Angew. Chem. Int. Ed.

2004, 43, 6042-6108

Integrated Nanoparticle-
Biomolecule Hybrid System:
Synthesis, Properties, and
Applications

• Synthesis and Properties of Biomolecule Functionalized

Nanoparticles
(生物分子改質奈米粒子)
• Biomolecule Functionalized Nanoparticles for Controlled
Chemical Reactivity
Introduction (生物分子改質奈米粒子調控化學反應)
• The Aggregation of Biomolecule Functionalized
Nanoparticles
(生物分子聚集改變奈米粒子功能性)
• Assembly of Biomolecule Nanoparticle Architectures on
Surfaces
(生物奈米粒子自組裝材料表面)

• Functional Biomolecule Nanoparticle Structure on

Surfaces for Application as sensors
(生物奈米粒子材料表面應用在感測器) Synthesis and Properties of
• Biomolecule Functionalized Magnetic Particles
(生物分子應用於磁性粒子) Biomolecule Functionalized
• Biomolecule Based Nanocircuitry Nanoparticles
(生物分子應用奈米迴路)
(生物分子改質奈米粒子)

1
 Biomolecule-nanoparticle
(生物分子結合奈米粒子)

Formation of the biomolecule-

nanoparticle (NP) hybrids
(生物分子與奈米粒子雜合)

Nanoparticles could be encapsulated

by some natural proteins to provide
their affinity binding
（一些蛋白質可能將奈米粒子封入提
供彼此結合性）

• a. Top and side view of

Chaperonin proteins as ATP- GroEL and T.th cpn
responsive barrels for the inclusion of • b. The formation of
GroEL-Cds NP
nanoparticles complexes by inclusion
of Cds NPs into the
cylindrical cavity of
GroEL, and ATP-
triggered release of the
guest

2
 Transmission electron micrographs

Properties of Nanoparticle-
Biomolecule Hybrid System
(奈米粒子與生物分子雜合系統的
特性)

T.th cpn-Cds NP Intact T.th cpn

complexes

flavin
The electrochemistry controlled
recognition of flavin by a
pyridineduamide-functionalized
nanoparticle

Biomolecule Functionalized
Nanoparticles for Controlled Chemical Biomolecule Functionalized
Reactivity Nanoparticles for Controlling DNA
(生物分子改質奈米粒子調控化學反 Reactivity
應)

3
 奈米粒子控制DNA反應 奈米粒子顯示DNA反應

Radio-frequency (1-GHz)

奈米粒子控制酵素反應 奈米粒子控制酵素反應
（CdS NP-capped mesoporous） （CdS NP-capped mesoporous)

Carboxylic acids modified Cds NPs

The convalent coupling of the Cds NPs

to the amine function of the matrix

Matrix by a siloxane anchoring site

The use of biotin-streptavidin (SAv )

interactions to build nanoparticle networks
End-to-end assembly

12: dibiotin TEM: SAv-interconnected Au nanorods

13: biotin disulfide 14: Ester derivative of biotin

Biotin-functionalized ferritin

15: bifunctional linker

4
 Nucleic Acid Functionalized NPs for TEM of gold nanorods that are
Controlled Aggregation organized by DNA hybridization
16: 3’-thiol-TACCGTTG-5’

17: 5’-AGTCGTTT-3’-thiol

18: A DNA linker

B: cuvettes with a mixture of

the Au NPs and the added DNA
strand responsible for the assembly
process
C: variations in the absorption spectrum
of the DNA-linked NP networks as
function of temperature
D: the satellite system consists of two
different sizes of nanoparticles

DNAzyme system for the analysis of DNAzyme system for the analysis of
metal ions metal ions

active 17E only

17E/17Ec is 1:20

Enzyme strand

a: the active 17E DNAzyme-NP b: an inactive 17Ec DNAzyme- c: Calibration plots for
sensor NP sensor the analysis of Pb2+ ions
1: absence Pb2+ ions 1: absence Pb2+ ions
2: presence Pb2+ ions 2: presence Pb2+ ions

Controlled association of Au NPs based on Controlled association of Au NPs based on

biocatalytic transformation of oligonucleotide biocatalytic transformation of oligonucleotide
Treatment with EcoRI
DNA-NP complex before DNA-NP complex and after subsequent
treatment with DNA ligase

• TEM analysis of the DNA-NP complexes before a.

and after b. treatment with EcoRI and after
subsequent with DNA ligase c.

5
 Enzyme-controlled distance between Composite Assemblies of Nucleic Acids,
DNA-bridged Au NPs Proteins, and Nanoparticles
(酵素控制奈米金粒子DNA橋) (奈米立子與核酸,蛋白質自組裝)
EcoRI
SAv
Biotinylated antibodies

The hybrid with 59° bend

Following the binding of EcoRI

The assembly of controlled

multiparticle composites upon
Hybridization on DNA template

The use of conjugate for the sensing of The construction of four-nanoparticle

antigens (偵測抗原) clusters (金奈米粒子四聚體)

Cowpea mosaic virus (CPMV)

labeled with 1.4nm Au clusters

Assembly of Layered Nanoparticle-

Protein Arrays on Surface

Assembly of Biomolecule-Nanoparticle
Architectures on Surface
(生物分子與奈米立子結構表面自組裝)

Construction of TiO2/cty c multilayers on a QCM Optical absorbance at

lamda=409 nm of assemblies
of different thicknesses

6
Nucleic Acid-Nanoparticle Architectures on Electrostatic deposition of CdS NPs on a DNA
Surfaces (核酸與奈米立子結構表面自組裝) chain at the air/water interface

Fig. B: AFM image of a DNA strand

With Au NPs that specifically bound
to the template through biotin-SAv
interaction

38. Cationic surfactant molecules

39. Positively charged CdS NPs(3 nm)
capped with thiocholine

The use of dip-pen lithography and DNA to

The 2D assembly of Au NPs by using a
produce a predesigned multinanoparticle
DNA-based method
pattern
43. TCTCAACTCGTAA10
44. A10CGCATTCAGGAT
45. TACGAGTTGA
-GAATCCTGAATGCG

AFM image of the resulting 2D Au

AFM image of the assembly ,
40. A gold NP is attached to oligonucleotide NP-DNA network
41. Hybridized with oligonucleotide which consists of large and
42. DNA network that consists of oligonucleotide small nanoparticles

Scale bar=1 um

The use of DNA as linker to construct

nanoparticle multilayer on surface
(利用DNA結合奈米粒子在表面形成多層連結)
46. Monolayer of an oligonucleotide
47. Composed of two domains (46 and 48)
48. Au NPs (13nm) that were functionalized
with oligonucleotide
Functional Biomolecule Nanoparticle
Structure on Surfaces for Application
as sensors
(生物奈米粒子材料表面應用在感
The absorbance and the fluorescence spectrum:
測器)
respectively, of the CdS NP multulayer assemblies:
a to d =1 to 4 layers

7
 Bioelectronic Systems Based on
Assembly of the CdS NP-AChE hybrid system for the
nanoparticle-Enzyme Hybrids as System photoelectrochemical detection of enzyme activity
(奈米粒子結合酵素生物電子感測系統)

Apo-glucose oxidase
FAD cofactor

acetylthiocholine
as the substrate
The reconstituted GOx electrode in the
presence of different concentration of
glucose

The enzyme generated

thiocholine

Photocurrent action spectra observed in Calibration curve of the photocurrent at

the presence of acetylthiocholine lamda=380nm at variable concentration

• a: 0mM
• b: 6mM
• c: 10mM
• d: 12mM
• e: 16mM
• f: 30mM
53: acetylthiocholine

Photocurrent spectra for the CdS-

AChE system

Electrochemical detection of DNA by

the deposition of catalytic silver
clusters on the DNA strand
55: dibromibe (inhibits the
photocurrent formation)

a: in the absence of inhibitor 55

b: upon addition of the inhibitor 55 (1X10-6 M)
c: after rinsing the system, exclusion of the inhibitor, and addition of 53

8
Hybridization of the complementary Loading of the Ag+ ions onto
target DNA with the DNA probe immobilized DNA

56: DNA probe

57: target DNA

Reduction of Ag+ ions by hydroquinone to form

silver aggregates on the DNA backbone

Dissolution of the silver aggregates

in acidic solution

Immunosensing at microsized Au
electrodes by the change of conductivity

PSA: potentiometric stripping analysis

Immunosensing at microsized Au
The use of a DNA-NP conjugate
electrodes
stabilized by an anionic protective

58: A probe nucleic acid

59: The target 27-mer nucleotide
60: Au NPs were functionalized
with a nucleic acid

9
 Stripping potentiograms measured upon the
sensing of different concentrations of DNA

Different concentrations of DNA that are

bound to magnetic particles and labeled with
CdS NPs

a: 0.2 mgL-1
b: 0.4mgL-1
c: 0.6mgL-1
d: control

Multitarget electrochemical DNA detection Stripping potentiogram measured upon

with different nanocrystal labels the sensing of different concentration

a: probe-modified magnetic beads The DNA molecular are labeled with

b: hybridization with the DNA targets ZnS NPs (T1),
c: second hybridization with the NP-labeled probe CdS NPs (T2),
d: dissolution of the NPs and the electrochemical detection PbSNPs (T3)

The amplified detection of DNA by using The amplified detection of DNA with
nucleic acid-Au NP-functionalized beads polystyrene(聚苯乙烯) beads

64: ferrocenecarboxaldehyde
(as a redox marker)

a: Hybridization of the NP-functionalized beads with the target DNA 65: the probe DNA linked to magnetic particles
b: the enhanced catalytic deposition of gold on the NPs 66: complementary nucleic acid
c: Dissolution of the gold clusters
d: the detection of the Au3+ ions by stripping voltammetry

10
The construction of CdS NP-DNA Dendritic amplified DNA sensing by the use
superstructures of oligonucleotide-functionalized Au NPs

The amplified detection of the 7249-

base M13mp18 DNA

The catalytic deposition of gold on

a Au NP conjugate

71: the DNA primer

72: M13mp18 DNA
73: streptavidin-Au

The analysis of a single-base Microgravimetric detection of a

mismatch in DNA single-base mutant

b: Arrows indicate
(single base mutant)

a: Normal DNA sequence

(1): the attachment of the SAv-Au conjugate

(2): the catalytic deposition of gold on the Au NPs

11
 The construction of mixed-metal
“barcode”

Nanoparticle-Biomolecule Conjugates
for Optical Sensing and Analysis

Au-Ag multistripe
“barcode” nanorod

Multiple immunoassay by using

Detection of DNA with “barcodes”
“barcodes”
78: a nucleic acid that is labeled 81: the antibodies with fluorescein- 82: Texas Red-labeled antirabbit IgG
with a fluorophore (TAMRA dye) labeled anti-human IgG 80: anti-rabbit IgG antibody
77: a primer nucleic acid that is linked 79: anti-human IgG antibody
to Ag
76: analyte DNA

Fluorescence images Reflectivity images

bound fluorescent DNA bound fluorescent DNA

Fluorescence images Reflectivity images

absence of analyte DNA absence of analyte DNA

a: Reflectivity images
b: Fluorescein (FITC)-labeled anti-human-igG
c: Texas Red-labeled anti-rabbit-IgG

Application of Au nanoparticle
labels that are encoded with DNA
for the amplified immunosensing
of prostate-specific antigen(PSA)

83: an antibody which is complementary to PSA was linked to magnetic beads

84: double-strain nucleic acids was linked to the magnetic beads-antibody-PSA
85: triple-component sandwich assay configuration
86: thermal dissociation of the nucleic acid duplex yielded the free nucleic acid
87: single-stranded-oligonucleotide-functionalized Au NPs

12
Surface plasmon resonance表面電漿共振(SPR)
Localized surface plasmon resonance
spectroscopy enhanced by Au NPs for DNA
analysis 局部表面電漿共振(LSPR)

Assembly of Au NP-bound reconstituted

glucose oxidase(GOx) on a dithiol monolayer

Assembly of Au NP-bound reconstituted

glucose oxidase(GOx) on a dithiol
monolayer that is associated with a SPR-
active surface

SPR spectra of the Au NP-GOx Calibration plot of the SPR spectra minimum
hybrid system shift as a function of glucose concentration

a: 0 mM
b: 0.3 mM
c: 1.6 mM
d: 8 mM
e: 40 mM
f: 100 mM

13
 Assembly of the Au NP-CdS NP-
acetyl choline hybrid system
Application of Raman dye-functionalized Au
NPs for amplified multitarget immunosensing
B: Flatbed scanner images of silver-
enhanced microarrays upon the
immunosensing of different
antibodies
Emission spectra upon the time-dependent
DNA replication(B) / telomerization(C)
a: 0 min b: 10 min c: 30min d: 60 min
Application of Raman dye-functionalized Au
NPs for amplified multitarget immunosensing
Replication and telomerization of nucleic
acid functionalized CdSecore/ZnSshell NPs
C: Typical Raman spectra that
correspond to the colored
dots in the immunosensing
array 88: primer (complementary M13mp18 DNA )
89: included Texas Red-functionalized dUTP
90: primer (recognized by telomerase )
Biomolecule Functionalized
Magnetic Particles
(生物分子應用於磁性粒子)
14

Au NPs are used for the deposition of silver
91: biotin-labeled nucleic acid
92: complementary biotinylated nucleic acid
The effect of gold enhancement upon the
stripping response for the DNA analyte
PSA signals prior to treatment of the system with gold enhancement
solution (a) and after 10 minutes of reaction (b)
Electrochemical analysis of DNA upon the
assembly of DNA molecules at magnetic
particles followed by their association with
Au NPs
The Au NPs chemically dissolved
93: the primer biotinylated nucleic acid was linked to magnetic
beads through an avidin bridge
94: functionalized with Au NPs
a: deposited gold is electrochemically stripped (path a)
b: the intermediate enlargement of the Au NPs results
in the future amplification of the signal (path b)
Application of Redox-functionalized
Magnetic Particles for the Triggering
and Enhancement of Electrocatalytic
and Bioelectrocatalytic Process
15

Linkage of 2,3-dichloro-1,4-naphthoquinone (95) to the
functionalized particles to yield the
aminonaphthoquinone (100)- functionalized magnetic
particles
Functionalization of magnetic particles with
the PQQ-NAD+ Dyed for the electrochemical
activation of NAD+-dependent enzymes
Key words
Carbodiimide coupling of the electron-relay
carboxylic derivatives (96-99) to the amino
groups of the siloxane layer
The electrochemical , electrocatalytic
and bioelectrocatalytic reaction of
functional magnetic particles which
are controlled by means of an external
magnet
Switching on or off the electrochemical
reaction of the redox-relay groups (R)
Covalently bound to the magnetic
particles and the electrocatalytic
function that is provide by the
redox groups
16
Differential pulse voltammograms of Switching on or off
a Au electrode
a: 接觸
b: 縮回未接觸

Magnetic activation

Magnetic deactivation
1. Bioelectrocatalytic oxidation of glucose in the presence of glucose oxidase
2. Oxidation of lactase in the presence of lactate dehydrogenase (LDH)

Cyclic voltammograms at a Au Enhanced bioelectrocatalytic oxidation of

electrode glucose in the presence of glucose oxidase (GOx)

upon attraction

retraction

a: 接觸 (on)
b: 縮回未接觸(off)

Enhanced bioelectrocatalytic oxidation of

glucose in the presence of glucose oxidase (GOx)

Magnetomechanical Detection
of Biorecognition Events

17
 Synthesis of the functionalized magnetic
particles for the biorecognition assay
Amplified detection of bioaffinity
recognition processes by mutilabeled
rotating magnetic particles

Labeling of the nucleic acid replica with biotin units

Amplified biorecognition assay upon the rotation
by thermal cycles for the amplified detection of viral
of the functionalized magnetic particles DNA by multilabeled rotating magnetic particles

Chemiluminescence intensities upon Schematic configuration of the instrumental setup

and concept for the magnetomechanical analysis of
the analysis of M13mp18 biorecognition processes on functionalizes cantilevers

B: at different rotation speeds a) 0, b) 60, c) 400, d) 2000, e) absence of DNA

C: Arrows indicate the time for switching the potential to -0.5V and to 0.0V

18
 Magnetomechanical deflection or retraction
of the cantilever in the analysis of M13mp18
TEM image with Pt NP aggregates
a: the cantilever is subjected to the external magnet
b: the external magnet is removed

B: using Taq polymerase and thermal cycles for replication and labeling process
C: Dependence of the deflection signal on the concentration of the M13mp18 DNA

Formation of a silver nanowire inside

a channel

Biomolecule Based Nanocircuitry 106: β-amyloid

(生物分子應用奈米迴路)

Formation of a silver nanowire Nanocircuitry produced upon

inside a channel (TEM images) biospecific interaction on surfaces

Electron micrograph

Fluoresecence images
at different
magnifications

19
 Structure of the Au NP-peptide
End –to-end interconnected peptide
complex on the histidine-rich
nanotubes
polypeptide nanotube template

The assembly of patterned actin-

AFM images
based Au nanowires
108: N-hydroxysuccinimidyl ester groups

B: AFM image of the Au wire/actin/Au wire filament

C: AFM image of the actin/Au wire/ actin filament

SFM images of nanoparticle networks AFM image of lamda-DNA

AFM image of Height profile of both

Lamda-DNA , the bare DNA and a
Which is partially decorated part
Linked with Au55
clusters

DNA fragment with

Au55 clusters along
The phosphate
Backbone of the DNA
Major grooves

20
 Assembly of a Au NP wire in the AFM image of the Au NP wire in the
pA/pT template by using Au NPs pA/pT template

pA/pT= polyadenylic acid / polythymidylic acid

The assembly of Au nanowires on a The assembly of Au nanowires on a

telomer template telomer template

The construction of a nanowire that bridges Molecular lithography based on homologous

two microelectrodes by deposition of Ag+ ions recombination processes carried out by the
on a bridging DNA strand RacA protein
Current versus voltage (I/V)
curves obtained with the structure
produced

21
 The fluid-flow-assisted molecular combing of
AFM image of the patterned DNA
DNA molecules on a surface to yield 1D or 2D
template after gold metallization arrays

AFM image of
AFM image of a a meshlike 2D
Pd nanowire , array of Pd
a section analysis nanowires
shows an average
Particle height of
Approximately 5 nm

Real-time conductance response from a si

AFM image nanowire device that is functionalized with a
PNA (peptide nucleic acid ) receptor
D: a DNA strand E: and F: AFM tip on the
cut by an AFM tip nanometer scale

The data points

Si nanowire device shown were obtained
with source (S) and from two independent
drain (D) Si nanowire devices

Image of the motility of actin/Au wire/

actin filament on a glass surface
The same frame imaged at 5-s time intervals

22