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Journal of Colloid and Interface Science 325 (2008) 356–362

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Journal of Colloid and Interface Science

www.elsevier.com/locate/jcis

Preparation and characterization of the biomineralized zinc oxide particles in

spider silk peptides

Zhongbing Huang a , Danhong Yan a ,MeiYang a , Xiaoming Liao a , Yunqing Kang a , Guangfu Yin a , ∗ , Yadong Yao a ,

Baoqing Hao b

a College of Materials Sciences and Engineering, Sichuan University, No. 24, South 1st Section, 1st Ring Road, Chengdu, Sichuan 610065, China

b College of Life Science and Technology, Southwest University for Nationalities, No. 16, South 4th Section, 1st Ring Road, Chengdu, China

article info

abstract

Article history:

Received 31 March 2008

Accepted 20 May 2008

Availableonline28May2008

In this work, hierarchical ZnO particles were prepared using a biomineralization strategy at room temper-

ature in the presence of peptides acidiﬁed from spider silk proteins. A mechanism of the mineralization

of the ZnO particles was that the anity of original ZnO nanoparticles and zinc ions in the peptide

chains played an important role in controlling the biocrystallizing formation of the pore ZnO particles.

The intensity of their visible green luminescence was enhanced with increases of the mineralization time

due to the porous surface defects. The hierarchical ZnO materials with biomolecules will facilitate their

photoluminescence spectra applications as biosensors or optoelectronic nanodevices in the future, when

covalently coupled with peptides or other biomolecules to achieve patterned growth over large areas of

substrate.

Keywords:

ZnO particles

Porous

Peptide

Biomineralization

1. Introduction

ture [19–21] . However, wet-chemical methods carried out under

mild conditions are attracting growing interest. In order to assem-

ble ZnO biosensors for the detection of biomolecules, the tech-

niques can cause no damage to the coupled biomolecules. If these

syntheses can be conducted under room temperature conditions,

they will reduce the production cost, the facility size (such as

cooling vacuum systems), and the manpower, which will have a

signiﬁcant impact on manufacturing zinc-based biodevices. Low-

temperature processing could also reduce nanoparticle aggregation

and enhance optical properties induced from special defects. Bio-

logical systems possess the ability to synthesize exotic materials

at room temperature via their enzymatic peptide activities [22] .

Recently, various room-temperature material syntheses have been

examined by the use of mimicked systems engaging in biominer-

alization [23–25] . Those biomolecular catalysts can cause mineral-

ization through speciﬁc interactions between chemical moieties in

unique bioconformations and inorganic solutes [26] .Forexample,

artiﬁcial peptides are applied to catalyze the hydrolysis and poly-

condensation of ZnO [27] . However, these special artiﬁcial peptides

are expensive and hard-won, and the size and shape control of

ZnO particles was hardly achieved. In general, spiral spider silk can

be hydrolyzed into peptides, which are composed of many amino

acids, including in particular nucleophilic hydroxyl of cysteine, as-

partic acid, and amine of histidine, for the biomineralization of

semiconductor metal oxides with the unique structure.

Here, we report that the amphiphile peptide templates acidiﬁed

from spiral silk protein of spiders ( Uloborus walckenaerius Latreille),

including cysteine, aspartic acid, and histidine, have chemical moi-

In nature, growth of crystals has typically been thought to oc-

cur by atom-by-atom addition to an inorganic or organic template

or by dissolution of unstable phases (ﬁne particles or metastable

polymorphs) and the more stable phases with reprecipitation [1] .

Recently, progress in biology has enabled us to identify peptides

through an anity for nonbiological materials, such as those that

mediate the mineralization of inorganic materials. In natural bio-

logical systems, proteins and peptide such as silicatein [2–5] and

ferritin [6–9] bring about the precipitation of inorganic materi-

als inside the cell, and can control their nucleation and growth.

These proteins are being used in vitro for the preparation of nano-

materials [10,11] . Now, these peptides are considered to have the

potential for the mineralization of inorganic materials [12] ,such

as silver, gold, and zinc sulﬁde, and can be utilized in preparing

crystalline micrometer- to nanometer-scale particles [13–15] .

Recently, wurtzite structure ZnO with a wide direct band gap

has been found to possess unique optical, acoustic, and electronic

properties. It is one of the most widely studied metal oxides for

use in blue-light-emitting diodes (LEDs) [16] , solar cells [17] , and

ultraviolet nanolasers [18] . These applications require the fabri-

cation of morphologically unique and functionally distinct ZnO

nanostructure materials, which are synthesized at high tempera-

* Corresponding author. Fax: +86 28 85413003.

E-mail address: nic0700@scu.edu.cn (G.F. Yin).

0021-9797/$ – see front matter

doi:10.1016/j.jcis.2008.05.040

Z.B. Huang et al. / Journal of Colloid and Interface Science 325 (2008) 356–362

357

were hydrolyzed in 2 ml of 6 M HCl in sealed ampoules at 130 ◦ C

for 6 h, and the amino acids were determined on an amino acid

AutoAnalyzer (HITACHI L-8800). A detailed protocol for the prepa-

ration of amphiphilic peptide templates is described in previous

reports [28] .

2.3. Gel electrophoresis

SDS-PAGE analysis was performed on all peptides collected

throughout the puriﬁcation using 10–20% Tricine gels (Invitrogen

Inc., Carlsbad, CA). The resulting gels were stained with Coomassie

Brilliant Blue (G250) and destained in 10% methanol, 3.5% acetic

acid.

2.4. Mineralization of ZnO in silk peptide template

Fig. 1. Illustration of ZnO mineralization and growth mechanism in the peptides of

silk protein.

Solutions of 2.0 ml of silk peptides (5.6 mg / ml) and 3.0 ml of

ZnO nanoparticles (5.6 mg / ml, prepared by ourselves) suspensions

were mixed with 20 ml of zinc nitrate solution (0.22 M) under ni-

trogen by gentle agitation for 30 min. Peptides bound to ZnO were

eluted after exclusion of unbound peptides from the peptide/ZnO

solution. The eluted peptides were mixed with the ZnO suspension.

After three rounds of screening, peptides bound to ZnO suspension

were obtained.

A solution of 20 ml of sodium hydrate (0.35 M) was added to

the above mixture to adjust its pH to 7.0–7.5 with very gentle ag-

itation for 30 min; the zinc nitrate was hydrolyzed to Zn(OH) 2

capped with the peptide template after 1 h. After 2 h of hydrolysis

in the dark at room temperature, both Zn(OH) 2 and ZnO micropar-

ticles bio-assisted with the peptide template were observed. After

10 h, most Zn(OH) 2 gels were converted to ZnO microparticles in

the peptide templates in uniform particle sizes. When precipitated

particles were observed, they were rinsed with distilled water af-

ter residual Zn(OH) 2 gel solution was excluded. After 3 days, all

Zn(OH) 2 in the peptide assemblies had grown to olivary ZnO crys-

tal particles with diameter about 200–500 nm. As a control ex-

periment, the same hydrolysis of the zinc precursor was examined

under the same experimental conditions without the peptide, and

this condition yielded only Zn(OH) 2 gels.

eties binding the original ZnO nanoparticles and bio-assisted crys-

tal growth of hierarchical olivary ZnO particles via the aggregation-

driven mineralization process ( Fig. 1 ). While original ZnO nanopar-

ticles were bound at the peptide chain and the zinc ions were

enriched to Zn(OH) 2 in weak alkaline solution at room temper-

ature, these nanoparticles were coated the Zn(OH) 2 gel particles.

Then Zn(OH) 2 further was condensed into ZnO nanoparticles by

the catalysis of the peptide chains. Mineralization with the pep-

tide of silk-protein could bio-assist to grow crystalline and ZnO

particles congeries selectively from Zn(OH) 2 in high yield. This

unique feature of the uniform and selective crystal growth would

be regulated by bio-assisted peptide templates, fusion of nucle-

ated particles, and nanoscale growth in the peptide assemblies.

Thus, a porous structure among nanoparticles would be formed

through Zn(OH) 2 gel dissolution during biomineralization. Finally,

the hierarchical ZnO particles with the olivary microparticles and

the irregular nanoscale particles would be formed by catalysis of

peptide chains of spider silks. The biomineralized ZnO materials

coupled with biomolecules under mild condition to achieve pat-

terned growth over large areas of the substrate will be expected

for potential application in the assembly of biological nanodevice

for biomolecular detection by photoluminescence (PL) spectra.

2.5. Characterization

The shapes and crystalline structures of synthesized nanocrys-

tals were studied by transmission electron microscopy (TEM,

JEOL-200, 160 kV, Philips TECNAI 20 high resolution (HR) TEM

at 400 kV) and accompanying selected area electron diffraction

(SAED) and scan electron microscopy (SEM, JEOL-5900LV, 20 kV).

The SAED patterns were obtained at a camera distance of 60 cm.

In the preparation of TEM specimens, 1.0 ml of the crystal sus-

pension was dropped on the Formvar/carbon-coated copper grid

and dried in air. The X-ray diffraction (XRD) pattern was recorded

on a Philips X’Pert MDP diffractometer with Cu K α radiation. The

rigid particles obtained from the peptide were rinsed with distilled

water before XRD measurement.

The PL measurements were performed at room temperature

using a Xe laser (320 nm in wavelength) as the excitation light

source on a Fluorescence Spectrophotometer (F-7000, HITACHI,

Japan). The laser pulse frequency was 15 Hz, with pulse duration

800psatanaverageenergyof50mW.

2. Materials and methods

2.1. Materials

The zinc precursor zinc nitrate (anhydrous, 99.99%) and tris-

(hydroxymethyl)methylamine-HCl (Tris-HCl) were obtained from

Chengdu Chemical Co. Ltd. (China). ZnO particle (diameter about

100 nm) suspensions were prepared in our laboratory. The spiral

silks of the spider Uloborus walckenaerius Latreille were collected

in the ﬁelds around Wangjiang Park, Chengdu city (China).

2.2. Hydrolysis of spider silk proteins

3. Results and discussion

Amphiphilic peptides with aspartic acid, glutamic acid, and ser-

ine were prepared by hydrolysis of the spiral silk. Samples weigh-

ing 0.4–0.5 mg were hydrolyzed in 1.5 ml of 5 M HCl in sealed

ampoules at 100 ◦ C for 6 h. In order to know the composition of

amphiphile peptides, samples of spiral silk weighing 0.2–0.3 mg

Adhesive properties of the cribellate spider’ viscid spirals rely

on the cribellate thread, including thousands of extremely ﬁne ﬁb-

rils (diameter 20–30) ( Fig. 2 a). This spiral silk of spiders is easily

acidiﬁed and hydrolyzed into peptides. The hydrolysis results of

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Z.B. Huang et al. / Journal of Colloid and Interface Science 325 (2008) 356–362

Table 1

Amino acid composition of spiral silk of Uloborus spiders

Amino acid Gly Ala Asp Glu Ser Phe Thr

Percentage (mol%) 13.32 14.83 8.70 11.74 13.01 7.33 3.59

Amino acid Leu Val Lys Ile Arg His Pro

Percentage (mol%) 5.14 5.07 4.33 4.65 2.54 2.50 2.55

Table 2

Amounts of residues in spiral silk of Uloborus spiders, expressed as residues per 100

total residues

Residues with small

side chains

Polar

residues

Acidic

residues

Basic

residues

Amounts (mol%) 41.16

36.41

11.74

9.37

(a)

in Fig. 3 d) shows the coarse structure particles, including ultraﬁne

particles with a size of 20–40 nm and interstices 2–10 nm in di-

ameter (pointed to by arrows), indicating that these ZnO particles

are a hierarchical structure containing ultraﬁne nanoparticles and

microscale olivary particles. The porous feature is apparent, where

the nanometer-sized grains are uniformly distributed and intercon-

nected to form a “netlike” structure.

When the zinc precursor was hydrolyzed for 3 days under the

same experimental conditions without the peptide, polydisperse

particles with diameters of 2–12 μm were observed, as shown in

Fig. 4 a, and their SAED reveals that these particles are an ambigu-

ously diffractive pattern of Zn(OH) 2 particles (insert of Fig. 4 a). The

alkalescent solution hydrolyzes zinc precursors to form Zn(OH) 2 .

However, it is not strong enough to drive the hydrolysis further

to form ZnO through condensation [30] . Therefore, these results

indicate that the peptides have the catalytic function to grow

nanometric ZnO particles at room temperature. To understand the

growth mechanism, the ZnO particles’ growth was monitored with

SEM and SAED at different growth stages. After half-day hydrol-

ysis of zinc precursor with the peptide, lots of Zn(OH) 2 particles

with diameter 1–2 μm were observed ( Fig. 4 b). Although some

ZnO nanoparticles with olivary shape emerged in Fig. 4 b, those

particles were shown to be Zn(OH) 2 from their SAED pattern, with

faint (101) and (002) spots of ZnO crystal particles. After 1–2 days

of growth, olivary particles started to appear in the aggregations

( Figs. 4c and 4d ). The amount of Zn(OH) 2 particles gradually de-

creases because some Zn(OH) 2 particles dissolved in solution to

form smaller ZnO particles, followed by the growth of olivary

ZnO particles. The process described above proceeds further af-

ter 3 days. The morphology of 3-day-old and 4-day-old samples

is shown in Figs. 4e and 4f . The dissipated Zn(OH) 2 particles were

not observed in the 4-day-old sample of Fig. 4 f and the ﬂower-

like ZnO particles were larger than those in the 1-day-old sample,

suggesting that most of the Zn(OH) 2 particles in Fig. 4 fshouldbe

biomineralized into larger ZnO particles in catalysis of the pep-

tide chains. The insert in Fig. 4 f clearly shows that a magniﬁed

ZnO ﬂower is composed of 1 olivary-like pistil and 10 petals with

3 layers. It could be observed that these particles were dissipated

in bound peptide chains to grow ZnO nanoparticle crystals with

mesoscale interstices. Aggregation-driven crystallization in various

biomineralization processes often happens in nature, and recently

this process was mimicked to grow crystals in aqueous media by

dissipating primary nanoparticle-building blocks in aggregated mi-

celles and capping enzymatic peptides [31] . In the same fashion,

it is plausible that highly crystalline ZnO particles are formed by

the transformation of Zn(OH) 2 at room temperature in the artiﬁcial

peptides [27] . To obtain a better understanding of the particle-

formation mechanism, SAED patterns of ZnO particles at different

growth stages were compared. SAED of biomineralizing nanoparti-

cles in the 1-day-old sample ( Fig. 4 c) shows faint and fused (100)

and (002) spots. This pattern indicates that those particles prefer-

(b)

Fig. 2. (a) SEM images of spiral silk of spider Uloborus walckenaerius Latreille. Insert

is a magniﬁed image. (b) SDS-PAGE of the hydrolyzed results of spider spiral silk

protein acidiﬁed in 5 M HCl for different times. The molecular weights are indicated

on the left of the bands.

2 h hydrolysis was about 45–64 kDa, near to the expected

molecular weight for the 16-unit construct.

From Tables 1 and 2 , we know that the amount of amino acid

residues with small side chains is less than that of ecribellate spi-

ral silk, whereas residues with polar and charged side chains are

more abundant than those of ecribellate spiral silk [29] ;especially,

the aspartic acid and glutamic acid content is very high. These re-

sults show that the obtained peptides are amphiphilic chains with

aspartic acid, glutamic acid, and lysine.

Due to the anity of aspartic acid/glutamic acid and lysine,

the amphiphilic peptide chains could absorb and immobilize the

original ZnO nanoparticles after 1 day at room temperature. TEM

images are given in Figs. 3a and 3c , showing the original nanopar-

ticles and a chainlike aggregation with an average diameter of

80 nm bound or coated by these peptides. The SAED pattern (inset

of Fig. 3 a) clearly shows the crystalline structure of ZnO particles.

TheSEMimagein Fig. 3 b shows aggregation of peptides with ZnO

nanoparticles and many peptide-bound nanoparticles, which are

partly exposed from peptide aggregation. After the zinc precur-

sor is mixed with peptides in the ZnO nanoparticle suspension for

2 days at pH 7.5–8.0, the typical sub-micrometer olivary particles

are formed (shown in Fig. 3 c). Their magniﬁed SEM image (shown

spiral silk protein acidiﬁed in 5 M HCl for different times are

shown in bands of Fig. 2 b. The molecular weights for obtained

peptides are 94, 66, 45, 35, 22, and 14.4 kDa, respectively. These

same results have been observed regardless of the different acidi-

ﬁcation times. Although more peptides with low molecular weight

(such as 20 and 14.4 kDa) can be obtained from 12 h hydrolysis

than from 8 h, most of the molecular weight of the peptides from

Z.B. Huang et al. / Journal of Colloid and Interface Science 325 (2008) 356–362

359

(a)

(b)

(c)

(d)

Fig. 3. (a) TEM image of original ZnO nanoparticles after the anity with the peptides (inset: SAED pattern); (b) SEM image of original nanoparticles capped by the peptides;

and (c, d) TEM and SEM images of the 2-day-old ZnO particle (inset: SAED pattern).

entially orient to their [100] crystallographic direction, and their

[010] and [002] directions are not aligned well. SAED of the fused

particles in the 2-day-old sample ( Fig. 4 c) shows the strong in-

tensities of the (100) and the (002) spots with the nanocrystallite

structure. These SAED patterns indicate that the particles are ag-

gregated along the [100] direction to grow crystalline ZnO particles

as shown in Fig. 4 d.

Fig. 5 ashowsaTEMimageofa3-day-oldsub-micrometer

ﬂower-petal top, revealing that this structure is likely to be lapped

over in a layer-by-layer fashion and consists of plates with a size of

20–50 nm. However, these plates are not well aligned, resulting in

the formation of an irregular hexagonal pattern, and some at least

do extend into the overlapping zone in Fig. 5 b. High-resolution

TEM (HRTEM) of ZnO nanoparticles at the particle-merging inter-

face also supports this growth mechanism. As shown in Fig. 4 b,

clear lattice fringes between (100) crystal planes with d spacing

of about 0.256 nm are observed, indicating that ZnO particles are

fused to the preferential orientation of their [100] crystal direction,

which is consistent with SAED results. It is also clearly shown that

there are obvious crystalline defects in the ZnO particles, suggest-

ing a low quality of crystalline structure prepared by biomineral-

ization process under this room temperature. In Fig. 4 b, the ZnO

particles are nearly obvious, with some pores embedded among

the nanosheets or on the surface of the particles (shown by three

open arrows), and some crystal dislocations and defects in those

nanosheets.

Fig. 6 demonstrates XRD patterns for the as-prepared products

obtained for different growth times. All diffraction peaks can be in-

dexed to the wurtzite phase of ZnO with trace amounts of Zn(OH) 2

present (Joint Committee on Powder Diffraction Standards (JCPDS)

Card No. 89-1397 and JCPDS 38-0385). In comparison with the XRD

pattern of original ZnO nanoparticles, the enhanced (100) reﬂec-

tion possibly results from the oriented growth of the constituent

nanorods in ZnO particles along the [100] direction, consistent

with SAED and HRTEM results.

Fig. 7 shows the PL intensities of the biomineralized ZnO parti-

cles for the different growth times. It was found that enhanced lu-

minescence was detected for the ZnO particles with greater growth

time. The ZnO particles with long growth time have stronger lumi-

nescent intensity than those with short growth time due to higher

grafting density. Furthermore, the luminescent intensity depends

on the growth time and exhibits a slight red shift (from 385.8 to

397.1 nm) when the growth time increases from 0.5 to 4 days.

The enhanced luminescence and red shift of these ZnO particles in

PL might be attributed to the polarization increase of the emitted

light from the lesser defects and larger size of ZnO particles due to

the crystallization of more Zn(OH) 2 [32] .

In Fig. 7 , the peak around 390 nm (3.19 eV) is usually at-

tributed to recombination of the typically reported free excitons,

that is, near band-edge emission. Other peaks at 450 nm (2.80 eV),

470 nm (2.68 eV), and 530 nm (2.30 eV) probably originate from

defect state luminescence. Most people think that visible lumines-

cence mainly originates from defect states such as interstitial Zn

(Zn i ) and oxygen vacancies. It is reported that oxygen vacancies

act as luminescence centers [33] .OxygeninZnOcrystalsexhibits

three kinds of charge states of oxygen vacancies, namely V O ,V O · ,

and V O ·· . In general, these oxygen vacancies are situated below the

bottom of the conduction band (CB) in the sequence of V O ,V O · ,

and V O ·· . Fig. 8 schematically shows the possible positions of the

defect levels. The peak around 390 nm should be related to the

defect levels. From Fig. 8 , we think it may be caused in the fol-

lowing ways: (1) transition from shallow donor levels of Zn i to

valence band (VB); (2) transition from CB to shallow acceptor lev-

els of interstitial O (O i ). [34] The density of Zn defects is very high,

360

Z.B. Huang et al. / Journal of Colloid and Interface Science 325 (2008) 356–362

(a)

(b)

(c)

(d)

(e)

(f)

Fig. 4. (a) SEM image of Zn(OH) 2 particles without the peptides grown after 3 days and the SAED pattern; (b–e) ZnO particles with peptides grown after 0.5, 1, 2, 3, and 4

days. Inserts in (a–d): SAED patterns; insert in (f): a magniﬁed ﬂowerlike ZnO particle composed of an olivary-like pistil and three layers of petals.

so the peaks around 390 nm are most probably related to the de-

fect levels of Zn i . The signiﬁcant green band in these PL spectra

peaks around 530 nm indicates a very high concentration singly

ionized oxygen vacancy (V O ·· ) from the oxygen deﬁciency [35] and

the surface defects in the ZnO particles biomineralized for 2–4

days, which is in agreement with the results obtained from the

HRTEM observation. The green emission (around 530 nm) results

from the recombination of a photogenerated hole with a singly

ionized charge state (V O · and V O ·· ) of the speciﬁc defect. The re-

sults of theoretical calculations on the energy levels of intrinsic

point defects indicated that O i and vacant Zn (V Zn ) produce shal-

low acceptor levels 0.3 and 0.4 eV above the top of VB, and that

Zn i produces a shallow donor level 0.5 eV below the bottom of

CB [34] .V O ·· produces a deep donor level 1.3 eV below the bottom

of CB, and V O · lies around 2.4 eV at the bottom of CB (shown in

Fig. 8 ) [36] .

According to the depiction in Fig. 8 , it seems that the emission

at 450 nm can be attributed to the recombination of an electron

at O i and a hole in the VB, and that the peaks at 470 nm can be

assigned to the recombination of an electron of Zn i and O i .Inthe

present case, the ZnO particles were biomineralized in an environ-

ment of peptide chains at room temperature. The surfaces of the

ZnO particles could link with the nucleophilic groups (such as hy-

droxyl and carboxyl) of peptide chains and adsorb peptides during

their growth. With the increase of the growth time and the forma-

tion of pores, the defects on the porous surfaces gradually emerge

and intrinsic point defects also form while Zn(OH) 2 are biomineral-

ized into the ZnO particles. It is reasonable to believe that various

defects and dislocations exist in the ﬂowerlike ZnO particles of 4

days growth, and especially that plentiful ionized oxygen vacancies

(V O · and V O ·· ) exist on their surfaces [37] , and therefore stronger

green light emission at 530 nm from them is observed. In compar-

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