This article was published as part of the

Nanomedicine themed issue

Guest editors Frank Caruso, Taeghwan Hyeon and Vincent Rotello

Please take a look at the issue 7 2012 table of contents to

access other reviews in this themed issue

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This journal is c The Royal Society of Chemistry 2012 Chem. Soc. Rev., 2012, 41, 2849–2866 2849

Cite this: Chem. Soc. Rev., 2012, 41, 2849–2866

Gold nanoparticle-enabled biological and chemical detection and

analysisw

Hilde Jansaand Qun Huo*

b

Received 10th October 2011

DOI: 10.1039/c1cs15280g

Gold nanoparticles (AuNPs) are some of the most extensively studied nanomaterials. Because

of their unique optical, chemical, electrical, and catalytic properties, AuNPs have attracted

enormous amount of interest for applications in biological and chemical detection and analysis.

The purpose of this critical review is to provide the readers with an update on the recent

developments in the field of AuNPs for sensing applications based on their optical properties.

An overview of the optical properties of AuNPs is presented first, followed by a more detailed

literature survey. As the last part of this review, we compare the advantages and disadvantages

of each technique, briefly discuss their commercialization status, and some technical issues that

remain to be solved in order to move the technique forward (151 references).

1. Introduction

Gold nanoparticles (AuNPs) are some of the most extensively

studied nanomaterials. Because they have many unique

optical, chemical, electrical, and catalytic properties, AuNPs

have attracted enormous amount of interest for biological and

chemical sensing applications. A literature search of key words

‘‘gold nanoparticles’’ and ‘‘detection’’ using SciFinder Scholar

returned a list of more than 5000 entries (journal articles and

reviews only, in English) from the year 2005. This research area

continues to quickly evolve, with innovative new techniques

and methods reported frequently in recent years. Although

there have been many review articles published in the past on

this topic,1–11 we feel it is time to write another review to cover

some of the most recent developments in the field. The emphasis

of this review is on different detection methods. With the

significant progresses and successes made in this field, many

techniques derived from this research area have advanced to or

very close to commercialization. A critical review will help

potential users to understand the options available and to select

the appropriate techniques for different application purposes.

For techniques that are at an early stage or require further

a Imec, SSET/Functional Nanosystems, Kapeldreef 75,B-3001 Leuven, Belgium

bNanoScience Technology Center, University of Central Florida,12424 Research Parkway Suite 400, Orlando, FL 32826.E-mail: Qun.Huo@ucf.edu; Tel: +1-407-882-2845

w Part of the nanomedicine themed issue.

Hilde Jans

Hilde Jans (PhD) graduated in2005 from the Catholic Univer-sity of Leuven (KULeuven) witha Master degree in Chemistry.After completing her thesiswork related to the synthesisand characterization of metalnanoparticles for nano-immunoassay applications,she obtained her PhD degreein 2010. During her PhDstudy, Hilde Jans was avisiting scholar in the Nano-Science Technology Center atUniversity of Central Florida(UCF). Dr Jans is now a

postdoctoral researcher at imec. She is currently involved inseveral European and national research projects aimed forbiosensing technology development using metal nanoparticles.

Qun Huo

Qun Huo (PhD) received herBSc degree in polymer sciencefrom University of Science andTechnology of China (1991),MSc degree in chemistry fromSun Yatsen University (1994),and PhD degree in Chemistryfrom University of Miami(1999). After spending twoyears as a postdoctoral fellowat University of Miami, shebecame an assistant professorat North Dakota State Univer-sity in 2001, and then joinedUniversity of Central Floridain 2005. She is now a tenured

associate professor in the NanoScience Technology Center atUniversity of Central Florida. Dr Huo’s primary research interestis gold nanoparticles for biomedical applications.

Chem Soc Rev Dynamic Article Links

www.rsc.org/csr CRITICAL REVIEW

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2850 Chem. Soc. Rev., 2012, 41, 2849–2866 This journal is c The Royal Society of Chemistry 2012

development, it is important to understand the major problems

that are remaining and need to be solved. This review also

serves the purpose of clarifying some confusing perceptions in

the field.

Many properties of AuNPs can be and have been used for

sensor and assay development. In this review, we will focus on

detection methods based on various optical properties and

surface chemistries of AuNPs. Optical methods are widely

used in the current analytical technologies and products.

Nanoparticle-enabled optical techniques can be readily developed

into commercial products and enter the marketplace. For detection

methods based on the electrical, catalytic, or other related

properties of AuNPs, there are a large number of comprehensive

reviews that have been written by the leading experts in this field to

which the reader may refer12–17 and therefore are not covered in

this review. One example we want to mention here in this review,

just as an illustration of the promising potentials of AuNPs as a

nanomaterial for sensing applications, is an electronic nose sensor

for cancer diagnosis based on the electrical conductivity change of

immobilized AuNP film upon detection of various cancer-related

volatile organic compounds (VOCs).18 This work has received

high profile publicity since it was first reported by the Haick

group. This group later reported that this new sensor technology is

able not only to detect cancer, but also to distinguish different

types of cancer.19–21

Although AuNPs have been used for both chemical and

biological detection and analysis, most of the effort has been

devoted to biological applications. In terms of chemical

detection, the main interest is more focused on applications

in environmental protection and homeland security, for example,

for the detection of toxic metal ions and explosive chemicals. For

biological and biomedical applications, the type of target analytes

spans a wide range from proteins, DNAs, small biomolecules, to

microorganisms including bacteria and viruses. Using AuNPs for

cancer-related detection and analysis has arisen as an extremely

active area in the last few years. A significant number of studies

discussed in this review are indeed aimed for cancer biomarker

research and applications. As one of the most challenging medical

problems, cancer causes economic burdens to society that

outweighs the effect of all other human disease. Innovative

technologies are needed to bring breakthrough advance to the

field. It is expected that the development of nanoparticle-based

technologies to improve cancer diagnosis and treatment will

continue to remain a hot topic in the future.

When biomolecular analysis is referred to, it is typically

perceived as concentration analysis of various biomolecules,

such as proteins, DNAs, and RNAs. But in fact, biomolecular

analysis is a much broader area than just a concentration

analysis. Biomolecular interaction study and biomolecular

complex detection comprise the major part of research effort

of biomolecular researchers. Although analytical techniques for

biomolecule concentration analysis are widely available, techniques

for protein–protein interaction and other biomacromolecule

complex study are rather limited.Many techniques such asWestern

blot analysis, co-immunoprecipitation, immunochromatography

that are used routinely in biomolecular research laboratories have

been around and have not been improved for decades. These

techniques are often labor intensive, time-consuming, and require

the use of large sample volumes. New technology that can improve

existing bioanalytical tools or reveal new molecular information

that cannot be observed using current techniques should bring

significant benefit to biomolecular and medical research.

Finally, we feel compelled to clarify one important concept in

the bioanalytical field: the classification of labeled and label-free

techniques. Label-free techniques, as the definition implies, have

advantages over techniques that require labeling. Labeled

techniques refer to techniques that require the labeling of the

capturing molecule or ligand (such as antibody or single strand

DNA probes that will capture the target analytes) or the

labeling of the target molecules. The immobilization of a

capturing molecule or target analyte on an optical substrate is

generally, in a less strict sense, not regarded as labeling. For

example, the surface plasmon resonance technique is a widely

known label-free technique, while ELISA, an immunoassay

that requires not only the immobilization of one antibody

on a substrate, but also a second antibody that carries a

signaling probe (the label), is a labeled technique. Most

fluorescence-based techniques are labeled techniques, because

they require the labeling of target molecules, and sometimes

both capturing molecules and target molecules. According to

the above definition, if a detection method is based on the

optical property change of the AuNP metal core, then it is a

label-free technique. If an additional dye molecule is required

to label either the capturing molecule (ligand) or the target

analyte, then it is a labeled technique. If an additional dye

molecule is involved in the detection, however, the dye molecule

is part of the AuNP signal transducing system, and it is not

conjugated to a specific capturing molecule (ligand) or target

analyte, then this technique should still be considered as a label-

free technique. We will point out explicitly whether a technique

is a labeled or label-free technique when specific examples are

introduced.

2. Overview of the optical properties of AuNPs for

sensing applications

AuNP absorbs and scatters light intensely at its surface plasmon

resonance (SPR) wavelength region and such properties make

AuNPs as one of the most valuable optical probes for sensing

applications.22–35 The SPR wavelength of gold nanoparticles can

be tuned from the visible to the near IR region by changing the

size and shape of the gold nanoparticles. The strong absorption

or scattering of AuNPs at the visible light region makes them

easily observable by the naked eye or detectable by inexpensive

instruments. The possibility of tuning the SPR band of AuNPs

(including nanorods, shells, stars, and other shapes) to the near

IR region makes them promising materials for in vivo imaging

and analysis. The optical properties of AuNPs are further

dependent on the surface chemistry and the inter-particle

interactions. As a matter of fact, a vast majority of techniques

developed so far involving AuNPs as optical probes are based

on the target analyte-induced optical property change of

individual AuNPs, or AuNP cluster formation, as summarized

in Fig. 1. Upon target analyte binding, the surface plasmon

resonance of the AuNPs will change due to the surface

chemistry change, or inter-particle interactions. The surface

plasmon resonance change of AuNPs can be detected either by

light absorption (Fig. 1A) (Section 3.1) or light scattering

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This journal is c The Royal Society of Chemistry 2012 Chem. Soc. Rev., 2012, 41, 2849–2866 2851

techniques (Fig. 1B) (Section 3.2). For absorption based

techniques, the surface plasmon wavelength shift of AuNPs

caused by target analyte binding is used as the transducer

signal. For light scattering techniques, there are more options

available and they are discussed in detail in Section 3.2.

A typical AuNP contains two structural components: the

metal core and the surface coating. The detection methods as

mentioned above are mainly based on the property and

property change of the metal core. Because nanoparticles have

a large surface area/volume ratio and the surface chemistry

can be well controlled, the surface coating layer of AuNPs

provides additional opportunity for detection and sensing.

Two specific examples are related to this concept (Fig. 1C):

one is the DNA probe based bio-bar-code amplification

platform developed by Mirkin’s group, and another one is the

surface chemistry change-induced sensing method developed by

Rotello’s group. In the bio-bar-code method, multiple copies of

DNA probes are attached to AuNPs and due to the amplification

effect of the large number of DNA probes, a high sensitivity was

achieved for both DNA and protein detection. In the surface

chemistry-controlled sensing method, different functional ligand

molecules were attached to AuNPs to create surface layers that

Fig. 1 Illustration of six detection mechanisms using the optical properties of AuNPs. (A) LSPR concept: (1) Irradiation of the gold nanoparticles

gives rise to a collective oscillation of their conduction band electrons resulting in a plasmon absorption in the visible range of the electromagnetic

spectrum. (2) This plasmon resonance is sensitive to changes in the local environment, for example the binding of biomolecules. (B) (1) When

nanoparticles are irradiated with laser light, they will scatter the light elastically in all directions. By monitoring the change in the scatter pattern the

diameter of the solid nanoparticles can be deduced. (2) When biomolecules are attached on the nanoparticle surface the size increase can be

monitored. (C) Detection using the surface chemistry and ligands of AuNPs. (1) Signal amplification based on the large number of DNA

strands (bar-codes) released from the AuNPs. (2) Surface chemistry-controlled reversible biomolecular binding to AuNPs in a competitive assay.

(D) (1) Normal Raman scattering is inherently weak. (2) When biomolecules of interest are bound to nanoparticles, the Raman signal can be

enhanced due to the enhanced electromagnetic field which is excited or due to a charge transfer mechanism. When molecules are attached to

AuNPs their properties can change. (E) Metal enhanced fluorescence can occur when a fluorophore is placed at a fixed position from the metal

nanoparticle surface. (F) When the fluorophore is in short or direct contact with the metal nanoparticle surface quenching will occur. In this case, a

competitive assay format is often used for the target analyte detection.

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2852 Chem. Soc. Rev., 2012, 41, 2849–2866 This journal is c The Royal Society of Chemistry 2012

can bind with proteins, enzymes and fluorescent polymers

reversibly. This reversible binding process provides a new

sensing platform for a wide range of target analytes. These

two examples are discussed in Section 3.3.

The unique interactions between the metal core and the

surface ligands of AuNPs provide several widely known signal

transduction systems for chemical and biological sensing

purposes. Three techniques can be considered as examples of

this sensing mechanism: the surface enhanced Raman scattering

(SERS) (Fig. 1D), metal-enhanced fluorescence (MEF) (Fig. 1E),

and metal-induced fluorescence quenching (Fig. 1F). Although

SERS is a light-scattering based technique, this is different from

the other techniques mentioned above, where the light scattering

is detected from the metal core. In SERS, the scattered light is

detected from the Raman active dye molecules located on the

surface of AuNPs. The AuNP metal core enhances the Raman

scattering of the organic molecule. When a fluorophore is attached

to a metal nanoparticle, the fluorescence of the fluorophore can be

enhanced or quenched, largely depending on the distance from the

fluorophore to the nanoparticle core. Competitive assays are

often designed using the metal-induced fluorescence quenching

phenomenon, while direct assays are conducted based on

the metal-enhanced fluorescence effect. These three detection

mechanisms and examples are discussed in Section 3.4.

3. Literature survey

3.1 Detection based on the localized surface plasmon

resonance (LSPR) property

The optical properties of AuNPs are governed by their unique

localized surface plasmon resonance (LSPR), which is the

collective oscillation of the nanostructure’s conduction band

electrons in resonance with the incident electromagnetic field.

The LSPR spectrum is strongly dependent on the nanostructure’s

size, shape, dielectric constant, and on the dielectric constant of

the surrounding environment (Fig. 2).22–35 The contribution of

the different parameters to the total extinction of the metal

nanoparticles is described by the Mie theory. The recognition

that the LSPR is sensitive to changes in these parameters has

resulted in intense development of noble metal nanostructures

for various biomedical applications such as biosensors.

Depending on the origin of the LSPR change, a distinction

between two types of LSPR sensors can be made. The first one

is based on the aggregation of the colloid which results in an

apparent color change from red to blue due to inter-particle

coupling. When metal nanoparticles approach each other

closer than their particle diameter, a red-shift of the LSPR is

observed. An extensive review on aggregation based LSPR

sensing was written on this topic by Ghosh et al.34

The other sensor type is based on the sensitivity of the LSPR

to changes in the dielectric constant of the surrounding

medium or adsorbents. This relationship is expressed by the

following equation:35

DlLSPR ¼ mDn 1� exp�2dld

� �� �

where m is the bulk refractive index response of the nanoparticle,

Dn is the change in the refractive index induced by the adsorbents,

d is the effective adsorbent layer thickness, and ld is the

characteristic electromagnetic-field-decay length. As such,

when molecules bind to a AuNP the refractive index will

change giving rise to a shift of the LSPR. It can be deduced

from this equation that the enhanced electromagnetic field will

decay when further away from the nanoparticle surface. As such,

the sensitivity towards refractive index changes is distance

dependent. Only changes in the refractive index at close

proximity to the nanoparticle surface will give rise to a shift

of the LSPR wavelength. This makes the refractive index

based biosensor specific for interactions close to the nanoparticle

surface. The thickness of the adsorbate layer inversely affects the

sensitivity of the plasmon band position to changes in the

refractive index of the surrounding medium or interacting

molecules which is limiting for the detection.

In the following, some examples of both types of LSPR-based

sensors are given and their advantages and disadvantages are

discussed in greater detail. All LSPR-based sensors are label-free

techniques.

3.1.1 Aggregation based LSPR sensing. In the early 1990’s,

biosensor technologies based on the aggregation/agglutination

of gold nanoparticles were successfully implemented to probe

biomolecular interactions. The first reports focused on the

detection of ssDNA by using two types of gold nanoparticles,

both coated with a complementary strand to a part of the

target ssDNA. Upon hybridization, the gold colloid changed

color from red to blue. Mirkin et al. were the pioneers in this

field and were able to detect femtomole levels of ssDNA using

this technology.36 Not only was this technology applied for

DNA detection, but also it was used to study protein–protein

interactions in detail. A well-known application of this technology

is the development of a pregnancy test. In this sensor technology,

AuNPs and micro-latex beads are used, both coated with specific

antibodies to b-hCG, a hormone released by pregnant women.

Upon mixing these particles with urine, containing the hormone,

pink aggregates could be clearly observed.37 In the early 1990s

the first AuNP-based pregnancy testing kits were marketed

by Carter-Wallace and are nowadays still available through

consumer healthcare giant Church and Dwight. Thanh and

Fig. 2 The plasmon resonance of metal NP is dependent on the size,

shape and material. Silver nanoparticles exhibit a plasmon resonance

around 400 nm whereas spherical AuNPs have their resonance around

520 nm. Special shaped AuNPs exhibit plasmon resonance at longer

wavelength.

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Rosenzweig reported on a AuNP-based aggregation assay to

determine the level of anti-protein A in aqueous and serum

solutions.38 Their technique shows comparable sensitivity to

the traditional ELISA. Hirsch and coworkers reported on the

use of gold nanoshells in an immunoassay that is capable of

detecting subnanogram levels of analyte within whole blood.39

Because nanoshells absorb light at the near IR region where

blood does not absorb significantly, antibody-conjugated

nanoshells and their aggregate formation upon binding with

the target analytes can be detected from whole blood samples

using near-IR light.

The aggregates-based LSPR sensing has many advantages as

compared to other techniques. It is a label-free, homogeneous

solution assay and the detection can be done by low cost, simple

instruments (spectrometer) or even by the naked eye. However

this technology has some drawbacks since AuNPs tend to

aggregate under various external perturbations, e.g. changes

of ionic strength, pH or temperature. Further, the color change-

based detection is limited to mostly colorless samples, or at

least, the color of the sample matrices should not overlap with

the color and color change of the gold nanoparticles.

3.1.2 Refractive index based LSPR sensing. Since the

LSPR of metal nanoparticles is sensitive to changes in

the refractive index close to the metal surface, monitoring

the biomolecular interactions on the metal nanoparticle surface

is feasible.40 Englebienne et al. have demonstrated that this optical

property can be used in homogenous biosensor technologies.41

They developed AuNPs which remain in suspension during the

experiments. As such, they were able to determine affinity

parameters of binding proteins for their ligands. In addition,

they used antibody-coated AuNP probes to quantitatively

sense the analyte binding. Also, they can monitor changes in

protein conformation using this technology.42,43 Recently,

PharmaDiagnostics has commercialized this LSPR-based

biosensing. This technology is named SoPRanot and it has

the advantage of being fast, specific, simple to perform,

requires no separation or purification steps and uses common

laboratory equipment for detection. As such, SoPRanot

enables sensitive, robust and reproducible screening in high-

throughput mode.

As the examples above suggest, homogeneous biomolecular

assay formats offer several advantages over conventional

heterogeneous assays. For example, the biomolecular inter-

actions are no longer governed by planar diffusion but by faster

radial diffusion. The active surface area can be significantly

increased by the use of suspended nanoparticles.44 Despite the

potential of this sensing principle, the homogenous detection of

biomolecules has only scarcely been investigated. On the other

hand, refractive index based LSPR biosensing using a hetero-

geneous assay format has been widely developed. As an example,

Frederix et al. have applied this sensing principle in the

construction of a transmission plasmon biosensor, whereby

metal nanoparticles immobilized on a transparent substrate

served as a sensing platform.45 Huang et al. used this technology

to develop an on-chip LSPR-based biosensor for label-free

monitoring of protein–protein interactions.46 To increase the

performance and assay sensitivity of this sensor platform, other

materials and nanostructured patterns on transparent surfaces

have been developed. For example the plasmon wavelength of

silver nanotriangles,47 gold nanoholes, nanorings,48 nanocrosses,49

and nanoshells showed an enhanced sensitivity for refractive

index changes and proved potential in monitoring biomolecular

interactions. Extensive work on silver triangular substrates was

performed by the group of Van Duyne35,47,50,51 and Liz-Marzan.31

Extensive work related to different gold nanostructures was

conducted by the research group of Voros (ETH Zurich)52 and

our research group at imec. Active research is going on in

various research groups to eventually bring this technology to

the market. Some major challenges such as improving the

detection limit, sensitivity and multiplex capabilities need to

be addressed. Notwithstanding these issues, LamdaGen

Corporation has already launched a high throughput (4–8

channels) bench-top LightPatht instrument combined with

pre-functionalized LSPR-based sensorships and software for

data-analysis.

3.2 Detection based on the light scattering properties of

AuNPs

There are many types of electromagnetic scattering and they

can be generally grouped into the categories of elastic and

inelastic light scattering. Elastic scattering involves no energy

transfer while inelastic scattering involves energy transfer

during the scattering process. Rayleigh scattering and Mie

scattering are elastic scattering, while Raman scattering,

inelastic X-ray scattering and Compton scattering belong to

inelastic scattering.53 Depending on whether the scattered light

intensity is proportional or not to the irradiance of the incident

radiation, light scattering can be further divided into linear or

non-linear scattering. Hyper-Rayleigh and hyper-Raman

scattering are two examples of nonlinear light scattering. A

majority of work that has been published on using AuNP

probes for chemical and biological detection is based on elastic

and linear scattering. Inelastic surface-enhanced Raman

scattering is covered in Section 3.4, because it is based on

the scattering of the organic molecule attached to AuNPs, not

directly from the metal core as mentioned above. In regarding

to the nonlinear hyper-Rayleigh scattering of AuNPs, P. C.

Ray has recently published a comprehensive review on this

topic and its application in chemical and biological detection.54

Our current review will focus on two general types of methods

of elastic and linear light scattering: one is based on the

scattered light intensity or wavelength change, and the second

one is nanoparticle-enabled dynamic light scattering assay

(NanoDLSayt).

The theory of light scattering of AuNPs and its potential

applications in biology was first systematically studied by

Yguerabide and Yguerabide and more recently by El-Sayed

et al.22,23 The light scattering cross section of AuNPs increases

with increased particle size. When the diameter exceeds 80 nm,

the light scattering cross section increases substantially compared

to smaller particles (Fig. 3).55 The scattered light intensity of

AuNPs is about 105 times stronger than a fluorescein molecule

and 100–1000s times stronger than a polymer bead. The

exceptionally strong light scattering of AuNPs is enhanced

by the surface plasmon resonance effect. As an optical probe

for light scattering-based biomolecular detection and analysis,

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the scattered light intensity from AuNPs must be substantially

stronger than the scattered light from the biological samples to

avoid the background interference from the sample matrix.

AuNPs can be easily observed or detected in biological samples

and matrices including whole blood, serum, cells, and tissues.

This property is essential for applying AuNP probes for both

in vivo and in vitro applications. Finally, it needs to be mentioned

here that all of the light scattering-based detection techniques

that have been reported so far are label-free techniques.

3.2.1 Scattered light intensity or wavelength change-based

methods. The most known light scattering detection method is

perhaps to directly observe and measure the scattered light

intensity or wavelength change of AuNP probes upon

chemical or biomolecular binding using a microscope, a

spectrophotometer, or sometimes just by the naked eye. In

traditional solid phase ligand binding assays or immunoassays,

AuNPs can replace other optical or enzymatic probes to

conjugate with the detecting molecule. Upon binding of the

detecting molecule–AuNP conjugate with the analyte, the

AuNPs can be visualized using an optical microscope, a scanner

or a spectrophotometer, and the observed scattered light

intensity or wavelength is correlated to analyte concentration.

Sometimes, a silver or gold enhancement may be used to

further enhance the scattering light intensity from the plate.

The bio-bar-code method developed by Mirkin’s group initially

for DNA detection56 utilized this detection mechanism to

achieve a high sensitivity that is two orders of magnitude better

than the analogous fluorophore system.

Storhoff et al. reported the detection of unamplified genomic

DNA sequences based on colorimetric scattering of AuNP

probes using a slightly different assay format.57 In this assay,

the binding of nucleic acid targets with the DNA-modified

AuNP probes is performed in a homogeneous solution, i.e.

the binding between AuNPs probes and the target analyte

occurs in the free solution. The target DNA binding introduces

AuNP clustering. The assay solution is then spotted on a glass

slide and the scattering light from the AuNPs is detected by a

simple waveguide device (Fig. 4). While individual AuNPs

scatter a green light, the AuNP clusters scatter yellow to orange

light, due to a plasmon band red-shift. The scattering light

change was detected and correlated to the target DNA concen-

tration. This scattering light-based method enables detection of

zeptomole quantities of nucleic acid targets without target or

signal amplification, a sensitivity that is over four orders of

magnitude higher than the typical absorbance-based method.

The authors further successfully applied this method to the

rapid detection ofmecA in methicillion-resistant Staphylococcus

aureus genomic DNA samples.

It has been further reported that the detection can be done

by directly monitoring the light scattering change of the assay

solution. According to Mie or Rayleigh scattering theory, the

scattered light intensity increases with increased particle size. For

Rayleigh scattering, the scattering light intensity is proportional

to the 6th power of the radius of the particle.22 When AuNPs

form clusters in solution due to target analyte binding, the

scattered light intensity could increase dramatically. This is the

reason why light scattering based detection techniques are

generally far more sensitive than light absorption-based assays.

Zhang et al. reported the detection of a small biomolecule,

adenosine, from human urine samples using resonance light

scattering of a AuNP modified structure-switching aptamer.58

The reported detection limit was 1.8 nM, with a dynamic range

of 6–115 nM. Xiang et al. used this method to study glycogen

biomacromolecule interactions.59 Glycogen can cause AuNP

aggregation and upon interaction with biomacromolecules, such

aggregation is disrupted, leading to decreased scattered light

intensity. Du et al. demonstrated the detection of human IgG

and DNA using this technique.60,61 Using specially designed

microscopes, Rashke et al.62 and Cao and Sim63 demonstrated

that protein antigen–antibody interaction may be detected from

the light scattering of individual AuNPs.

3.2.2 Nanoparticle-enabled dynamic light scattering assay

(NanoDLSayt).Dynamic light scattering (DLS) is a well-known

Fig. 3 Calculations of the contributions of scattering and adsorption to

the total extinction cross-section of AuNPs of different sizes at their plasmon

band position. The calculations were performed using MiePlot v.3.4.12

(Philip Laven, Geneva, Switzerland). All calculations were performed for

spherical AuNPs in water (5 1C, density: 0.99996382 kg m�3).

Fig. 4 Colorimetric detection of nucleic acid sequences reported by

Storhoff et al. Step 1: DNA–gold nanoparticle probes (A and B) are

hybridized to a DNA target in solution. Step 2: The samples are spotted

onto a glass slide which is illuminated with white light in the plane of the

slide. The evanescent induced scatter from the gold nanoparticles is

visually observed. Individual 40- to 50-nm diameter gold probes scatter

green light, whereas complexed probes scatter yellow to orange light

because of a plasmon band red-shift (Reprinted by permission from

Macmillan Publishers Ltd: Nature Biotechnology, copyright 2005).

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analytical technique for particle size analysis, particularly for

particle sizes in the nanometre scale range.64 It has been used

routinely in polymer characterization, nanomaterial research

and the pharmaceutical industry to study drug formulation and

biopharmaceuticals. While DLS is occasionally used to monitor

the scattered light intensity change of a sample solution, most

frequently its purpose is to determine the average particle size

and size distribution of a nanoparticle solution. DLS measures

the particle size by monitoring the scattered light intensity

fluctuation caused by the Brownian motion of the particles in

solution. Because of the familiarity of DLS to most researchers

in the nanomaterial field, the background of this technique is

not explained here. Even though DLS is also a scattering light

detection technique, the technique we discuss at the following is

not based on scattered light intensity change like the examples

presented in the previous section. Instead, NanoDLSayt

technique detects target analytes based on the size change of

AuNPmolecular probes upon interaction with analyte molecules.

There were two principal assay formats developed (Fig. 5):

one is based on the analyte binding induced AuNP cluster or

aggregate formation; and a second one is based on the individual

AuNP size change upon analyte binding. The two assay formats

can be modified and adapted for different application purposes.

The aggregation assay format (Fig. 5A) can be applied to any

target analytes, small or large, including proteins and DNAs,

small chemicals, metal ions, etc. The individual particle size

change format is more suitable for the detection and analysis

of large biomacromolecules, mainly proteins (Fig. 5B).

(1) Aggregation-based average particle size change. In 2008,

Huo’s group reported a nanoparticle aggregation assay for

protein and DNA detection and analysis using AuNPs

coupled with dynamic light scattering.65–67 This assay is based

on the same principle that numerous other assays are based

upon: the AuNP cluster formation due to target analyte

binding. The nanoparticle cluster formation causes the average

particle size increase of the assay solution. Instead of detecting

the SPR wavelength shift (color change), or scattered light

intensity change, the average nanoparticle size change of the

assay solution is measured using DLS and the size information

is correlated to the target analyte concentration. This technique

has been applied for both protein and DNA detection. The

initial study demonstrated that the sensitivity of NanoDLSayt

is comparable to most other assay techniques: the sensitivity

for a cancer biomarker protein, PSA, could reach a low ng per

mL range, and the detection limit for DNA is in the pM range.

Later, Chun et al. reported that by using a gold-coated

magnetic nanoparticle, a detection limit of 0.01 ng mL�1 can

be achieved for a-fetoprotein.68

Other groups have reported the use of this concept for toxic

metal ions, small chemicals and biomolecules, DNA, and virus

detection. Ray’s group reported the detection of toxic metal ions

including arsenics and lead, and explosives TNT using small

ligand-modified AuNPs with extremely high sensitivity and

excellent selectivity.69–71 For arsenic or lead detection, metal ion

chelating ligands glutathione, dithiothreitol, or cysteine were

attached to the AuNP surface.69,70 For TNT detection, p-ATP-

functionalized AuNPs were used as the probe.71 The sensitivity of

this method for arsenic detection can reach 10 ppt, a concen-

tration that is three orders of magnitude lower than the WHO

guidelines. This sensitivity is two orders of magnitude more

sensitive than the colorimetric techniques. This assay has been

applied successfully to the analysis of environmental samples such

as contaminated well water and consumer products including

paints and toys. For TNT detection, the detection limit can reach

100 pM, which is 5 orders of magnitude more sensitive than the

typical colorimetric methods. The use of this technique is not

confined to only AuNPs, Zhang et al. reported the use of

aza-crown-ether-modified silver nanoparticles for lead(II) ion

detection in surface water with pM sensitivity.72

Miao et al. reported an equally sensitive detection of Pb2+

using Pb2+-specific DNAzyme controlled AuNP aggregation

coupled with DLS. This assay could also be used to detect

Cu2+ and UO42+.73 The same group recently also reported

the detection of double-stranded DNA with a detection limit

of 0.5 fM with excellent sequence specificity.74

Using a similar approach, Yang et al. reported an assay

for adenosine detection.75 Split aptamer fragments were

conjugated to AuNPs to form the probe. Again, the binding

of the aptamer-conjugated AuNPs causes AuNP aggregate

formation. The size change of the assay solution is detected by

DLS and correlated to the adenosine concentration. The

reported limit of detection (LOD) was around 7 nM, 5 orders

of magnitude lower than the colorimetric methods.

Fig. 5 The principle of NanoDLSayt. Two assay formats are illustrated here: A—analyte binding induced nanoparticle cluster formation; and

B—analyte binding induced individual nanoparticle size increase. In both cases, the particle size change of the assay solution can be measured by

DLS and correlated to the analyte information. While the first assay format can be applied to any type of target analytes, small or large, the second

assay format is primarily used for the detection of larger analytes, such as proteins, protein complexes, aggregates and viruses.

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More recently, the Han group developed an innovative

approach for DNA detection using true label-free citrate-

protected AuNPs coupled with DLS.76 Most prior work on

DNA detection involving the use of AuNP probes requires the

conjugation of a single strand DNA probe attached to AuNPs.

The assay developed by the Han group is based on the

different binding affinity of single strand DNA and double

stranded DNA to the citrate-protected AuNPs. Single strand

DNA can bind (or adsorb) to AuNPs through Au–N bonding

between AuNPs and the base moiety on DNA to form a

protective layer on the AuNP surface. Citrate-protected

AuNPs tend to aggregate at a salt concentration higher than

10 mM due to the disruption of the electrical double layer.

When the citrate-AuNPs are protected by a single strand DNA

layer, the AuNPs are stabilized and remain monodispersed in

high salt content solution. In contrast, when a single strand

DNA probe is hybridized with a target DNA to form a double

stranded DNA, the double stranded DNA lost its binding

affinity to citrate-AuNPs. Therefore, citrate-AuNPs are not pro-

tected by double strand DNA molecules against salt-induced

aggregate formation. Using this difference, the authors demon-

strated the detection of the sequence-specific nopaline synthase

(NOS) gene from transgenic plants. The detection limit can reach

the 10 fM range. This sensitivity is 5 orders of magnitude better

than the colorimetric method. A recent side-by-side comparison

study conducted by Pylaev et al. on DNA sequence detection also

confirmed that the dynamic light scattering detection is significantly

more sensitive than the colorimetric assay.77

Driskell et al. reported the detection of influenza virus using

specific anti-influenza virus antibody conjugated AuNPs and

DLS.78 After optimization, they demonstrated a detection

limit of o100 TCID50 per mL, which is 1–2 orders of

magnitude improved over commercial diagnostic kits. This

sensitivity increase is achieved without increasing the assay

time or complexity. Additionally, this assay was demonstrated

to perform equivalently for the influenza virus prepared in

different biological matrices.

As demonstrated by these published works, AuNPs coupled

with DLS detection is a highly promising new technique for

biological and chemical detection and analysis. The sensitivity

of this technique is on the average of 1–5 orders of magnitude

better than many of the other existing techniques. It is a single-

step homogeneous solution assay and it is extremely easy to

use. The target analytes that can be detected by this technique

range from ions, small chemicals and biomolecules, proteins,

DNAs to virus particles. Essentially, any target analytes that can

introduce AuNP cluster or aggregate formation can be detected

using this technique. As demonstrated from publications from

different groups on different target analytes, the reproducibility

of NanoDLSayt is excellent, and as a matter of fact, it is better

than many other existing techniques. Based on all the published

work, the CV% (coefficient variation) of NanoDLSayt is

generally below 10%, and with simple sample matrices such as

buffer solutions, it is often below 5%. As a comparison, the

CV% of a typical ELISA assay is 5–20%.

(2) Individual particle size change. Biological molecules

such as proteins and DNAs are macromolecules. For example,

bovine serum albumin, a protein with a molecular weight of

60–70 KDa has a hydrodynamic diameter around 5–7 nm, and

a typical IgG molecule (150 KDa) has a hydrodynamic

diameter around 7–10 nm. When a complete layer of proteins

is adsorbed or specifically bound to the AuNPs, the diameter

of the nanoparticle will increase by as much as twice of the

diameter of the protein molecule as illustrated in Fig. 6.

Because of the relative large size of the proteins, analyte

proteins may be detected by monitoring the individual particle

size increase without the need of introducing nanoparticle

aggregation by protein analytes. At a saturated binding level,

the size of a target protein may be deduced from the average

particle size increase of the assay solution. This unique feature

of NanoDLSayt to reveal the ‘‘size information’’ of a target

analyte brings new analytical capabilities in biomolecular

detection and analysis, as to be seen from the following

examples. It is important to be aware that in order to use this

assay format correctly, the assay needs to be designed to avoid

nanoparticle–nanoparticle interaction. This can be conveniently

done using a low concentration of AuNP probes. The strong

light scattering property of AuNPs is particularly important for

this application.

A. Label-free and real-time protein–protein interaction

study in solution. For this application, a capture protein or

binding ligand is immobilized or conjugated to AuNPs. Upon

mixing with a sample solution that contains the target binding

protein, the nanoparticle size will increase gradually and this

size increase can be monitored continuously in real time by

DLS to generate a binding curve as illustrated in Fig. 6. Huo’s

group first applied this technique on a model system, protein A

Fig. 6 Real-time monitoring of target analyte binding to the AuNPs

using NanoDLSayt. Assays can be designed so that a saturated

binding level is reached. At a saturated binding level, the size of the

target protein analyte can be estimated. The net particle size increase is

approximately twice of the diameter (D) of the analyte. Such information

can be used to deduce the complexing status of a protein analyte. Because

a protein complex is larger than an individual protein, the binding of a

protein complex to the AuNPs will cause a larger particle size increase at

the saturated binding level than a small individual protein. Protein

complexes and aggregates may further cause nanoparticle cluster

formation. These different scenarios can be distinguished according to

DLS analysis.

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(prA)–human IgG interaction.79 Protein A was immobilized

on AuNPs to form the molecular probe. The subsequent

binding of human IgG to the prA-conjugated AuNPs was

monitored directly by DLS according to the nanoparticle size

change. The different binding activities of the prA–AuNP

conjugate under different conditions (crosslinking versus

non-crosslinking of the prA layer, and pH effect) with human

IgGwere revealed from the assay. Later this assay was applied for

antibody isotyping and quality control analysis.80 Although this

application is not widely known among researchers, antibody

isotyping is a very important step in antibody production and

biopharmaceutical industry. In this study, NanoDLSayt analysis

of several antibody products purchased from commercial sources

led to the same results and conclusions as revealed by using

a commercially available test kit, however, at a substantially

lower cost.

B. Protein complex detection and binding partner analysis.

Protein–protein interactions play an essential role in almost all

cellular functions.81 Many intracellular biochemical processes

are triggered by the assembly of proteins into macromolecular

complexes, providing a means to control the myriad of

biochemical processes for the efficient management of vital

biological responses. A daily task for most biomolecular

researchers is to study various protein complexes and their

biological functions. While numerous methods and technologies

are available for protein concentration analysis, the number of

techniques for protein complex analysis and study is rather

limited and these techniques have not been improved for decades.

One of the most commonly used techniques in biomolecular

research laboratories for protein complex detection and binding

partner analysis is co-immunoprecipitation followed by immuno-

blotting. This technique is time-consuming (1–2 days), labor-

intensive (multiple assay steps) and requires substantially a large

amount of samples (typically 100s mL of sample solution at

reasonably high protein concentrations).

Using a two-step assay format and the assay principle as

outlined in Fig. 6, the Huo group developed a simple method

for protein complex detection and binding partner analysis.82

To conduct the assay, a specific monoclonal antibody for a

target protein is conjugated to AuNPs to form the immuno-

probe. The AuNP immunoprobe is then used to catch the

target protein or protein complexes from the sample solution.

When the binding reaches a saturated level (such a saturated

binding can be achieved by choosing an assay condition so

that the target protein concentration is substantially exceeding

the AuNP concentration), the average particle size increase

can be used to deduce whether the detected target protein is in

a monomer or the other complexed form. Typically, the size of

a target protein is known. If the protein is in a monomer state,

the maximum particle size increase that can be observed

should be approximately twice of the diameter of the protein.

If the observed particle size increase of the assay solution

exceeds substantially this value, the possible existence of

protein complex should be considered. In a second step of

the assay, an antibody that is specific to a suspected binding

partner of the complex is added to the assay solution. If the

suspected binding partner is part of the complex, further

increase of the average particle size should be observed; and

if the suspected protein is not part of the complex, then the

average particle size of the assay solution will remain unchanged.

Using this assay, we discovered for the first time a heteromeric

protein complex that contains EGFR, Stat3 and Src from a

pancreatic cancer cell line, Panc-1.82 Compared to co-immuno-

precipitation, NanoDLSayt requires only a minute amount of

samples (1–2 mL) and results are obtained in minutes instead of

hours or days.

In addition to the two applications discussed here, this

technique has also been applied for label-free protein aggregate

detection83 and cancer biomarker research.84,85 From these

studies, new molecular information on protein biomarkers that

had not been or cannot be revealed by other existing techniques

was discovered. Because of the complexity of the biological

problems addressed in these work, a separate review is being

prepared to examine these issues.

3.3 Detection based on the surface chemistry of AuNPs

Compared to many other nanoparticle materials, the surface

chemistry of AuNPs is significantlymore advanced and understood.

AuNPs form stable chemical bonds with S- and N-containing

groups. As to citrate-protected AuNPs, the presence of positively

charged Au+ ions attracts negatively charged proteins and DNA

molecules, a main force driving the adsorption of biomolecules

to AuNPs. Small organic ligands and polymers with a wide

variety of chemical functionalities may be conveniently attached

to AuNPs through thiol or N-containing linker molecules. The

unique ligand place exchange reaction of AuNPs allows one to

readily modify and switch the chemical functional groups on the

AuNPs.86–91 Furthermore, like other nanoparticle materials, the

large surface area of AuNPs allows one to use the surface ligands

as a way of amplification in chemical and biological detection.

3.3.1 DNA bio-bar-code technique. A most representative

example of using the surface chemistry of AuNPs to amplify

analytical signal transduction is the widely referred DNA

bio-bar-code method developed by the Mirkin group (Fig. 7).

Initially this method was reported for the detection of

protein analytes,92 but later was also applied for DNA target

detection.93 For protein detection, the AuNPs are conjugated

with a capturing molecule, which is an antibody for the specific

protein analyte, along with multiple copies of DNA strands.

The DNA strands are the bio-bar-codes. Another capturing

molecule, typically an antibody that binds to a different

epitope of the protein analyte, is conjugated to a magnetic

microparticle (MMP). The target protein analyte is first

captured by the MMPs, and then the AuNP probe is added

to form a sandwich structure with the captured target protein

and MMPs. By applying a magnetic field, the sandwiched

assay product can be isolated from the assay solution to

eliminate unbound AuNPs. The bio-bar-code DNA molecules

are then released from the AuNPs and analyzed by PCR

methods, or scanometric (PCR-less) methods amplified by

the enhanced light scattering of metal nanoparticles as the

authors reported previously.56

The DNA bio-bar-code technique combines two essential

elements to create ultra-high sensitivity of detection: (1) the

ability to attach a large number of DNA probes onto AuNPs;

and (2) the detection of DNAs with extremely high sensitivity

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using currently available techniques. Furthermore it is a

homogeneous solution assay. One has the option to add high

concentration of AuNPs and MMP probes to increase the

kinetics of the binding. The addition of more probe particles

(both AuNPs and MMPs) will push the equilibrium toward

the complex formation, increasing the detection limit. Without

PCR amplification, this method can achieve a 30 aM (attomolar)

detection limit for prostate specific antigen, a protein biomarker

for prostate cancer. If PCR is applied, the sensitivity can be

boosted to 3 aM. This bio-bar-code assay represents a six-order

of magnitude higher sensitivity than most conventional assays.

For target DNA detection, PCR-like sensitivity can be achieved.

Later this technique was reported for the detection of cyto-

kines at a 30 aM concentration range,94 a soluble pathogenic

biomarker for Alzheimer’s disease at a concentration around

100 aM,95 and more recently, used to detect trace amounts of

PSA from prostate cancer patients after radical prostatectomy

surgery.96

3.3.2 Controlled surface chemical functionality of AuNPs

for biological sensing and detection. Another approach that

uses the surface chemistry of AuNPs for sensing has been

developed by the Rotello group. Prior to sensing applications,

this group has conducted extensive studies to understand and

control the interactions between AuNPs and biomolecules,

primarily proteins, by controlling the surface chemical func-

tionality of AuNPs.97–103 The surface chemistry of AuNPs can

be conveniently modified using different synthetic methods.

Small chemical ligands or polymers may be attached to

AuNPs through Au–S or Au–N bonding. The AuNP surface

can thus be made to contain positive, negative, mixed charges,

or to be neutral. Different proteins, with different amino acid

residues and charges on the surface, tend to bind with

these surface-modified AuNPs with different affinities through

non-covalent interactions.

In 2007, the Rotello group made an important finding that a

fluorescent polymer (or green fluorescent protein) may reversely

bind and dissociate from the functionalized AuNPs through

controlled non-covalent chemical interactions between the

polymer and AuNPs.104 When the fluorescent polymer is

bound to the AuNPs, its fluorescence is quenched; and once

it is dissociated from the AuNPs, the fluorescence property is

restored. Based on the different binding affinity of this fluorescent

polymer and proteins with AuNPs, a competitive assay was

developed for protein detection (Fig. 8). The target protein is

detected according to their different binding patterns to variously

functionalized AuNPs. By using an array of surface-modified

AuNPs, a target protein will cause different fluorescence

responses upon binding with the AuNPs, therefore, a fluores-

cence response pattern is generated. By analyzing the fluores-

cence response pattern of the assay solution using a suite

of statistical analysis tools, different molecular targets or

biological species may be detected and distinguished from

one to another. This approach is based on a ‘‘chemical nose’’

sensing mechanism. The most unique and exciting aspect of

chemical nose sensing is that one does not need to know the

true identity or nature of the target analyte in order to detect

this analyte. There is also no need to generate a specific

antibody for the detection.

Initially, this method was reported for the detection and sensing

of proteins. Later, it was further successfully applied for the

detection and sensing of bacteria and cancer cells.105–109 More

recently, the Rotello group further reported the use of an enzyme,

b-galactosidase (b-Gal), to replace the fluorescent polymer as the

signal transducing molecule and to enhance the sensitivity of

the technique.110 Because of the amplifying capability of the

enzyme through catalytic reactions, the sensitivity is improved

substantially. For example, a detection limit of 1 � 102

bacteria per mL in solution and 1 � 104 bacteria per mL in

a test strip format was reported using this method based on a

simple color change of the assay solution.

Fig. 7 The DNA bio-bar-code method for protein detection developed by the Mirkin group. (A) An AuNP probe is functionalized with antibody

and bar-code DNAs, while a magnetic microparticle (MMP) probe is conjugated with a capture antibody for the target protein. The MMP probe is

first used to capture the target protein analyte and then the AuNP probes are added to form a sandwich-structure with the captured target proteins.

The AuNP–MMP clusters are then separated from the assay solution by applying a magnetic field. The bar-code DNAs are then released from the

AuNP–MMP clusters, and analyzed. The quantity of the DNA bar-code is correlated to the target protein concentration (Science by American

Association for the Advancement of Science. Reproduced with permission of American Association for the Advancement of Science in the format

Journal via Copyright Clearance Center).

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3.4 Detection based on the metal core–surface ligand

interaction of AuNPs

Colloidal AuNPs exhibit strong enhancement of Raman scattering

and fluorescence emission from molecules adsorbed on their

surface. As already outlined in the Introduction, the enhance-

ment mechanisms that are relevant to SERS are also present in

surface-enhanced fluorescence (SEF). Many applications as a

method to enhance existing fluorescence based techniques are

also relevant to SERS and vice versa. For example, many

SERS probes are, indeed, fluorescent dyes.

3.4.1 Surface-enhanced Raman scattering (SERS). Mole-

cular vibrations can be probed by infrared absorption spectro-

scopy or by inelastic scattering of photons from vibrational

quantum states, called Raman scattering. The frequency shift

between incoming and Raman scattered light is determined by

the energy of molecular vibrations. In general, Raman scattered

light is inherently weak. However, the intensity of Raman scattering

can be enhanced dramatically when the inelastic scattering process

takes place in very close vicinity of metal nanostructures. This effect

of surface-enhanced Raman scattering (SERS) elevates vibrational

spectroscopy to new limits in sensitivity and molecular structure

selectivity.

Surface-enhanced Raman scattering (SERS) was discovered in

the early 1970’s. Since then, this field developed explosively, reflected

by the enormous amount of research papers, reviews and analytical

books that have appeared in the literature. The interest in SERS

shifted from fundamental research to applications governed by the

ability to reliably control the size, shape and surface properties of

nanostructured materials, making SERS a recognized analytical

technique for the sensitive and selective detection of molecules in

close vicinity to metal nanostructures.111,112

Although the SERS effect has been studied extensively, the

mechanism leading to the surface enhancement is still not

Fig. 8 Protein detection using a nanoparticle–fluorescent polymer ‘‘chemical nose’’ sensor developed by the Rotello group. (A) Illustration of the

sensing mechanism. An anionic, fluorescent polymer is first adsorbed to the cationic gold nanoparticles through electrostatic interactions. This

adsorption causes fluorescence quenching of the polymer. Displacement of the quenched fluorescent polymer by a target protein restores the fluorescence

of the polymer. By using different types of gold nanoparticles, a protein sensor array is generated. Different target proteins will show different binding

affinity towards the nanoparticle array. (B) The chemical structure of the cationic gold nanoparticles and anionic fluorescence polymer. (C) The response

profile (expressed as a canonical score plot) of different target proteins to the nanoparticle–fluorescent polymer sensor (Nature Nanotechnology by Nature

Publishing Group. Reproduced with permission of Nature Publishing Group in the format Journal via Copyright Clearance Center).

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completely understood. It is generally believed that there are

two main contributions to the SERS effect:111–113 the electro-

magnetic mechanism or chemical enhancement. The electro-

magnetic mechanism arises from the optical excitation of

surface plasmon resonances in the metal nanoparticles, which

leads to significant increase in the electromagnetic field

strength at the metal nanoparticle surface. In the chemical

enhancement, molecules adsorbed onto the metal nanoparticle

surface are believed to couple electronically with the surface

leading to an enhancement effect similar to resonance-Raman

scattering or via resonant charge-transfer. The extent of the

relative contribution to SERS from electromagnetic and

chemical effects remains a subject of discussion. Independent

research in several groups has shown that Raman scattering

can be enhanced up to 1014 relative to normal Raman scattering.

As such, SERS opens up opportunities for monitoring biologically

relevant molecules even at the single molecule level. SERS also

offers several other advantages for the detection of biomolecules.

First of all it is a rapid non-destructive tool and it yields

compound-specific information. Furthermore it has the potential

for multi-component analysis. Additionally, SERS spectroscopy

can be performed in the presence of water, and as such this

technique can be applied for the investigation of organic and

biochemical substances in their natural environment.

Different roughened or nanostructured metals such as coinage,

transition and alkali metals have been evaluated as SERS

substrates. The commonly used substrates for SERS are the

coinage metals Ag, Au, Cu. These metals support plasmons in

the visible region.114 Transition metals are generally not

considered as SERS-active, because it is more difficult to excite

the plasmon in the visible region. The lack of attention to

alkali metals, although they have tunable plasmon resonances

in the visible region, is due to their high chemical reactivity,

which makes their exposure to air impossible.115 The SERS

effect using Cu, Ag, Au, Li, Na, K substrates is more intense

compared to that of transition metals. The typical enhance-

ment achieved using these substrates can be as high as 104–106

whereas transition metal substrates (Fe, Co, Ni Rh, Pd and Pt)

only show enhancement up to 101–104.116 Despite their lower

SERS enhancement, transition metals do have potential in

modern industries and technologies such as electrochemistry,

corrosion and catalysis and may as such not be neglected.

There are a few comprehensive reviews that have been written

by the leading experts in this field to which the reader may

refer.117–119

Towards biological applications and chemical inertness

roughened or nanostructured Au and Ag substrates are favor-

able. Several researchers have reported on the use of AuNP

immobilized substrates for efficient biomarker detection by

SERS and others use colloidal AuNPs as SERS labels for

analysis.120,121 The research group of Van Duyne developed

gold or silver coated polystyrene nanosphere substrates func-

tionalized with a self-assembled monolayer for glucose detec-

tion (Fig. 9).122,123 The potential of AuNP array substrates as

SERS substrates was also shown by Liu et al. in the develop-

ment of a sandwich assay using crystal violet as a reporter

molecule.124 On the other hand, AuNP-modified gold sub-

strates showed improved performance as SERS substrates as

demonstrated by Cheng et al. for the detection of dipicolinic

acid, a biomarker for bacterial spores.125 Very recently, Lee

et al. reported a highly reproducible SERS-based immunoassay

for cancer marker detection by the use of hollow AuNPs

combined with an AuNP-patterned surface in a sandwich

approach. Using their SERS-based approach, a three to four

orders of magnitude increase in detection sensitivity was

obtained compared to that of conventional ELISA.126,127

The Mirkin group mainly focused on using AuNPs function-

alized with various Raman dye-labeled oligonucleotides to

derive Raman spectroscopic fingerprints for the multiplex

detection of oligonucleotide targets. After Ag enhancement,

reproducible SERS responses were obtained.128 On the other

hand, Vo-Dinh and co-workers made use of the distance

dependency of SERS caused by the rapid decay of the field

enhancement around metal spheres. In their technology,

sentinel nanoprobes (Raman-labeled DNA hairpin probe immo-

bilized on colloidal AgNPs) are used to detect oligonucleotide

targets by SERS.129 In the absence of the complementary

oligonucleotide target, a strong SERS signal is observed due

to the hairpin conformation. Upon hybridization, the hairpin

conformation is disrupted and the SERS signal is quenched

due to the physical separation of the Raman label from the

nanoparticle surface. Recently, this research team demonstrated

the feasibility of using this technology for qualitative multiplex

oligonucleotide target detection.130 Besides oligonucleotide detec-

tion, assays for protein detection have been developed using

immuno-AuNPs. In most cases the AuNPs are biofunctionalized

with the protein of interest and afterwards, a Raman-active

reporter molecule is co-immobilized. With these ‘so-called’ SERS

nanotags, superior detection limits (subpicomolar concentrations)

and sensitivities are obtained (by Proter and co-workers).131 How-

ever a major limitation of this approach is that these nanotags are

prone to spectral changes and colloidal aggregation because they

are not shielded from their environments. To overcome this

problem, several groups have employed various encapsulation

strategies. These core–shell SERS nanotags have a metallic core

Fig. 9 (A) The fabrication of silver-coated polystyrene nanosphere

substrates; known as film over nanospheres (FON) substrates.

(B) Functionalization of the gold or silver FON substrates with a self-

assembled monolayer of decanethiol–mercaptohexanol for glucose detec-

tion via SERS developed by the group of VanDuyne (The publisher for this

copyrighted material is Mary Ann Liebert, Inc. publishers).

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for optical enhancement, a reporter molecule for spectroscopic

signature and a shell for protection and conjugation.132–135

Researchers are currently exploring the use of these SERS

nanotags as biocompatible nanosensors that can be placed

inside small structures such as live cells.136 These mobile

sensors can probe the chemistry taking place in different cell

compartments by revealing SERS spectra of cellular molecules

in their close vicinity.137 Moreover, these SERS-based nano-

sensors allow structural characterization of molecules in live

cell and as such providing key information for monitoring

cellular processes such as enzymatic activity. AuNPs with reporter

molecules attached, which exhibit a known and calibrated pH

dependent SERS signal, can also act as intracellular pH sensors.

Oxonica, a nanomaterials group, has developed commercial

Nanoplext Biotags. These are nanoparticles with a diameter

of 90 nm composed of a gold core, an adsorbed layer of SERS

active molecules and a silica shell. Oxonica has developed

various nanoparticles composed of the same layer structure

but with different embedded SERS active molecules. Since

each organic molecule has its own fingerprint-like spectra,

these nanoparticles can be used for multiplexed biomarker

quantitation. In addition, Oxonica claims that their particles

are extremely stable and can be used in any biological matrix,

including whole blood. These products of Oxonica are currently

being investigated by several research groups in order to ascertain

their potential for in vitro and in vivo diagnostics. There are

currently no SERS-based diagnostic products on the market.

However, as a leading supplier of Raman instrumentation,

Renishaw has now invested in D3 Technologies Ltd. This

company is currently developing a complete SERS-based

molecular diagnostic system. They explore the use of nano-

engineered Karites SERS substrates comprising of a gold

covered regular array of holes on a silica wafer. By using these

disposable substrates they are able to identify and characterize

minute amounts of material without the use of surface chemistry.

In addition they are working on all other facets of the system

like the instrumentation to perform the measurements and the

software to evaluate the sensor output.

3.4.2 Metal-enhanced fluorescence. Fluorescence is an

established and a dominant sensing technology in medical

diagnostics and biotechnology. Although fluorescence is

known as a highly sensitive technique, where single molecules

can readily be detected, there is still a drive for further

reducing detection limits.138 The detection of a fluorophore

is usually limited by its quantum yield, photostability and the

autofluorescence of samples. To overcome some of these

constraints, intense recent research is devoted to the use of

metallic nanostructures to favorably modify the spectral properties

of fluorophores. The use of fluorophore–metal interactions has

been termed radiative decay engineering, metal-enhanced fluores-

cence or surface-enhanced fluorescence.

The current interpretation of metal-enhanced fluorescence

(MEF) is that it is comprised of two cooperative mechanisms,

a near-field effect and an induced plasmon effect.139 In the

near-field effect, the generated increased electric field between

or around the metal nanoparticles results in an increase in the

absorption cross-section of a nearby fluorophore and sub-

sequently an increase in the fluorescent emission. Partially energy

transfer from the fluorophore to the surface plasmon (induced

plasmon effect) results in enhanced plasmon scatter that

positively influences the fluorescent emission with or without

reducing the excited-state decay time. As such, MEF is also

supported by the scattering component of the metal extinction,

i.e. the ability of fluorophore-coupled Plasmon’s to radiate.

The first experimental and theoretical reports on metal-

enhanced fluorescence in the 1970’s and 1980’s have however

been overshadowed by the large signal enhancements seen

with SERS. In SERS, enhancement factors of 1014 have been

frequently reported whereas the increase in fluorescence signal

of appropriate fluorophores near metal nanostructures is only

a factor of B10.140 Although both technologies require the

proximity of a metal nanostructure to enhance the Raman or

fluorescent properties, they do have different requirements.

For example, efficient SERS requires close contact of the

studied molecules with the metallic surface, at distance below

20–30 A whereas at this distance, the fluorescence of the

molecules is significantly quenched, primarily by energy transfer

to the metal surface. In MEF, the optimal separation distance of

the fluorophore and the metal nanoparticles is larger, but very

limited.When the fluorophores were placed at leastB11 nm from

the nanostructured metals or from shell protected nanoparticles,

enhanced fluorescence has been reported.140,141 For example,

Aslan et al. used an additional silica coating around the metal

nanoparticles to provide the surface nanostructure functionality

(Fig. 10), the surface plasmon absorption and to tune and

ensure the distance of the fluorophores from close-range metal

quenching.142,143 Also other spacers, including DNA strands

or antibodies, have been used to obtain efficient fluorophore–

metal separation for enhanced fluorescence observations.144

The fluorophore emission near metal nanoparticles depends

not only the fluorophore–particle surface distance but also the

molecular dipole orientation versus particle surface. Zhang

et al. also reported on the wavelength dependency of MEF.145

Fig. 10 Comparative emission spectra of a fluorescent (doped with

Rhodamine 800) core–shell Ag@SiO2 nanoparticle and a control nano-

particle containing no Ag. The optimal thickness of the shell is around

11 nm. These core shells Ag@SiO2 show potential for applications in

MEF solution-based sensing (Journal of Fluorescence by Springer

New York LLC. Reproduced with permission of Springer New York

LLC in the format Journal via Copyright Clearance Center).

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There is a correlation with the scattering portion of the

extinction spectrum of metal nanoparticles and MEF.

Furthermore, several research groups have reported on the

effect of AuNP size and shape on the fluorescent properties of

a fluorophore.146,147 Particles with large scattering cross-section

increase the fluorescence yield, as is the case for gold nanoshells,

nanorods, and nanoparticles of larger size. This can be simply

explained by the angular dependent nature of plasmon scattering

and the scattering vs. absorption contribution to the gold

extinction spectrum favoring the fluorescent emission.

The observation of fluorescence enhancement has been

limited to silver nanoparticles and the effect of AuNPs on

fluorophores can only be found sporadic.148 Gold serves as an

ultra-efficient quencher of the light exciting fluorophores as

demonstrated for several AuNP–fluorophore composites and

used in various homogenous assays for DNA and protein

detection.149 Nevertheless, Matveeva et al. demonstrated a

strategy to reverse the quenching of fluorescence by AuNPs

to enhancement by using an additional coating of silver.141

Also Aslan et al. and Cheng et al. demonstrated that AuNPs

of sufficient large size (Z 100 nm), where the fluorophore is

separated by a spacer layer of at least 10 nm from the AuNP

surface, ensure MEF.147,150 There are still many indications

for using gold instead of silver for MEF because gold has

better chemical stability and the surface chemistry is well under-

stood, while surface modification and binding of biomolecules

can easily be accomplished. In this respect, some development of

homogeneous assays for the detection of DNA and proteins can

be found. For example, a label free DNA sensing by using the

fluorescence quenching and enhancement was shown by Cheng

et al.150 They developed a homogenous assay for the detection of

DNA based on fluorescently labeled DNA hairpin probes and

reported a detection limit below 100 pM of target DNA. Most

recently the detection of terbium-levofloxacin, a synthetic

chemotherapeutic antibiotic derived from a fluorinated quinolone,

was demonstrated in pharmaceutical preparations,151 opening the

potential application of this technique for pharmaceutical research.

4. Discussions and future perspective

As illustrated from the literature survey, many techniques have

been or are being developed for chemical and biological

sensing and analysis based on the optical properties of AuNPs.

Briefly, the following is a summary of the advantages and

disadvantages of each detection method and the remaining

challenges.

Detection based on the LSPR property change of AuNPs is

perhaps the easiest technique to use. It requires low cost, well-

known instruments such as a UV-Vis spectrophotometer, or

sometimes, even no instrument. Similar to the SPR technique,

LSPR techniques can be considered as ‘‘label-free’’ detection.

Sensing molecules such as antibodies or chemical ligands do

need to be immobilized on AuNP probes (while in SPR, they

are immobilized on a gold monolayer film), however, there is

no need to label the target analyte or use an optically-labeled

sensing molecule or binding ligand. This detection can be used

for both homogeneous solution or heterogeneous assay formats.

The major downside of this technique is the relatively low

sensitivity. Although some improvements can lead to higher

sensitivity, most reports have shown the detection limit for protein

in the ng per mL regime and DNAs down to nM. The LSPR

technique has also been used for small chemical detection. Another

problem for homogeneous solution based LSPR detection is the

color of the sample. Because LSPR is in principle a colorimetric

method, this technique is not suitable for analyte detection

from intensely colored samples. Although AuNPs with a

LSPR wavelength at near-IR can be used for deeply colored

samples such as blood, they require the use of gold nanoshells,

nanorods, or other non-spherical AuNPs. However, their

synthesis method is much more complicated than the citrate-

protected spherical AuNPs and associated with this, it is a

significant challenge to functionalize them with biomolecules.

LSPR-based techniques have been successfully commercia-

lized. The most known one is the pregnancy test kit. Also the

SoPRanot product, which makes use of a homogenous assay

format with spherical AuNPs, has been commercialized by

PharmaDiagnostics. A heterogeneous analog product, Light-

Patht was recently launched by LamdaGen Corporation.

Overall, light scattering-based techniques have a substan-

tially higher sensitivity compared to adsorption-based techni-

ques. A number of highly sensitive techniques were all based

on the light scattering of gold and silver nanoparticles. The

light scattering cross-section of AuNP increases dramatically

when the diameter of AuNPs exceeds 80 nm. Many biological

sample matrices such as blood and serum also scatter light

intensely, since it contains a high concentration of colloidal

particles. By using large AuNPs (exceeding 80 nm), the light

scattering detection can be used for a wide variety of samples,

including blood and serum. The instrument used for light

scattering detection can be a typical spectrophotomer, or

dynamic light scattering (DLS) instrument. The cost of a

DLS instrument is only slightly higher than most spectro-

photometers. The light scattering from AuNPs does not suffer

from photobleaching and instability issues as in the case of

fluorescence-based techniques.

The nanoparticle-enabled dynamic light scattering assay

(NanoDLSayt) provides unique advantages and capabilities

for biomolecular research. NanoDLSayt not only can be used

for target analyte concentration analysis, it can reveal the

‘‘size’’ information of the analyte directly from the assay. This

capability is particularly useful for studying protein–protein

interaction and for complexed protein detection and analysis.

In terms of simple concentration analysis of protein analytes,

NanoDLSayt is on par with most other existing immunoassay

techniques. For the detection of DNA, metal ions, and virus,

NanoDLSayt has shown 1–5 orders of magnitude of improved

sensitivity compared to many other existing techniques. Nano

Discovery Inc. is commercializing NanoDLSayt. The company

has developed a new DLS instrument, NDS1200, that is

especially designed for high throughput analysis and also

simultaneous kinetic study of multiple samples automatically

using a NanoDLSayt technique.

There have been mainly two forms of detection methods based

on the surface chemistry of AuNPs. The DNA bio-bar-code

method developed by Mirkin’s group has the distinct advantage

of an ultra-high sensitivity of detection. This method probably

provides the best sensitivity for protein detection among all

existing analytical techniques. The DNA bar code attached to

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the AuNPs leads to very unique integration of both high

sensitivity detection and multiplexing analysis capability.

The drawbacks of this method are the relatively high cost

associated with AuNP probe preparation, the multiple steps

that are required for conducting the assay and the assay

format that is still based on a heterogeneous solid–liquid

interface assay. The other surface chemistry-related detection

method is based on the reversible binding of a fluorescent

polymer introduced by Rotello’s group. The most appealing

advantage of this technique is that one does not need to know

the specific molecular identity of the target in order to detect

the target from a sample matrix. This capability should be

particularly useful for detecting new pathogens and targets of

which the identity is not known or completely known.

As already stated, light scattering based technologies have

the advantage of high sensitivity and this is also the case for

SERS-based detection and analysis. This technique relies on

the interaction of the metal core and the surface ligands. Upon

excitation of the plasmon the electromagnetic field in close

neighborhood of the AuNP surface is enhanced, and in

addition also resonant-charge transfer to chemisorbed molecules

can occur, giving rise to an extreme enhancement of their

corresponding Raman signal. SERS-based biosensing relies

mainly on heterogeneous assay formats, although homogenous

formats do exist. This technology provides specific molecule

information and is not influenced by the sample matrix. More-

over, the SERS technique can be conveniently adapted for

multiplexing analysis. A SERS-based diagnostic product is being

commercialized by D3 Technologies Ltd. Other companies have

commercialized modified AuNPs (Nanoplext Biotags from

Oxonica Inc) or nano-engineered substrates (Karites from

Renishaw Diagnostics) for research purposes.

The last optical property of AuNPs that was described in

this review is metal-enhanced fluorescence. This technology is

perhaps the leastmature of all optical sensing technologies described

here. This has mainly to do with the various constraints related to

the technology. To achieve fluorescent enhancement, the distance

between the fluorophore and the metal nanoparticles needs to be

well controlled. Additionally, the molecular dipole orientation, the

applied excitation wavelength, the AuNP size or shape can all affect

fluorescent enhancement. Nevertheless this sensing technology can

improve the detection limit of the widely used fluorescence-based

detection methods both in homogenous and heterogeneous assay

formats, therefore, is worth of continuing investigation.

5. Conclusions

In summary, research has been extremely productive in AuNP-

enabled biological and chemical sensing and analysis. In the past

ten years, many new techniques have been developed and a

significant number of these techniques have been or are very

close to be commercialized. These new techniques not only

substantially improved the detection limit, but further brought

new analytical capabilities that enable scientists to discover

molecular information related to diseases and other biological

processes that cannot be revealed by existing techniques. All these

accomplishments clearly demonstrate and testify the significance

of nanotechnology in the advancement of modern science and

knowledge.

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