high

selectivity thanks to their strong interaction. This finding is consistent with our initial design of

peptide sequences and binding to GO sheet through biomolecule-materials interface

interactions.

 8

Figure 3. AFM characterizations of materials: (a) GO nanosheets, (b) GO/PNF nanohybrids,

(c) section analysis of GO, (d, e) GO/PNF/Au-Pt nanocomposites with different magnifications,

and (f) section analysis of the components of GO/PNF/Au-Pt nanocomposites.

The as-prepared binary GO-PNF nanohybrids served as biological templates for

biomimetic synthesis of bimetallic Au-Pt NPs to create GO/PNF/Au-Pt nanocomposites finally.

The theoretical isoelectric point of KIIIIKYWYAF peptide sequence is 9.94, and therefore the

peptide molecules (or PNFs) can adsorb negatively charged AuCl4- and PtCl62- ions in a neutral

solution. After the overnight reaction, the excess ions in the solution were centrifuged out. Then

NaBH4 was added to reduce the adsorbed ions to prepare ternary GO/PNF/Au-Pt

nanocomposites. Figures 3d and 3e show typical AFM height images of the synthesized

GO/PNF/Au-Pt nanocomposites. It can be seen that there are a large number of PNFs and metal

NPs on the GO nanosheets. The height of the components, like GO nanosheets, PNFs, and Au

Pt NPs, are measured with AFM section analysis, and the data is shown in Figure 3f. It can be

found that the average height of PNFs is 5.1 nm, which is not much different from the data

measured in Figures 2c and d. In addition, the height of biomimetic Au-Pt NPs is measured to

be about 8.73 nm.

 9

Figure 4. TEM, SEM and XPS characterizations of GO/PNF/Au-Pt nanocomposites: (a, b)

TEM images, (c) SEM image, (d-f) XPS spectra: (d) survey spectrum, (he) Pt4f, and (if) Au4f

spectra.

The formed GO/PNF/Au-Pt nanocomposites were further characterized with transmission

electron microscopy (TEM), scanning electron microscopy (SEM), and x-ray photoelectron

spectroscopy (XPS). Figure 4a presents the typical TEM image of the peptide-induced

GO/PNF/Au-Pt nanocomposites. It can be seen that a lot of NPs are formed along the self

assembled PNFs that adsorbed on GO nanosheets. From the zoomed TEM image in Figure 4b,

the binding of Au-Pt NPs on GO sheets is further identified, which indicates that PNFs and Au

Pt bimetallic NPs have been loaded onto the surface of GO nanosheets successfully. We suggest

10

that GO nanosheets, as the supporting base of the whole materials, can make the whole ternary

hybrid materials more stable, and the existence of bimetallic NPs can also be seen in the SEM

image (Figure 4c), which is also consistent with the presented TEM images. Finally, in order

to determine the elements in GO/PNF/Au-Pt nanocomposites and the electrical state of Au and

Pt, XPS was applied. The obtained XPS data (Figure 4d) indicates that the created

GO/PNF/Au-Pt nanocomposites include C1s, N1s, O1s, Pt4f and Au4f. The detailed XPS

spectra of Au (4f7/2 in 84 eV, 4f5/2 in 87.7 eV) and Pt (4f7/2 in 71 eV and 4f5/2 in 74 eV) further

confirmed the formation of Au-Pt NPs in GO/PNF/Au-Pt nanocomposites, as shown in Figure

4e and f.

2.4. Formation mechanism of ternary GO/PNF/Au-Pt nanocomposites

In this section, the formation mechanism of PNFs via the motif-designed peptide sequence

KIIIIKYWYAF is further discussed. Figure 5a shows the 3D molecular structure formula of

the used peptide sequence in this study. The KIIIIK sequence and YWYAF are linked by amino

groups. GO nanosheet is a monolayer of carbon atoms with dense honeycomb structure and

contains a large number of reactive oxygen species functional groups, which can provide a

number of functional active sites. Therefore, GO nanosheets can be used as precursors for

specifically binding peptides and self-assembled PNFs. Previously, through the combination of

experiments and computer simulation, the Naik team confirmed that the peptides with YWYAF

groups show preferential binding on the edge and plane of graphene, and the anchoring binding

of this peptide seems to be more reliable than other binding methods [33, 41] . Therefore, two Y

and one F amino acids in the YWYAF motif have aromatic groups, showing strong π-π stacking

between the aromatic groups and graphene surface, which makes it easier for PNFs to connect

to the surface of GO nanosheets through noncovalent interactions (Figure 5b). In addition, in

the study of Zorbas et al., it has been shown that the aromatic residues of peptides may play an

important role in the dispersion of carbon nanotubes in aqueous solution through π-π stacking

11

interaction[42]. Therefore, we suggest that the existence of PNFs can also promote the dispersity

of GO and the formation of stable GO-PNF nanohybrids.

Figure 5. Binding mechanism of PNFs onto GO: (a) 3D molecular structural formula of

KIIIIKYWYAF peptide, and (b) schematic diagram of the graphene-specific binding of PNFs.

PNFs not only provide nucleation and growth templates for biologically induced synthesis

of Au-Pt NPs, but also serve as bridges between GO and biomimetic Au-Pt NPs, thus forming

GO-PNFs-Au-Pt nanocomposites with high loading density of NPs. The isoelectric point of the

peptide motif KIIIIKWYAF is 9.87, while the reaction solution is weakly acidic. When the pH

of the solution is less than the isoelectric point of peptide, peptides are more likely to absorb

the negatively charged metallic ions.

2.5. Application of H2O2 Sensing

In order to detect explore the application of electrochemical biosensor, GO/PNF/Au-Pt

ternary nanocomposites were used to modify glassy carbon electrode (GCE) to prepare

nonenzymatic electrochemical biosensors for the detection of H2O2. Figure 6a shows the cyclic

voltammogram (CV) of GCEs that modified with Au NPs, Pt NPs, Au-Pt NPs, and

12

GO/PNF/Au-Pt nanocomposites (the electrodes are named as Au/GCE, Pt/GCE, Au-Pt/GCE

and GO/PNF/Au-Pt/GCE, respectively) at a scanning rate of 50 mV s-1 . It is clear that both

Au/GCE and Pt/GCE show a smaller current response than other two GCEs. Moreover, from

the comparison of the materials of single metal NPs, the electrochemical sensing performance

of Au/GCE is slightly higher than that of Pt/GCE. The response current of Au-Pt/GCE is

obviously higher than that of Au/GCE or Pt/GCE, which depends on the contribution of

bimetallic structure. It has been reported that the addition of a second metal, such as Au, to Pt

could not only improve the electrochemical catalysis and sensor performance of Pt, but also

prevent Pt catalytic poisoning effectively. [43]

It is worth noting that our GO/PNF/Au-Pt/GCE showed excellent catalytic effect for the

detection of H2O2, and the peptide composite-modified GCE showed a stronger current

response and a wide oxidation peak between 0.5 - and 0.8 V. We suggest that GO/PNF/Au-Pt

nanocomposites have excellent electrochemical performance, and the existence of GO materials

makes the electrode materials more orderly, while providing a larger specific surface area for

material exchange. Meanwhile, PNFs can be adsorbed onto GO sheets through molecule

material interaction, which can be used as the gap between GO sheets, thus making the diffusion

of detection substrates in the electrode materials faster.[23] In addition, there are many active

sites in PNFs, which can adsorb a large amount of Au-Pt NPs, which and further improved the

catalytic oxidation performance of the synthesized materials. Although the electrode material

prepared in this way is not in a three-dimensional3D structure, the ternary nanocomposites can

make the whole material to form a conductive network-like structure, which is also beneficial

to the material contact and current transfer.

The current response of the fabricated GO/PNF/Au-Pt/GCE at different H2O2

concentration (0-20 mM) is further measured. It can be seen that the current response value

increases obviously with the increasing of the H2O2 concentration. Through further linear fitting

 13

between peak current and H2O2 concentration, a good linear relationship between peak current

and H2O2 concentration is found.

Figure 6. Electrochemical tests of various GCEs: (a) CVs Pt NP/GCE, Au NP/ GCE, Au-Pt

NP/GCE, and GO/PNF/Au-Pt/GCE, (b) current response of GO/PNF/Au-Pt/GCE towards H2O2

with different concentrations, (c) current response of GO/PNF/Au-Pt/GCE towards H2O2 with

different scan rates, and (d) peak currents versus the square root of the scan rates.

In order to further explore the kinetic of catalytic oxidation of H2O2 by GO/PNF/Au

Pt/GCE, CV curves of GO/PNF/Au-Pt/GCE with different scanning rates were analyzed in the

electrolyte solution with 5 mM H2O2. As shown in Figure 6c, it can be seen that there are

obvious oxidation peaks between 0.5-0.8 V, and the peak current increases with the increasing

of the scanning rate. Through linear fitting between the peak current and the square root of the

scanning rate (Figure 6d), it can be seen that there is a linear relationship between the oxidation

peak current and the square root of the scanning rate, and the linear correlation coefficient is

 14

0.994, which indicates that the reaction on the electrode surface is a diffusion control process,

and the diffusion control process is conducive to the quantitative detection and analysis of H2O2.

Figure 7. Electrochemical sensing of H2O2: (a) I–T response and (b) linear calibration (1 μM-

1 mM) of GO/PNF/Au-Pt/GCE in 0.1 M NaOH with successive addition of H2O2 at 0.69 V

versus SCE. The inset in Figure 7b shows the linear calibration of I-T response with H2O2

concentration from 1 to 20 mM.

The chronoamperometry is one of the commonly used methods for electrochemical

detection. As can be seen from Figure 6b, the highest peak potential reaches at 0.69 V with the

increasing of the H2O2 concentration, and therefore 0.69 V is chosen as the applied potential

for the current-time (I-T) measurement. Figure 7a shows the I-T curve of the prepared

GO/PNF/Au-Pt/GCE with continuous addition of different concentrations of H2O2 at the

working potential of 0.69 V. With the addition of H2O2 solution, obvious change of current can

be observed by only adding 1 μM H2O2, and the response speed is faster and more stable.

Through the fitting (Figure 7b) of the response current versus the added H2O2 concentration,

the linear fitting degree is good and the sensitivity is high in the ranges from 1 μM to 20 mM,

and the detection limit is calculated to be about 0.379 μ M. It should be noted that the linear

ranges include two separated parts, from 1 μM to 1 mM, and 1 mM to 20 mM, respectively.

Compared with other previously reported electrochemical nonenzymatic H2O2 biosensors

[44-48], the ternary GO/PNF/Au-Pt/GCE-based H2O2 biosensor exhibits lower detection limit and

15

wider detection range, as shown in Table 1. The potential application scenarios for

electrochemical sensors with this detection range are discussed further. First, the selective and

sensitive detection of H2O2 is becoming increasingly important in several fields such as

environmental and industrial analysis. The electrochemical sensor with low detection limit and

wide detection range can adapt to the requirements of environmental detection for different

H2O2 concentrations. [49] . Secondly, H2O2 is widely chosen as a strong oxidant in food additives.

The electrochemical H2O2 sensors are highly resistant to interference, selective, and can

accurately detect the H2O2 content in food products. [50] . In addition, the sensitive and selective

determination of H2O2 plays an important role in biological systems. The presence of H2O2

promotes a series of life activities such as intracellular signal transduction, cell proliferation

and protein synthesis. [51, 52] . Excessive H2O2 concentration can lead to cellular damage, which

can cause a series of diseases. The wide detection range and low detection limit of our prepared

chemical sensor is of great importance for the accurate and reliable detection of endogenous

H2O2.

Table 1. Comparison of the H2O2 sensing performances of the GO/PNF/Au-Pt nanocomposites

with other materials.

Materials

Linear range

Detection limit

Ref.

GQD-PNF-GO nanohybrids

0.01-7.2 mM

0.055 μM

[23]

RGO-GSP- PtNP

nanohybrids

0.2-8.0 mM，

8.0-15.0 mM

28 μM

[44]

GO-AgNPs nanohybrids

0.02-18 mM

0.13 μM

[45]

RGO-PNF-AgNPs

nanohybrids

0.05-5 mM

10.4 μM

[46]

RGO-Au NP hybrid

membranes

0.25 to 22.5 mM

6.2 μM

[47]

hollow CuO/PANI fibers

0.001-19.899 mM

0.45 μM

[48]

GO/PNF/Au-Pt

nanocomposites

0.001-1 mM，

1-20 mM

0.379 μM

This work

16

Figure 8. (a) Selectivity of biosensor: amperometric responses upon successive addition of

H2O2, UA, AA, UALys, and H2O2. (b) Long-term stability towards the detection of 5 mM H2O2.

Other oxidizable organic compounds will affect the detection of H2O2. In order to test the

anti-interference of the modified GCEs, uric acid (UA), ascorbic acid (AA), and lysine (Lys)

were selected to test the selectivity and anti-interference of the fabricated H2O2 sensor. As

shown in Figure 8a, the response current value increased rapidly after the addition of H2O2,

and the current signal almost did not change when adding UA, AA, and Lys, respectively. When

H2O2 was added again, the current value increased clearly, indicating that the added

interferences could not affect the detection performance of the fabricated H2O2 sensor, which

proved that the GO/PNF/Au-Pt/GCE had good selectivity and anti-interference ability.

Furthermore, the long-term stability of the GO/PNF/Au-Pt/GCE was checked (Figure 8b).

The fabricated H2O2 sensor was stored at 4 ℃ for different periods and measured every 2 days.

The result indicates that the current response value of the electrode in 5 mM H2O2 solution is

still more than 94.3% of the initial current response value after storing for 7 days, which proves

that the GO/PNF/Au-Pt/GCE has high stability for sensing H2O2.

3. Conclusions

17

In summary, we report a biomimetic method for the green synthesis of GO/PNF/Au-Pt

nanocomposites based on peptide self-assembly and biometallization. AFM experiments show

that the GO has a strong affinity with the surface of PNFs, and a uniform GO/PNF nanohybrid

material was formed by directly mixing GO nanosheets and PNFs together within 2 hours. The

bound PNFs served as versatile binding sites for adsorbing metal ions, and further mediated the

biomimetic synthesis of bimetallic Au-Pt NPs with uniform size and morphology. In addition,

PNFs provided flexible bridges for connecting GO nanosheets and Au-Pt NPs. Electrochemical

experiments showed that the prepared GO/PNF/Au-Pt nanocomposites have high

electrocatalytic activity towards H2O2, and the prepared GO/PNF/Au-Pt-based electrochemical

H2O2 sensors exhibited the characteristics of low detection limit, high selectivity, good

reproducibility, and long-term stability. We believe that the facile functionalization of GO

nanosheets with motif-designed and self-assembled PNFs will contribute to the functional and

structural tailoring of materials for the fabrication of high-performance biosensors for

biomedical and bioanalysis application in the future.

4. Experimental section

Reagents and materials: The peptide with a sequence of KIIIIKYWYAF was bought

from the SynPeptide Biotechnology Co., Ltd. (Nanjing, PR China). Chloroauric acid (HAuCl4

3H2O, ≥49.0% Au basis), Potassium hexachloroplatinate (IV) (K2PtCl6, 99.9%), Ascorbic acid

(AA), uric acid (UA) and L-Lysine (Lys) were obtained from the Macklin Biochemical Co.,Ltd

(Shanghai, China). H2O2 (analytical grade, 30% aqueous solution), Trifluoroacetic acid (TFA,

99%), ethanol, trifluoroethanol (TFE, 99.5%), and NaOH (96%) were provided by the

Sinopharm Chemical Reagent Co., Ltd. (Beijing, PR China). GO (10 mg/g in water) was

purchased from the GaoxiTech Co., Ltd. (Hangzhou, PR China).

Self-assembly of peptide monomer to form PNFs: In short, 6 mg peptide powder was

dissolved in 6 mL 0.1% TFA solution to form a uniformly dispersed peptide solution. which

was then diluted to 0.2 mg/mL with TFE, and then diluted to 0.1 mg/mL with ethanol. After 5

days of reaction, PNFs were prepared.

18

Preparation of GO/PNF/Au-Pt nanohybrids: 5 mL as-prepared PNFs were mixed with

5 mL 0.1 mg/mL GO, which were then reacted at room temperature for 2 hoursfor the formation

of GO/PNF nanohybrids. Afte that, 500 μL HAuCl4 (10 mM) and 500 μL K2ClPt6 solution (10

mM) were added into the formed GO/PNF nanohybrids for the adsorptio of metallic ions onto

GO/PNF nanohybrids. After reacting overnight at room temperature, the mixed solution was

centrifuged to remove the up solution and obtain the precipitation. The obtained solid mateiral

was further dissolved with ultrawater. Finally, 10 μL 1% NaHB4 solution was added to reduce

the metallic ions to form GO/PNF/Au-Pt nanocomposites.

Preparation and characterization of Au/GCE, Pt/GCE, Au-Pt/GCE, and GO/PNF/Au

Pt/GCE: The glass carbon electrodeS (GCE, diameter 4.0 mm) were polished with alumina

powder of 0.05 mm, and then cleaned by ultrasonic in ultra-pure water and ethanol solution,

respectively. Then 50 μL Nafion solution was added to 1 mL solutions of Au NPs, Pt NPs, Au

Pt NPs, and GO/PNF/Au-Pt, respectively. After that, the mixed solution was put in ultrasonic

bath for 15 minutes, and then 6 μL prepared solution was dripped onto the surface of GCE for

the preparation of Au/GCE, Pt/GCE, Au-Pt/GCE, and GO/PNF/Au-Pt/GCE, repectively.

Characterization techniques: All atomic force microscopy (AFM) samples were

prepared by dropping 10 μL samples onto the freshly cleaved mica substrates and dried in air

for characterization. AFM measurements were performed in air using the AFM-Nanoview 6800

AFM (FSM-Precision, Suzhou Flying Man Precision Instrument Co., Ltd, PR China ) with

tapping mode. Silicon probes of type Tap300Al-G (300 kHz, 40 N/m) were used for AFM

image capturing. The tapping mode images were recorded and analyzed with Gwyddion

software (Version 2.57).Transmission electron microscope (TEM, Tecnai G2 F20, FEI Co.)

was used to observe the structure and morphology of Peptide nanocomposites.Scanning

electron microscope (SEM, Regulus 8100, Hitachi, Japan) was used to observe the

microstructure of the Peptide nanocomposites. The X-ray photoelectron spectroscopy (XPS)

characterization of samples was performed on a PHI 5000 VersaProbe III spectrometer

(UlVAC-PHI Company, Japan). All the electrochemical experiments were carried out at room

temperature using an electrochemical workstation (CHI660E, Shanghai Chenhua). Using the

traditional three-electrode system, the working electrode is modified glassy carbon electrode,

the auxiliary electrode is platinum wire, and the reference electrode is saturated calomel

19

electrode. Electrochemical experiments were carried out after deoxidizing 20 min in high purity

nitrogen using 0.1M NaOH solution as electrolyte solution.

Note

The authors declare no conflict of interests.

Acknowledgements

The authors thank the financial support from the National Natural Science Foundation of China

(No. 51873225), the Taishan Scholars Program of Shandong Province (No. tsqn201909104),

and the High-Grade Talents Plan of Qingdao University.

Received: ((will be filled in by the editorial staff))

Revised: ((will be filled in by the editorial staff))

Published online: ((will be filled in by the editorial staff))

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22

We demonstrate the self-assembly of motif-designed peptide monomers for the creation of

peptide nanofibers (PNFs) with multiple functions, including strong graphene oxide (GO)

binding and biomimetic metallization. Bimetallic Au-Pt nanoparticles are formed along the axis

of PNFs that bound on GO, and the formed GO/PNF/Au-Pt nanocomposites exhibit enhanced

electrochemical catalysis and nonenzymatic sensing towards H2O2.

B. Liu, P. He, H. Kong, D. Zhu, G. Wei*

Title: Peptide-induced synthesis of graphene-supported Au/Pt bimetallic nanoparticles for

electrochemical biosensor application

ToC Figure

Table of Contents

We demonstrate the self-assembly of motif-designed peptide monomers for the creation of

peptide nanofibers (PNFs) with multiple functions, including strong graphene oxide (GO)

binding and biomimetic metallization. Bimetallic Au-Pt nanoparticles are formed along the axis

of PNFs that bound on GO, and the formed GO/PNF/Au-Pt nanocomposites exhibit enhanced

electrochemical catalysis and nonenzymatic sensing towards H2O2.

B. Liu, P. He, H. Kong, D. Zhu, G. Wei*

Title: Peptide-induced synthesis of graphene-supported Au/Pt bimetallic nanoparticles for

electrochemical biosensor application

ToC Figure

Journal's Table of Contents (ToC)

Click here to access/download;Journal's Table of Contents

(ToC);TOC.docx