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rsc.li/analyst
Analyst
www.rsc.org/analyst
ISSN 0003-2654
PAPER
Michele Zagnoni et al.
Emulsion technologies for multicellular tumour spheroid radiation assays
Volume 141 Number 1 7 January 2016 Pages 1–354
Analyst
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This article can be cited before page numbers have been issued, to do this please use: A. Ghasemi, N.
Rabiee, S. Ahmadi, S. hashemzadeh, F. Lolasi, M. Borzogomid, A. Kalbasi, B. Nasseri, A. Shiralizadeh
Dezfuli, A. Aref, M. Karimi and M. R. Hamblin, Analyst, 2018, DOI: 10.1039/C8AN00731D.
1
Optical Assays Based on Colloidal Inorganic Nanoparticles
Critical review Article
Amir Ghasemi
1,3
, Navid Rabiee
2
, Sepideh Ahmadi
3,4
, Shabnam Hashemzadeh
5,6
, Farshad Lolasi
7,8
,
Mahnaz Bozorgomid
9
, Alireza Kalbasi
10
, Behzad Nasseri
11,12
, Amin Shiralizadeh Dezfuli
3,13
,
Amir Reza
Aref
13
,
Mahdi Karimi
14,15,16*
, Michael R. Hamblin
16,17,18*
1 Department of Materials Science and Engineering, Sharif University of Technology, Tehran, Iran.
2 Department of Chemistry, Shahid Beheshti University, Tehran, Iran
3 Advances Nanobiotechnology and Nanomedicine Research Group (ANNRG), Iran University of
Medical Sciences, Tehran, Iran
4 Department of Biology, Faculty of Basic Sciences, University of Zabol, Zabol, Iran
5 Department of Medical Physics and Biomedical Engineering, Faculty of Medicine, Shahid Beheshti
University of Medical Sciences, Tehran, Iran
6 Research Center for Pharmaceutical Nanotechnology, Biomedicine Institute, Tabriz University of
Medical Science, Tabriz, Iran
7 Department of Biotechnology, Faculty of Advanced Sciences and Technologies, University of Isfahan,
Isfahan, 81746-73441, Iran.
8 Department of Agronomy and Plant Breeding, College of Agriculture, Isfahan University of
Technology, Isfahan, Iran
9 Department of Pharmaceutical Chemistry, Islamic Azad University of Pharmaceutical Sciences Branch,
Tehran, Iran
10 Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA
11 Departments of Microbiology and Microbial Biotechnology and Nanobiotechnology, Faculty of Life
Sciences and Biotechnology, Shahid Beheshti University, Tehran, Iran
12 Chemical Engineering Deptartment and Bioengineeing Division, Hacettepe University, 06800,
Beytepe, Ankara, Turkey
13 Belfer Center for Applied Cancer Science, Dana-Farber Cancer Institute, Harvard Medical School,
Boston, MA, USA
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14 Cellular and Molecular Research Center, Iran University of Medical Sciences, Tehran, Iran
15 Department of Medical Nanotechnology, Faculty of Advanced Technologies in Medicine, Iran
University of Medical Sciences, Tehran, Iran
16 Wellman Center for Photomedicine, Massachusetts General Hospital, Harvard Medical School,
Boston, MA, 02114, USA
17 Department of Dermatology, Harvard Medical School, Boston, MA 02115, USA
18 Harvard-MIT Division of Health Sciences and Technology, Cambridge, MA, 02139, USA
*
Corresponding author: m_karimy2006@yahoo.com, karimi.m@iums.ac.ir (Mahdi Karimi)
*
Corresponding author: Hamblin@helix.mgh.harvard.edu (Michael R. Hamblin).
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Contents
1- Introduction .......................................................................................................................................... 5
2- Au NPs: Synthesis, Functionalization, Properties and Application ...................................................... 7
3- Colorimetric Detection Based on AuNPs ....................................................................................... 10
3-1- Colorimetric Detection Based on Modified AuNPs ....................................................................... 11
3-1-1- Cross-Linking Approach ......................................................................................................... 11
3-1-2- Non-Cross-Linking Approaches .............................................................................................. 21
3-2- Colorimetric Detection Based on Unmodified AuNPs ................................................................... 28
4- Silver nanoparticles (AgNPs): Synthesis, Functionalization, Properties, and Application ............. 36
5-Colorimetric Detection Based on AgNPs ............................................................................................ 41
5-1-Colorimetric detection based on combinations of AuNPs and AgNPs ........................................... 46
6- Magnetic Nanoparticles (MNPs): Synthesis, Functionalization, Properties and Applications ....... 53
7- Colorimetric Detection Based on MNPs ......................................................................................... 58
7-1 Colorimetric detection based on combinations of AuNPs and MNPs ............................................. 59
7-2 Colorimetric detection based on combinations of AgNPs and MNPs ............................................. 61
8- Colorimetric detection based on other inorganic nanoparticles ...................................................... 62
9- Conclusion and Future Outlook ...................................................................................................... 69
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Abstract
Colloidal inorganic nanoparticles have wide applications in the detection of analytes and in
biological assays. A large number of these assays rely on the ability of gold nanoparticles
(AuNPs, in the 20-nm diameter size range) to undergo a color change from red to blue upon
aggregation. AuNP assays can be based on cross-linking, non-cross linking or unmodified
charge-based aggregation. Nucleic acid-based probes, monoclonal antibodies, and molecular-
affinity agents can be attached by covalent or non-covalent means. Surface plasmon resonance
and SERS techniques can be utilized. Silver NPs also have attractive optical properties (higher
extinction coefficient). Combinations of AuNPs and AgNPs in nanocomposites can have
additional advantages. Magnetic NPs and ZnO, TiO
2
and ZnS as well as insulator NPs including
SiO
2
can be employed in colorimetric assays, and some can act as peroxidase mimics in catalytic
applications. This review covers the synthesis and stabilization of inorganic NPs and their
diverse applications in colorimetric and optical assays for analytes related to environmental
contamination (metal ions and pesticides), and for early diagnosis and monitoring of diseases,
using medically important biomarkers.
Keywords. Colorimetric assays; environmental contaminants; disease biomarkers; gold and
silver nanoparticles; cross-linking and non-cross-linking; surface plasmon resonance
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1- Introduction
Early detection of diseases such as cancer and infection is an effective way to prevent them from
spreading. Diagnostic procedures based on serological, biochemical, molecular, and staining
methods have been used to detect many diseases, such as cancer, infectious agents, proteins, and
potentially toxic metal ions [1-6]. Such methods, however, have many disadvantages, including
the need for expensive equipment, lengthy procedures, inefficient detection rates, and poor
detection thresholds. Biochemical tests are usually time-consuming and expensive to implement.
Moreover, they can have low sensitivity and specificity, giving false positive and negative
reactions along with insufficient accuracy. Diagnosis based on serological methods, may not be
suitable for early detection of disease due to inadequate production of antibodies, the required
long time to obtain results, and the need for complex facilities [7-9].
Nanotechnology is concerned with the production, investigation, and application of materials at
the nanometer scale. When the size of the particles is in the nanometer range, their physical and
chemical properties vary markedly from the properties of the same bulk material [10].
Nanoparticles are considered to be one of the most important types of nanomaterials, which have
diameters less than 100 nm, and can take various forms depending on their material type and
synthetic procedure [11]. Inorganic nanoparticles have played an important role in development
of nano-biosensors [12]. The properties of inorganic nanoparticles, such as their optical (metal
nanoparticles), electrical, magnetic (iron oxide or cobalt nanoparticles), and catalytic (metal
nanoparticles, oxide and quantum nanoparticles) properties, depend strongly on the type of
material they are composed of [13, 14]. Their optical properties, especially fluorescence-based
techniques, have been investigated due to their tunable features and conditions. These techniques
are based on the fluorescence resonance energy transfer (FRET), photobleaching mechanism and
tuning the luminescence quantum yield. To be exact, conjugation of metallic NPs to an active
fluorophore molecule (sensitizer) has led to the development of accessible, inexpensive and
environmentally-friendly devices for early diagnosis and monitoring treatment procedures[15-
17]. Because of these beneficial features, these devices have gathered a varied range of
applications in diagnosis and treatment [18], gene therapy [19], drug delivery [20], etc. Gold
nanoparticles (AuNPs) and silver nanoparticles (AgNPs), which have properties such as surface
plasmon resonance (SPR), undergo visible color and spectral changes, and unique optical
features, are widely used in colorimetric techniques [21]. Magnetic nanoparticles (MNPs) are
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also important in the setting of drug and gene delivery due to their non-toxic nature, high field
irreversibility, and super-paramagnetic properties [22-24]. Metal oxide nanoparticles and
semiconductor quantum dots (QDs) have unique photocatalytic properties for detection of trace
contaminants, making them relevant for water purification and wastewater treatment [25].
Among mineral nanoparticles, AuNPs are widely used in colorimetric techniques because of
their rapid and easy synthesis, and facile control of size and shape. AuNPs possess surfaces that
can be functionalized, and are sustainable and biocompatible [26-28]
The National Institute of Standards and Technology (NIST) has discussed AuNPs as “standard
nanoparticles” for biological studies and testing [28]. In the current review, we discuss the use of
colorimetric techniques based on inorganic nanoparticles to detect diseases, infections, proteins,
analytes, and ions. The properties, synthesis, and applications of these nanoparticles are also
surveyed. Three types of approaches are discussed based on cross-linking, non-crosslinking, as
well as unmodified nanoparticles. Figure 1 summarizes the properties and functionalization of
inorganic nanoparticles, as well as their use in colorimetric techniques.
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Fig.1. Properties and functionalization of inorganic nanoparticles in colorimetric assay.
2- Au NPs: Synthesis, Functionalization, Properties and Application
The current method of synthesis for metal NPs relies on the reduction of cations. This leads to
nanostructures with the ability to tune the size and shape. In chemical synthesis methods, ions of
gold in a salt are reduced to form AuNPs. Reduction of gold salts is achieved by using reducing
agents such as citrate [27, 29-33], ascorbate [32, 34], borohydride [34] or amines [35]. In these
methods, the use of stabilizers is necessary to prevent aggregation of AuNPs. Among various
stabilizing agents, citrate [27], and alkanethiols [36] are considered to be all-purpose agents.
Since AuNPs with different sizes and shapes have diverse optical and electrical properties, size
control is critical to obtain particles with uniform characteristics. To this end, changing the pH
and the ratio of chemical reagents [37] or using physical parameters in the synthetic procedure
(e.g. temperature [35], microwaves [36], or UV [38] irradiation) have been deployed to control
the size and shape. Recently, Alkilany and co-workers used grafted polyethylene glycol-g-
polyvinyl alcohol as a reducing, capping and stabilizing agent combined in a one-step synthesis
of AuNPs. The results of this study demonstrated that the spherical NPs produced by this method
were highly stable and monodisperse with tunable size between 23-79 nm [39].
Another important method for the preparation of metal NPs is electrochemical synthesis. During
this procedure, the electrolysis of an appropriate salt of the appropriate metal (usually HAuCl
4
for AuNP synthesis) in an electrochemical cell, leads to electro-reduction of cations on the
cathode. In contrast to chemical synthesis, this method is suggested to have a better ability to
control size, shape, and purity. Despite these advantages, the method suffers from two
drawbacks: deposition of metal NPs on the cathode and accumulation of NPs around the
proximity of the cathode. These problems however, have been solved by using stabilizers with
polyfunctional groups and a rotating cathode, respectively [40].
One example of the physical synthesis of NPs is laser ablation in liquid media. AuNPs can be
prepared by exposure of a gold target to pulsed laser ablation in water or an aqueous solution of
an acid, base or salt. In this method, laser irradiation can result in fusion of the produced Au NPs.
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Because of the dependence of particle size on Zeta potential, the size of AuNPs can be adjusted
using various surfactants [41].
Chemical and electrochemical synthesis methods both require and produce toxic chemicals.
Physical methods, also may have high costs and a low rate of product formation [42]. To
overcome these drawbacks, researchers have exploited biological systems for the synthesis of
metal NPs in a so-called “green approach” [43]. In green approaches, plant leaf extracts have
often been employed as reducing and stabilizing agents, for the synthesis of AuNPs with a
variety of sizes and shapes [42-44].
The surface chemistry of NPs plays a critical role in their interactions. Hence, metal NP surfaces
are usually modified and functionalized according to their intended usage. The general goals of
functionalization include improvement of in vivo stability[45], prevention of aggregation, and
avoidance of uptake by the reticuloendothelial system [45, 46], control of toxicity [47], and
optimization for clinical diagnostic and targeting applications [48, 49]. Chemical or biological
agents can attach to AuNPs by electrostatic adsorption or by chemical reactions [45-52]. Because
the surface of AuNPs is negatively charged, positively charged agents such as cysteine and β-
amyloid peptides in acidic pH, would be adsorbed by AuNPs [46]. Thiol groups are known to
form chemical bonds with gold atoms. Therefore different chemical and biological molecules are
often linked to AuNPs using thiol groups as a mediator or linker [45, 50, 52].
The ligand exchange reaction is a potential process to functionalize metal NPs with thiolated
molecules [48, 51]. For instance, NPs coated with thiol-terminated polyethylene glycol (PEG)
possess high stability and long blood circulation times [45]. Thiolated DNA and glucose
modified AuNPs have been used to design sensing systems [50, 52]. Kloper et al. [47] studied
the effects of the type of modifier ligand and its charge on the toxicity of AuNPs. They reported
that small AuNPs modified with cationic ligands displayed more acute toxicity when compared
with NPs modified with negatively charged ligands.
Like all nanostructures, AuNPs have a high surface area to volume ratio. As a matter of fact, the
most important feature of AuNPs is their size-tunable optical properties. In AuNPs, size
confinement results in different behavior from the bulk form. When exposed to appropriate
wavelengths of electromagnetic radiation, AuNPs display the surface plasmon resonance (SPR)
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phenomenon. SPR is the collective and coherent resonance of free electrons caused by energy
absorption at a distinct wavelength of light, This wavelength depends on the particle size. This
effect generates extreme electromagnetic fields at the surface of the NP and enhances the
absorption and scattering of incident radiation. Spherical AuNPs with a diameter of 20 nm show
an absorption peak at about 520 nm. As the size increases, it leads to the shift of the absorption
peak to longer wavelengths, so the color of AuNPs changes from red to blue. Nanorods possess
two absorption peaks according to their length and diameter. While the weaker peak in the
visible region is not affected by size, the sharper peak in the near infrared (NIR) region shifts to
longer wavelengths, with increase in aspect ratio. The aspect ratio of Au nanorods prepared by a
seed-mediated synthesis method is usually controlled by the concentration of Ag ions [26, 53-
55].
Due to the local SPR characteristic of AuNPs, Raman scattering by these particles can be
magnified up to 12 orders of magnitude. This is called “surface enhanced Raman scattering”
(SERS) [56]. AuNPs with an average size of 60-70 nm show highly efficient SERS with periodic
light emission on the millisecond to second time scale [57]. The high light to heat conversion
ability of AuNPs, which results from excited electron-electron collisions, is a useful property of
AuNPs for applications in photothermal therapy [53]. AuNPs also can mediate redox and
electrocatalytic reactions for electrochemical applications [58, 59]. Whereas electron transfer
efficiency is more pronounced in smaller particles, particle size does not affect the optocatalytic
activity [55]. Electrostatic interactions between these nanostructures and target cells make them
potential candidates for killing pathogens and cancer cells [43, 54, 60]. Some studies have also
indicated that AuNPs can have antioxidant activity [44, 54].
AuNPs have been used for diverse applications from diagnosis to therapy. The relative ease of
synthesis and their biocompatibility make them excellent candidates for biological research and
clinical applications. Since the properties of AuNPs depend on their size and shape, it is
necessary to choose the appropriate size NPs for each specific application. For example, since
the scattering to absorption ratio increases for larger NPs, these larger particles are more suitable
for detection, while smaller particles are preferable for photothermal therapy. Additionally, in
comparison to gold nanospheres, nanorods show longer blood duration times and may have
higher cell affinity [53].
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3- Colorimetric Detection Based on AuNPs
In recent years, the development of novel colorimetric techniques has attracted considerable
attention thanks to the developments of nanotechnology. Recently, AuNP-based colorimetric
biosensing assays have been significantly employed in various applications due to their
simplicity and versatility [61]. Because of their ability to be biologically functionalized,
biological compatibility, and spectral properties, AuNPs have been used in colorimetric assay
techniques [62, 63]
Functionalization is necessary for the stability, performance, and biocompatibility of AuNPs.
Furthermore, functionalization is also needed to preserve the AuNPs as well to maintain the
properties of surrounding biological molecules. Similarly, the AuNPs must also be stabilized to
maintain their unique properties such as surface plasmon resonance and light dispersion [64, 65].
The color change of a suspension of NPs occurs when the NPs are converted from a single
dispersed suspension to an aggregated state in response to the presence of specific analytes [21,
66-68]. Usually, the maximum absorption peak of 20 nm AuNPs is approximately 520 nm
resulting from the SPR, and this is responsible for their red color. The aggregation of AuNPs is
responsible for a color change of the colloidal solution from red to blue, and the re-dispersion of
the aggregated NPs reverses the color change from blue to red [69-71]. When AuNPs aggregate,
they undergo a plasmon-plasmon interaction. This causes a color change of the Au colloidal
solution from red to blue, and leads to shifting of the SPR towards longer wavelengths, up to 620
nm. The exact process of aggregation has been discussed in detail [72, 73]. Moreover, assays
based on AuNPs can also be monitored using UV-vis spectrophotometry [74]. In recent years,
diagnostic and detection methods for analytes at trace concentrations, based on novel
electrochemical systems[75], specifically AuNPs, have been widely applied as colorimetric
sensors for the detection of DNA [74, 76], RNA [74, 77, 78], proteins [71, 79], ions [80],
enzymes [81], pathogens [82, 83], various cancers [84, 85], parasites [86], food and water
pollution [87, 88], hydrogen peroxide [89], and single nucleotide polymorphisms [90]. These
colorimetric techniques based on AuNPs are quick and easy to perform. They also have many
advantages including low cost and the ability to detect color changes with the naked eye [91-93].
In colorimetric detection techniques based on AuNPs, both modified and unmodified NPs can be
used. Modified nanoparticles, are functionalized via connection to various biological groups,
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including nucleic acids fitted with thiol groups, which have strong binding affinity to the gold
surface. Nanoparticles without functionalized surfaces can be also used in colorimetric technique
as unmodified nanoparticles. These two techniques are described in the following sections.
3-1- Colorimetric Detection Based on Modified AuNPs
AuNPs can functionalized by various molecules including nucleic acids (DNA, RNA, and
aptamers), peptides, proteins, carbohydrates, antibodies, peptide nucleic acids (PNA) etc. Several
articles have been recently published describing colorimetric methods based on AuNPs
functionalized with different types of molecules to detect pathogens [94] and ions [95].
In this section, we summarize different types of pathogens, cancer biomarkers, enzymes, ions,
and environmental pollutants that have been detected with functionalized AuNPs. AuNPs can be
functionalized with oligonucleotides; this attachment can be performed with a head-to-head, tail-
to-tail, or head-to-tail arrangement of hybridization [96, 97]. Moreover, based on the
accumulation or dispersion of the NPs, colorimetric techniques can be subdivided into two types,
which are “cross-linking” and “non-cross-linking” approaches.
3-1-1- Cross-Linking Approach
Cross-linking or inter-particle bond formation leads to a development of a polymeric network
which results in inter-particle attachment [98]. Such connections can be formed by chemical
cross-linker molecules, by direct linking between antibodies and antigens [99], via aptamer
interactions with target molecules [100], or by the interaction between streptavidin and biotin
[101], and so on [102].
In addition, cross-linking connections can be formed by formation of Au-S or Ag-S bonds, in
which two single-stranded DNAs (ssDNA) probes are attached to the surface of the NPs and
create a polymeric network. Consequently, the color of solutions containing AuNPs changes
from red to blue when the target molecule binds to the probe upon salt addition [103].
The ssDNA and double-stranded DNA (dsDNA) sequences have different electrostatic
properties. The basic difference is that while ssDNA can uncoil its spirals, which can be then
attached to AuNPs, dsDNA molecules have a stable double helix structure which always displays
a negative charge related to the phosphate groups on the exterior. Thus, a strong repulsion
between the negative charges of dsDNA and AuNPs occurs; this attracts negative citrate ions and
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inhibits the absorption of nanoparticles by dsDNA. On the other hand ssDNA has sequence
flexibility; so after the spiral opens up, the bases can bind to the AuNPs and the resulting AuNPs
will be stable [104].
Oligonucleotides conjugated to AuNPs were used to detect DNA for the first time in 1996 by
Mirikin et al [105]. In this study, two complementary thiolated probes were attached to AuNPs to
detect the target by hydridization. The authors showed that this system created a polymeric
network based on cross-linked target binding. They also observed a change in the color. In order
to detect pathogens and viruses, functionalized AuNPs can be used with two complementary
probes. Many nucleic acid-based targets such as RNA, genomic DNA, or amplified nucleic acid
sequences can also be used in comparable approaches.
Storhoff et al. used a “spot-and-read” colorimetric detection assay for the detection of genomic
DNA with high sensitivity. This technique can be easily implemented in a biological laboratory
by placing hybrid NPs functionalized with a nucleic acid probe and mixed with genomic DNA
on a glass surface that created chromatic dispersion. This method enables the detection of trace
amounts of DNA down to the zeptomole level (10(-21) mol). This method was used to detect
Staphylococcus aureus based on the mecA gene sequence [106]. In other studies, AuNP based
colorimetric techniques have been used for pathogen detection[107]. To be more precise, a
fluorescent dye can be used as a sensitizer for bioassay detection of the pathogens along with
conjugation of AuNPs to the DNA or mRNA strands. In this system, a light beam introduced into
the microfluidic device excites the AuNPs. The metallic NPs can provide signal amplification in
smart-phone based microfluidic devices as well. Majdinasab et al. developed a novel
colorimetric detection method to identify a food pathogen, Salmonella typhimurium, using two
thiol probes for the invA gene with 20 nm AuNPs. In this study, both genomic and amplified
DNA sequences were used. The results demonstrated that the efficiency and sensitivity of
detection of the AuNP-based assay was relatively low when non-amplified target DNA was used,
as compared to Polymerase Chain Reaction (PCR) amplified DNA samples [108]. In a similar
study, several pathovars of Pseudomonas syringae bacteria (the causal agent of different
agricultural diseases) were detected using two thiol probes attached to AuNPs via cross-linked
hybridization. The detection method was based on amplified DNA.
In recent years, AuNPs have been widely used to detect pathogenic viruses. Viruses can have
either RNA genomes or DNA genomes, and both types can be detected by this approach. DNA
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viruses include human papillomavirus type 16 (HPV-16), and type 18 (HPV-18). They are major
causes of cervical cancer in women. Chen et al. established a colorimetric technique based on the
immobilization of two thiol probes on the surface of 13 nm AuNPs. In this nanobiosensor, when
amplified genomic DNA and probe sequences were mixed, color variations could not be
observed because of the too wide gap between the probes attached to the nanoparticles and the
target DNA [109]. Influenza virus A (IAV) is one RNA virus composed of single-stranded RNA
(ssRNA) and two membrane proteins HA and NA. Several colorimetric techniques were
developed based on AuNPs functionalized with monoclonal antibodies (mAb) that recognized
these proteins. Liu et al. showed that hybridization mAb-AuNPs with hemagglutinin type A
(HA) changed the color of NPs and their absorption spectrum. These authors used also
transmission electron microscopy (TEM) and also dynamic light scattering (DLS) to verify the
coagulation and sedimentation of the NPs [110] .
Bacteriophage or simply “phage” is another virus that attacks bacteria and kills them. Lesniewski
et al. proposed the use of AuNPs functionalized with antibodies through covalent binding, for the
detection of phage T7. In this colorimetric approach, in presence of T7 virions, they formed an
immunological complex with the antibody modified AuNPs which caused them to aggregate and
color changed from red to purple. They used M13 phage as a negative control. It was also shown
that this detection technique was capable of detecting all variants of phage T7, and not only those
that were biologically active, as would be the case for a conventional biological plaque assay.
They showed a detection limit of 1.08 × 10
10
PFU/mL (18 pM) T7 [111] (Fig. 2A).
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Fig. 2 Schematic illustration of crosslinking based colorimetric assays. (A) The detection of
bacteriophage T7 using AuNPs modified with covalently bonded anti-T7 antibodies and color
change based on antibody antigen interaction which causes them to aggregate [111]. (B)
Colorimetric detection of Hg
2+
based on chelation reaction between Hg
2+
and chitosan and
observed color changes in AuNPs [112]. (C) LCR amplification and colorimetric assay of CpG
methylation in DNA: In the presence of methylated genomic DNA a red to purple color change
can be detected [113]. (D) Colorimetric assay for detection of protein kinase activities based on
hybridization between STV-AuNPs and biotinylated peptide (biotin-LRRASLG), and the PKA
catalyzed phosphorylation of biotin-peptide prevented AuNPs crosslinking and the monodisperse
AuNPs remained red [81]. Reprinted with Permission
The incidence of cancer has increased exponentially in recent years, so rapid and accurate
diagnosis followed by timely treatment has become necessary. Today, nanobiosensors based on
AuNPs have been widely used for the diagnosis of cancer and malignant cells. In order to detect
cancer, NPs modified with aptamers that recognized 7-MCF cells (human breast cancer cell
lines) were described by Sadat Borgheia et al. [84]. These authors used aptamers in combination
with other AuNPs to enhance the detection of cancer cells. In their method, two complementary
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thiolated ss-DNA probes were attached to AuNPs. In the presence of 7-MCF cells, binding
occurred between soluble nucleolin aptamers (AS 1411) and nucleolin receptors that are over-
expressed in cancer cells. As a result, binding between AS 1411 and cancer cells resulted in the
removal of all aptamers from the solution. Due to the lack of aptamers in the environment no
binding occurred to the ssDNA-AuNP probes, and the color of the solution did not change.
Colorimetric techniques have been developed in order to detect contamination of food and water
[87, 88], factors involved in plant diseases [114], and presence of heavy metals ions [112, 115-
117]. For instance, it is known that pollution due to heavy metal ions, especially Pb
2+
, represents
a threat to the environment and human health. In a new study, Chai et al. [115] demonstrated that
glutathione functionalized AuNPs (GSH-AuNPs) could be exploited to develop a colorimetric
sensor for Pb
2+
detection. In this method, the presence of Pb
2+
ions and the binding to chelating
ligands on the surface of NPs, led to changes in color and wavelength. A similar study was
conducted by Zhang et al. [116] to detect the metal ions Cd
2+
, Ni
2+
and Co
2+
using NPs
functionalized with a probe peptide (P-AuNPs) instead of chelating ligands with comparable
results. Chen et al. [112] used NPs functionalized with chitosan to detect Hg
2+
. This technique
approach is illustrated in Fig. 2B. Another study based on cross-linked functionalized NPs with
MMT-AuNPs (5-mercaptomethyltetrazole) was performed by Xui et al. for detection of Al
3+
ions
[117]. In all these studies listed for the recognition of metal ions, the difference in the type of
cross-linker and the binding molecules on the surface of AuNPs may affect the time of detection.
Another application of colorimetric technique is concerned with the detection of mutations such
as single nucleotide polymorphisms [90], as well as detecting CpG methylation in genomic DNA
[113]. The methylation of DNA is considered to be an indicator of DNA epigenetic modification,
and occurs in a wide range of organisms ranging from eukaryotes to prokaryotes. Methylation
plays a key role in the regulation of gene transcription in embryonal development, and is
implicated in different diseases. A ligase chain reaction (LCR)-based colorimetric method to
detect CpG methylation was developed by Su et al. [113] who used four probes, A, B, A′, and B′.
In the presence of only 0.01 fM methylated DNA, probes A and B were joined together by
binding to methylated DNA in order to form the AB DNA strand that was amplified by the LCR
technique. Next probes A′ and B′ became connected through the DNA strand AB. Under
denaturing and annealing temperature cycles, the DNA strand, AB and the methylated DNA
hybridized thus connecting probe A′/B′ with probe A/B. As a result, A′B′ ssDNA was hybridized
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with DNA strands a and b immobilized on 13 nm AuNPs, Subsequently aggregation and change
in color was observed as shown in Fig. 2C.
Colorimetric technique based on cross-linking hybridization has also been used for the detection
of proteins and enzymatic activity. For example, AuNPs functionalized with peptides have been
used by Wei et al. in order to detect proteins [118]. In another study, Sun et al. detected protein
kinase activity using a colorimetric technique based on NPs functionalized with streptavidin
(STV-GNPs). It involved binding between STV-AuNPs, and a biotinylated peptide (biotin-
LRRASLG), which acts as a specific substrate for the enzyme protein kinase A (PKA). In the
presence of the enzymatic activity of PKA, the biotinylated peptide was destroyed thus
preventing the crosslinking of STV-GNPs and the color of AuNPs remained red [81] (Fig. 2D).
Table 1 summarizes studies where cross-linking colorimetric detection was used to identify
pathogens, viruses, cancers, metal ions, proteins, etc.
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Table 1. AuNP-based colorimetric detection using interparticle-crosslinking mediated aggregation
Ref Size of
AuNPs
LOD Linear Range Functionalizatio
n
Shape of
AuNPs
Target of
Nucleic Acids
Examples Target
]119[ 13.7 ±0.8
nm
1 pmol 20-0.5 pmol Thiol DNA-
probe
NR Amplified target
DNA.
Mycobacterium
tuberculosis (MTB) and
Mycobacterium
tuberculosis complex
(MTBC)
Pathogens
]120[ 13 nm 10
8
CFU/mL
−1
2.91×10
8
-
16×10
8
CFU/mL
−1
MEA-AuNPs Spherical NR
E,coli O157:H7
]69[ 13 nm 15 ng/L 200-2 ng/l Thiol DNA-
probe
Spherical Amplified target
DNA.
Pseudomonas syringae
]108[ 20 nm 21.78 ng/L NR Thiol DNA-
probe
Spherical Amplified target
DNA.
Salmonella
Typhimurium
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]92[ 13 nm 10 CFU/mL
-1
NR Thiol DNA-
probe
Spherical Amplified target
DNA
Helicobacter pylori
]110[ 13 nm 7.8 HAU NR mAb-AuNPs Spherical Viral RNA Influenza A
Viruses
]114[ 19 nm 7.2 ng NR Thiol DNA-
probe
Spherical Amplified DNA
target
Tomato
leaf curl New Delhi
virus (TolCNDV)
]109[ 13 nm 1.4×10
-4
M 1.4×10
0
-1.4×10
−5
µM Thiol DNAprobe Spherical amplified DNA
target
papillomavirus type 16
and type 18
]63[ 20, 50 and
100 nm
1000 cell 0-40000 cell Ap-AuNPs Spherical -
Cancerous Cells
Cancer
]84[ 25 nm 10 cell 10
1
-10
5
cell ssDNA-AuNP
probes
Spherical -
MCF-7
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]121[ 14 nm
length and
18 nm
width
100 cells/mL NR AuNPs-
monoclonal anti-
HER2/c-erb-2
antibody
AuNPs- RNA
aptamer
Oval-shape -
Breast Cancer Cells
]122[
NR
0.05 M Cd
2+
,
0.03 M Ni
2+
,
2M Co
2+
0-3 M Cd
2+
,
0-5 M Ni
2+
,
0-10 M Co
2+
peptide- AuNPs Spherical
- Cd
2+
, Ni
2 +
and Co
2 +
Metal Ions
]115[ 5-8 nm 100 nM NR GSH-AuNPs Spherical - pb
2+
]112[ 16 nm 1.35 M 9-50 M Chitosan-AuNPs Spherical - Hg
2+
]117[ 12 nm 0.53 M 1-10 M MMT-AuNPs Spherical - Al
3+
]123[ 13± 2 nm Hg
2+
=30 nM,
Melamin=80
nM
0.05-3 M Hg
2+
,
0.08-1.6 M Melamin
Cysteamine-
AuNPs (CA-
AuNPs)
Spherical
Melamin, Hg
2+
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]124[ 15 nm 0.2 µmol L
−1
0.1-2.0 µmol L
−1
Tryptophan-
AuNPs
Spherical
Mg
2+
]81[ 13 nm 0.0005-0.02 U
µL
−1
0.0005-0.05 U µL
−1
. STV-AuNPs Spherical
Protein kinase activity
Enzymes and
Protein
]118[ 13± 2nm 0.2 nM 0.2-10 nM p-AuNPs Spherical - Flt-1 protein
]88[ 20 nm 5-0.1 nM NR PEG –AuNPs,
Eutravidin-
AuNPs
Spherical - Botulinum neurotoxin
serotype A light chain
(BoLcA)
Others
]125[ 13 nm 0.2 M 3–10 M AE-AuNPs Spherical - H
2
S
]126[ 13 nm 11.6 nM 0.1–2 M ctDNA-AuNPs
assembly
Spherical -
Spermine
]127[ 10 nm 50 nM 50 nM-1 M Cysteamine
AuNPs
Spherical
Clenbuterol
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3-1-2- Non-Cross-Linking Approaches
Another type of NP-based colorimetric technique is based on non-crosslinking aggregation. This
technique is based on steric or electrosteric aggregation of nanoparticles including AuNPs and
AgNPs without creating cross-linked interparticle bonds (Fig. 3). Moreover, in this technique
(unlike the cross-linking technique where crosslinks are created between two binding moieties
attached to the NPs) a single target-binding probe attached to AuNPs is used. The outstanding
pioneer scientist in this field (Pedro Baptista) for the first time, developed an inexpensive and
easy-to-use assay which provided a visible change of color, blue shift, when the AuNPs bound to
trace trace amounts of analyte. To achieve this result, different salt concentrations were
investigated and tuned by the color changes which came from the aggregation in the presence of
a complementary target, and not with mismatched targets.[62, 128-130]. Bakthavathsalam et al.
[131] described a colorimetric technique based on thiol-functionalized NPs for the detection of
E.coli. The binding of the probe to AuNPs was investigated in the presence of E.coli genomic
DNA. The presence of genomic DNA ensures the nanoprobes remain stable and prevents them
from salt-induced aggregation. In the absence of genomic DNA, the nanoprobes are unstable and
the AuNPs aggregate. The authors investigated the attachment method using atomic force
microscopy (AFM), and the size of the AuNPs (Fig. 4A). Ultra-sensing detection of E. coli was
possible by conjugating specific antibodies against E. coli to the AuNPs for colorimetric-
biosensing. In this system, the conjugation was applied in a lateral flow assay (LFA) with a pre-
determined detection time. In addition, this capillary flow allowed the pathogen to increase the
agglomeration of the antibodies conjugated to AuNPs which led to a red color shift. In a similar
study by Castilho et al, a colorimetric technique based on non-cross-linked AuNPs
functionalized with a thiol nano probe was developed to detect Paracoccidioides brasiliensis (a
South American pathogenic fungus). In this study, DNA coding for the p27 antigen was used as
the target. In the presence of the target aggregation of NPs and a color change was prevented,
while in the absence of target aggregation and a color change occurred [132].
Colorimetric techniques based on non-cross-linking hybridization have been used for the initial
screening of cancer. An AuNP-based colorimetric technique based on non-cross-linked
aggregation and peptide substrate that could be phosphorylated by protein kinase (PKCα) was
developed to detect breast cancer [133]. In the presence of the peptide substrate, phosphorylation
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prevented the aggregation of the AuNPs. In the absence of phosphorylation aggregation and
changes in color were observed. It is known that in cancer cells there is an increase in the levels
of PKCα. In target breast cancer cells where phosphorylation of substrate peptide occurred, the
color red remained; while with normal cells where phosphorylation was absent, a red to purple
color changes was observed (Fig. 4B).
Single nucleotide polymorphisms (SNP) occur widely across the human genome, and are related
to the predisposition to several diseases, including cancers, diabetes, and allergies. Various
techniques have been developed in order to detect SNPs, including AuNP-based colorimetric
assays.
Wang et al. [134] used gold nano-spheres (AuNS), nanorods (AuNR) and nanostar-shaped
nanoparticles (AuNT) functionalized with ssDNA to detect genotype SNPs. They used ssDNA-
AuNRs with aspect ratios of 2, 3 and 4 and a different type of anisotropic gold core. In this
approach, fully matched dsDNA-modified anisotropic AuNPs aggregated after salt induction.
However, in the case of a single base mismatch at the DNA level, AuNPs did not aggregate upon
salt induction, and remained dispersed. The same study showed that ds-DNA AuNT aggregated
faster than AuNS and AuNR, suggesting that aggregation of nanoparticles based depended on
shape in addition to aspect ratio. In this investigation, individual SNPs were detected using only
a single Au-nanoprobe (Fig. 2C).
AuNP-nanoprobes can also be used for the detection of small molecules relevant to toxicological
investigations. In a recent study, a colorimetric assay based on non-cross-linked AuNP
aggregation was developed by Sharma et al. using NPs functionalized with antibodies for the
rapid detection of phenylurea herbicide. which is a harmful environmental contaminant. In the
presence of antigen (Diuron), the interactions between the antigen and the antibody-
functionalized AuNPs led to aggregation, and as the concentration of antigen increased, the color
gradually turned blue. Upon addition of increasing salt concentrations, changes in the surface of
the nanoparticles occurred and, and the nanoparticles aggregated. The conjugated hapten-protein
(DCPU-BSA) was used as a control. DCPU-BSA prevented the aggregation of anti-diuron Ab–
AuNPs and the color remained red. The detection limit was~5 ng/mL and the method had the
advantage of not requiring washing steps. (Fig. 2D).
One of the main differences between the cross-linking and non-cross-linking approaches, is that
the non-cross-linking mechanism does not require receptors and binding sites. The non-cross-
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linking approach is faster than the cross-linking approach, and relies on interactions between NPs
due to their surface charge changing in the presence of the target analyte [135].
Studies where non-crosslinking colorimetric detection has been used for the identification of
pathogens, viruses, fungi, cancer biomarkers, metal ions, etc. are summarized in Table 2.
Fig.3. A schematic illustration of non-crosslinking AuNP and AgNP aggregation-based
colorimetric detection
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Fig.4. Schematic illustration of the non-crosslinking based colorimetric assay. (A) colorimetric
detection of E. coli genomic DNA based on AuNPs probes; in presence of genomic DNA,
AuNPs probes do not aggregate and the color remains red; in contrast, in the absence of genomic
DNA, AuNPs probes lose stability and tend to aggregate [131]. (B) Non-crosslinking
aggregation-based colorimetric detection of cancers. This assay is based on the non-crosslinking
aggregation using a cationic PKC-specific peptide substrate [133]. (C) Colorimetric detection of
SNP based on DNA-functionalized anisotropic AuNPs and showing a mixture of AuNR AuNT
and the AuNS probe after mixing with the primer
solution and color changes of NPs [134]. (D)
Colorimetric detection of phenylurea herbicide, in the presence of antigen, color change is
visible upon aggregation of Ab-AuNPs [136]. Reprinted with permission
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Table 2. AuNP-based colorimetric detection based on non-crosslinking aggregation mechanism
Ref Size of
AuNPs
LOD Linear
Range
Functionalizatio
n
Shape of
AuNPs
Target of
nucleic acids
Examples
Types
]131[
20 nm 54 ng
215-27 ng
Thiolated DNA
probe
spherical
unamplified
genomic DNA
E. coli
Bacteria
]137[ 17-23 nm 5 CFUs
10
0
-
0.5×10
4
CFUs/mL
Thiolated probe-
AuNPs
_
Amplified by
NASBA
Salmonella
spp
]138[ 30 nm 10
3
CFU/g
NR mAb-AuNPs “Popcorn”
shaped mAb-AuNPs
Salmonella
typhimurium
DT104
]139[
15 nm
1×10
1
TCID5
0 units
1×10
1
-
1×10
6
TCID50
units
DDZ-AuNP - genomic RNA
Dengue virus
(DENV)
Viruses
]140[
10-20 nm
1 pg
total
RNA
100 pg–1
fg
Thiolated-TSV
oligonucleotide-
AuNPs
spherical
RT LAMP
Products
Shrimp Taura
syndrome
virus (TSV)
]133[ 20 nm 0.05
g/mL
0-0.2
g/mL
peptide substrate spherical
- Breast cancer
Cancer
biomarkers
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s
]141[ 12 nm 0.4 nM . 25 nM to
1
µ
M,
Ds-DNA spherical
-
Hg
2+
Metal Ions ]142[
∼
47nm
∼
9 nM
1-1000 nM DTT-AuNPs Anisotropic
AuNPs and
spherical
AuNPs
- Pb
2+
]143[ 23 nm 20
ng/L
-1
NR Thiolated probe-
AuNPs
spherical
complementary
DNA (cDNA)
Paracoccidioi
des
brasiliensis
Fungus
]134[ AuNS: 40 (d)
nm,
AuNR: 20
(d)×42 nm,
AuNR: 15
(d)×45 nm,
AuNR: 13
(d)×51 nm,
AuNT: 20
(th)×50nm
NR
NR
ssDNA-
nanoparticles
Nanospheres
nanorods,
nanotriangle
s
complementary
DNA
SNPs and
DNA
mutations
Others
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]144[
NR
<1 ppm
0.25-5 ppm 4-Amino
thiophenol-
AuNPs (4-ATP)
nanorods
- Nitrite
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3-2- Colorimetric Detection Based on Unmodified AuNPs
The unmodified AuNPs-based colorimetric technique utilizes nanoparticles with unmodified
surfaces. In these methods, changes made to the surface of the NPs by binding of the analyte
affects their electrostatic charge, leading to instability and aggregation of the NPs [145].
Attachment of single-stranded thiolated nucleic acids (RNA, DNA, and aptamers) to the AuNPs
results in stabilization [146-148]. It should be noted that binding of ssDNA leads to opening of
the coiled structure and exposure of the bases leads to their absorption on the surface of the
AuNPs, therefore contributing to their stability. dsDNA, however, because of the repulsion
between the negative phosphate charges and the surface of AuNPs, is unable to bind to the
surface of nanoparticles. In the case of nucleic acid targets, however, binding to the
complementary probes upon salt addition, results in accumulation of nanoparticles and a color
change from red to blue (Fig. 5).
Fig.5. Schematic of colorimetric assays based on unmodified AuNPs
In the past years, colorimetric techniques based on unmodified nanoparticles have been widely
used to detect pathogens, biomolecules, antibiotics and other drugs, metal ions, and enzyme
activities, because of their simplicity, speed, and accuracy.
The surface of NPs can have a positive or negative charge depending on the method of synthesis.
For example, the use of sodium citrate in the synthesis of AuNPs results in negative charges on
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their surface; whereas the use of hexadecyl trimethyl ammonium bromide (CTAB) [149], sodium
borohydride in the presence of cysteamine [150], HAuCl4, and polydiallyl-dimethyl ammonium
chloride (PDDA) create positive charges on the surface of AuNPs [151].
Pan et al. [152] used unmodified AuNPs for detecting apoptosis based on caspase-3 enzyme
activity. In this study, a specific peptide, Ac-Gly-Asp-Glu-Val-Asp-Cys-Cys-Arg-NH2
(GDEVDCCR, GR-8) was used as a substrate for the enzyme. The peptide had a negative charge
and was attached via its thiol groups to the AuNP surface to stabilize them. After GR-8 was
treated with caspase-3 a smaller positively charged peptide - Cys-Cys-Arg-NH2 (CCR, CR-3) -
was produced by the enzymatic cleavage of GR-8. When this positively charged peptide binds to
the AuNPs via thiol groups, the negative surface charge density of NPs is reduced which leads to
their aggregation.
In another study, Cao et al. [150] developed unmodified AuNPs with positively charged surfaces
synthesized by sodium borohydride and cysteamine to detect nuclease activity. In this study, the
reaction between polyanionic ssDNA and cationic AuNPs led to electrostatic binding and AuNP
aggregation and a color change. The activity of the nuclease enzyme, s1, produced smaller
ssDNA fragments, thereby preventing AuNP aggregation.
One of the most important applications of unmodified nanoparticle-based colorimetric assays is
the detection of bacteria and viruses. One example is provided by the report of Hussain et al.to
detect Mycobacterium tuberculosis infection [153]. These authors developed a system based on
unmodified AuNPs with an average size of 14 nm, and 21-mer oligonucleotide probes that
recognized the sequence of M. tuberculosis IS6110 DNA duplex. The ssDNA oligo-probes
underwent electrostatic binding to the AuNP surface and stabilized them. In the presence of the DNA
target, the ssDNA oligo-probe hybridized to its complementary DNA and upon addition of salt
aggregation occurred. In this study, the detection limit for the PCR products and genomic DNA
was about 1 and 40 ng DNA respectively.
Another study reported the detection of Maize chlorotic mottle virus, which causes a lethal
necrosis disease of maize. Liu et al. [154] extracted RNA virus from infected leaves and
amplified it by RT-PCR. In the existence of probes, RT-PCR target products, and salt induction,
color changes and aggregation of AuNPs were observed.
Biomarkers can be used to diagnose pathogens, detect various disease processes, or predict
susceptibility to a certain disease as a prognostic indicator for patients. Lee et al studied a
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biomarker for malaria. The authors used Plasmodium vivax lactate dehydrogenase (PvLDH) and
Plasmodium falciparum LDH (PfLDH) proteins, as well as pL1 aptamers as probes and AuNPs.
When PLDH proteins were present in human serum, binding occurred between aptamers and
protein. In the presence of the cationic detergent cetyltrimethyl ammonium bromide (CTAB) the
negatively charged AuNPs aggregated and a color change occurred. In the absence of the target
proteins, the unbound pL1 aptamers bound to the CTAB and the AuNPs remained dispersed. The
serum protein detection limit of PvLDH and PfLDH was 1.25 PM and 2.94 pM, respectively.
The authors proposed that this colorimetric aptasensor represented a rapid, highly sensitive, and
accurate biosensor for the diagnosis of PvLDH and PfLDH proteins [155].
Luo et al. used a thiolated aptamer as a probe for the detection of carcinoma embryonic antigen
(CEA), a tumor marker. Aptamers with a thiol group can attach to the surfaces of AuNPs. In the
presence of CAE that hybridizes with aptamers, the addition of NPs and salt, results in NP
aggregation. A shift in the spectrum from 520 to 650 nm was observed. They concluded that the
technique can be deployed to identify cancer biomarkers, given the high sensitivity and rapid
detection [146].
Drug testing is performed as a forensic procedure to check for possible abuse of drugs, and to
ensure patients comply with instructions and avoid addiction. For this reason, drug screening
tests are required to be highly specific and rigorous. Recently, these tests have been facilitated by
the introduction of AuNPs. Shi et al. [156] detected the presence of methylamphetamine
(METH) using unmodified AuNPs and a METH-specific aptamer in approximately 20 minutes
and with a sensitivity of 0.82 µM. The results indicated that in presence of METH in the urine,
the AuNPs aggregated upon salt addition, and a change in color from red to purple was observed.
The rapid detection of antibiotics and drugs is one of the benefits of colorimetric assays based on
unmodified NPs. For example, the antibiotic streptomycin was detected in milk and in animal
serum because of the presence of antibiotic residues in the animal feedstuffs. Using fluorescence
quenching aptasensors combined with unmodified NPs, the fluorescent dye (5’,6-fluorescein)
FAM-labeled double-stranded (dsDNA) and aptamers, Sarreshtehdar Emrani et al. [157] reported
that, in the presence of antibiotics and aptamers, FAM-labeled complementary strands were
attached to the surface of the NPs, leading to NP stability and no change in color. In the absence
of a sufficient concentration of antibiotics, the complex aptamer/FAM-labeled complementary
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strand dsDNA remained stable and AuNPs aggregation was observed. The antibiotic detection
limit in this technique was 73.1 and 47.6 nM using colorimetric and fluorescence readouts
respectively. In another study, Teetoet et al. [158] used AuNPs with an average size of 13-23 nm
to identify ramoplanin (an actinomycetes-derived antibiotic). After binding to the surface of the
NPs, aggregation and a color change was observed. This was due to the binding of antibiotic
amino groups to the AuNPs creating a positive charge and aggregation. The authors also
concluded that NPs of 13 nm in size were more effective compared to 23 nm NPs. This was
because the surface area of AuNPs increases with the decrease in particle size.
The advantages of unmodified AuNP assays include:
1. Addition of NPs after binding of probe to target has already occurred; the binding process
sometimes employs elevated temperatures. High temperature can lead to the aggregation
of NPs and to color changes, thus causing false positive or negative results.
2. No need to perform functionalization of the NP surface.
3. High rapidity with the ability to detect within minutes.
Studies on unmodified NP assays are summarized in Table 3.
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Table 3. AuNP-based colorimetric detection based on unmodified AuNPs
Target Examples Target of Nucleic
Acids
Shape of
AuNPs
Linear Range LOD Size of AuNPs Ref
Bacteria
Acinetobacter
baumannii
Amplified ITS
regions
Spherical 13–0.406 ng/l
-1
0.8125 ng/L 13±2 nm ]91[
Mycobacterium,
tuberculosis complex
PCR product and
genomic DNA
Spherical 14.1–0.44 ng PCR
product,
40–10 ng genomic
DNA
1 ng for PCR
product 40 ng for
genomic DNA
14±2 nm ]153[
Viruses
Maize chlorotic mottle
virus
RT-PCR target
products
Spherical 2.4 ng/L-1.875
pg/L
<30 pg/L of RNA 13±2 nm ]154[
Hepatitis C HCV RNA Spherical 25–2000 copies 50 copies reaction 15±2 nm ]159[
Cucumber green
mottle mosaic virus
(GMMV)
RT-PCR target
products
Spherical 120-3.375 pg/µL 30 pg/µL 13±2 nm ]160[
Biomarker
s
Plasmodium vivax
lactate dehydrogenase
- Spherical 0 pM–1 M 1.25 pM and
2.94 pM
NR ]155[
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(PvLDH) ,
Plasmodium
falciparum LDH
(PfLDH)
Carcinoma embryonic
antigen (CEA)
- Spherical 0-120 ng mL
_1
3 ng/mL 13 nm, ]146[
Proteins
and
Enzymes
VEGF protein - Spherical NR 185 pM - ]161[
α-fetoprotein (AFP) - Spherical 50-1000 pg/mL 33.45 pg/mL 13 nm ]162[
Tryptophan - Spherical 0.2-10 M 0.1M 13 nm ]163[
Thrombin - Spherical 1 pM-5 M
1 pM 15 nm ]151[
Protein kinase - Spherical 0-1 U/L 0.232 mU/L 13 nm ]164[
Caspase-3-activity - Spherical 0- 0.3 g mL
_1
0.01 and 0.005g/
mL
13±1 nm ]152[
α-Glucosidase activity
and α-Glucosidase
inhibitor
- Spherical 0.0025-0.05 U mL
-
1
0.001 U/mL 13nm ]165[
hepatomaupregulated - Spherical NR 2.4 nmol/L 15nm ]166[
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protein RNA
Drugs
Ramoplanin - Spherical 0.30-1.30 ppm 0.01 ppm 13nm and 23nm ]158[
Methomphetamin - Spherical 2-10 M 0.82M 13 nm ]156[
Steptomycin - Spherical 0-4000 nM 73.1 nM 15 nm ]157[
Metals
Ions
Hg
2+
Spherical NR 30 nM 13 nm ]167[
Cd
2+
- Spherical 0-50 M 5 M
∼
13 nm ]168[
Cu
2+
- Spherical 0.05-1.8 µmol.L
-1
30 nmol.L
-1
∼
13 NM ]169[
Melamine - Spherical 1 mg/L-8×10
-2
g/L 0.4 mg/L 13±1 nm ]170[
Cu
2+
- Spherical 0.5-10 µM 250 nM 13nm ]171[
Hg
2+
- Spherical 50-300 nM 15 nM 13 nm ]172[
Hg
2
- Spherical 0.5-10 nM 0.26 nM 15 nm ]173[
Others
Sulfide - Spherical 0-10 M 80 nM 8.1± 1.1 nm ]174[
Thiocyanate - Spherical 0-2 M 1 M 13 nm ]175[
Hypochlorite - Spherical NR 1.5M 13 nm ]176[
Urea - NR 20-150 mM 20 mM NR ]177[
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17β-estradiol - - 1-10
5
ng/mL 0.1 ng/mL 13 nm ]178[
Adenosine
triphosphate (ATP)
- Spherical 50-1000 nM <50 nM 13 nm ]179[
Adenosine
triphosphate (ATP)
- Spherical 0-5 µM 0.1 µM 14 nm ]180[
Pork Adulteration Genomic DNA Spherical NR
6 µg/mL
20 nm ]181[
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4- Silver nanoparticles (AgNPs): Synthesis, Functionalization, Properties, and
Application
Currently reported methods for the physical synthesis of AgNPs rely on two main approaches:
(a) evaporation-condensation and (b) laser ablation. In the evaporation-condensation method,
bulk silver is heated at a high temperature in a tube furnace. After evaporation, silver atoms
condense from the gas phase and form Ag clusters by rapid cooling. The spherical AgNPs
produced can be sorted according to their size and charge using a differential mobility analyzer
and, subsequently, a narrow particle size distribution is obtained [182]. The laser ablation of Ag
metal in an appropriate solvent results in formation of AgNPs with different sizes that can be
controlled by the type of the solvent used and by the concentration of surfactant employed [183,
184].
The chemical synthesis of colloidal AgNPs is commonly carried out using a combination of two
reductants and a stabilizing agent in a two-step process. Briefly, Ag salt is reduced by agents like
NaBH
4
and trisodium citrate [185, 186] or by sodium citrate and tannic acid [187]; citrate also
acts as a stabilizing agent. The size and shape of the particles are controlled by the reaction
conditions including the type and the concentration of chemical reagents, pH, and temperature.
For example, triangular NPs can be obtained using ascorbic acid as a mild reductant [186] and
Ag ions reduced on Au seeds can result in the synthesis of larger and more uniform AgNPs
[188]. Gamma radiation can be used to produce reactive species in solvents that act as reducing
agents to form stable colloidal Ag nanoclusters from Ag cations [189].
In the electrochemical method, electro-reduction occurs at the interface of the cathode and Ag
+
solution. This method has been accomplished without any stabilizer [190] and with a polymeric
stabilizer for the control of size and shape [191].
In recent years, green biosynthesis methods have attracted the attention of various workers in the
field. These methods are applied to produce AgNPs using plant extracts (e.g., aloe vera) both as
reducing and stabilizing agents [192-199]. Microorganisms like Bacillus koriensis can be utilized
for the same purpose [200, 201]. These methods are eco-friendly, inexpensive and suitable for
medical applications [45-54].
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The surface modification of AgNPs may impact their size, stability and function. Ligand
exchange is one functionalization method that is deployed for citrate stabilized AgNPs. Studies
have shown that the presence of long chain molecules on these NPs decreases their catalytic
activity, as a consequence of diffusion restriction [187, 191]. AgNPs have been functionalized
with different molecules such as carbohydrates, in order to modulate their cellular uptake and
control their toxicity to human and microbial cells [202-206]. Recent studies have shown that
AgNPs embedded in multi-walled carbon nanotubes (MWCNTs) show anticancer and
antibacterial properties [207]. AgNPS have been functionalized with a range of molecules to
allow specific and sensitive detection of various analytes. These molecules have included
macrocyclic polyammonium cations, bis-acridinium lucigenin, and ascorbic acid [208-210]. The
surface of AgNPs can be also functionalized for targeted drug delivery [211, 212].
Silver is considered to be the most conductive metal both thermally and electrically. Another
important property of AgNPs is their catalytic activity [179]. AgNPs can release Ag ions as
antimicrobial agents. At the nanoscale level, the antimicrobial effect is more impressive than the
bulk form.
All of these properties are influenced by the size and shape of AgNPs; the smaller the size, the
better the activity [187, 193-195]. Similar to AuNPs, AgNPs also have interesting optical
properties. Compared to AuNPs, the SPR effect in AgNPs projects further from the surface, and
is tunable over a broader range of wavelengths (350-450 nm). Larger AgNPs show effective
SERS properties and are excellent candidates for the detection of single molecules [186, 188,
213].
AgNPs have been used in different fields including catalysis, sensing and optoelectronics,
although the most common application still relates to their use as antibacterial, antiviral and
antifungal agents [184, 185, 189, 213]. As mentioned above, it is necessary to choose NPs of
appropriate shapes and sizes for the intended applications.
Table 4 compares the applications of both AuNPs and AgNPs in biology and medicine.
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Table 4. Applications of AuNPs and AgNPs
GNP/AgNP
Shape/Size (nm)
Scientific base Application Ref.
Au Nano sphere
13 nm
SPR DNA methylation detection [214]
Au Nano rod
Aspect Ratio(AR) = 4
SPR Drug delivery [215]
Au Nano rod SPR Photothermal therapy [216, 217]
Au Nano sphere
40,60,100 nm
SERS Embryonic stem cell differentiation imaging [218]
Au Nano sphere
100 nm
Fluorescence quenching DNA sensing [219]
Au Nano sphere
15-100 nm
Electrochemical (voltammetry) Hydroquinone detection, amitrole sensing [58, 220]
Au Nano rod
A.R.= 3.9
SPR, SERS Cancer cell imaging, photothermal therapy [221]
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Au Nano sphere
35 nm
SPR, SERS Oral cancer detection and sensing [222]
Au Nano sphere
12 nm
Scattering and fluorescence Cancer imaging [223]
Au Nano sphere
47 nm
SPR, scattering and fluorescence Tumor cell imaging, photothermal therapy [224]
Au Nano rod
AR = 3.5
Photoacoustic, SERS Ovarian cancer detection [225]
Au Nano sphere
13-21 nm
Electrochemical, SPR Cholesterol sensing [55]
Au Nano sphere
10-20 nm
Electrostatic interaction with pathogen cell,
Immune response activation
Antimicrobial activity [60]
Au Nano sphere Electrostatic interaction with target cell Anticancer activity, drug delivery [226]
Ag Nano sphere
5-192 nm
Interaction with pathogen cell membrane Antimicrobial and antifungal
[185, 189, 193-
195, 197-199,
201, 203-205]
Ag Nano gate Ag binding to thiol of GSH, fluorescence
quenching
Targeted intracellular controlled drug delivery
and tumor therapy, delivery monitoring [211]
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Ag Nano sphere, triangular SPR Ascorbic acid quantitative detection [186]
Ag Nano sphere
2.5-28 nm
SPR Phosphate, glutathione, Cu
2+
assay [208, 210, 213]
Ag Nano sphere
10-200 nm
Electron transfer Catalyst [187]
Ag Nano particle SERS Organochlorine pesticide, endosulfan sensing [209]
Ag Nano sphere
60 nm
SERS, cell penetration Sensing, therapeutics [212]
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5-Colorimetric Detection Based on AgNPs
As mentioned above, AuNPs and AgNPs, both noble metal NPs, demonstrate LSPR (local SPR),
which is the basis of label free colorimetric assays. When these NPs are exposed to light, their
free electrons absorb energy at appropriate wavelengths producing high extinction coefficients.
The smaller NPs show absorption peaks at shorter wavelengths; the absorption peak shifts to
longer wavelengths with the increase in the size of NPs. The aggregation/dispersion of these NPs
also has a similar effect to that seen with AuNPs. Researchers exploit this phenomenon for
chemical and biochemical analyses when a fast response is required. The interaction between
functionalized AgNPs and target molecules causes NPs aggregation or dispersion that results in a
change in the color of the solution. This color change can be also detected by eye (Fig. 6). The
linear relationship between absorbance and concentration is applied to quantify analytes. This
method is very simple, sensitive, selective, specific and inexpensive [227-230].
Fig. 6 Aggregation and color change of modified AgNPs in the presence of lead ions [231].
Reprinted with permission
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The change in expression levels of specific genes might represent a disease hallmark, especially
in the cancer setting. Detection of mRNAs and proteins in the early stages of disease is crucial
for rapid diagnosis and treatment. Li et al. detected C-Myc mRNA using PNA oligomer
functionalized AgNPs at nanomolar concentration with single base mismatch resolution [228].
Trypsin is a protease, produced from the pancreas, and changes in its concentration suggest
abnormal pancreatic activity. Han and co-workers have used negatively charged AgNPs for the
quantitative determination of this enzyme. Appropriate peptide chains were immobilized on the
NP surface acting both as a stabilizer and as a substrate for trypsin. When exposed to trypsin,
peptide chains are cleaved so that positively charged fragments remain on the AgNP surface.
Thus, the neutralization of the native negative surface charge decreases the electrostatic
repulsion, with consequent NP aggregation. The resultant absorption changes provide the basis
for quantitative analysis of trypsin over the range of 2.5-200 ng/mL
and a detection limit of 2
ng/mL
[232].
DNA modified AgNPs have been used for protein sensing. Transcription factors are proteins that
bind to specific DNA sequences. Researchers at National University of Singapore modified the
surfaces of two sets of AgNPs with dsDNA containing single stranded complementary
sequences. These sequences also displayed good affinity for estrogen receptor α. The interaction
between these DNA sequences, under certain salt concentrations, induced NP aggregation and a
shift in the color of solution towards blue. Because of the high affinity, protein-DNA binding
prevented NP aggregation by stabilizing transient structures via steric protection forces. The
resulting accuracy was better than that achieved with colorimetric sensors based on analyte-
induced aggregation mechanism [233].
Drug determination in clinical samples is an important task in analytical chemistry. Because of
the affinity of sulfur atoms to Ag, unmodified AgNPs have been utilized for the detection of two
different thioamide drugs. The anti-thyroid drugs, propylthiouracil and methimazole were
analyzed based on covalent bond formation with AgNPs, causing aggregation and a decrease in
absorbance at 400 nm. The assay response was linear in the range of 0.02-0.8 µg/mL for
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propylthiouracil and of 0.05-1.2 µg/mL for methimazole with corresponding LODs of 0.007
µg/mL and 0.01 µg/mL respectively [234].
Enzyme immobilization on AgNPs allows the design of nanosensors for the colorimetric
determination of appropriate substrates [235]. Coralyne (a small molecule drug that intercalates
into DNA) was quantitatively analyzed using homo-polyadenine adsorbed onto AgNPs in the
range of 0-10 M. In the presence of coralyne, the polyadenine chains separated from the surface
of NPs to form a duplex. Consequently, the NPs aggregated and the A550/A397 (absorbance at
550nm/absorbance at 397nm) changed according to the coralyne concentration [236].
A colorimetric assay based on green synthesized AgNPs was used for the determination of
ammonia in biological samples. This sensitive, selective, simple, and rapid response method
showed good potential for ammonia detection at low concentrations for medical applications
[237].
Anisotropic triangular AgNPs have been applied for the colorimetric detection of ascorbic acid at
M levels[238]. The surface modification of AgNPs with pH sensitive or thermosensitive
chemicals can be used in order to detect the acidity or the temperature of solutions. For instance,
Korean researchers utilized cytochrome c (Cyt c)-modified AgNPs for the determination of pH
within a broad range (3-11). At an acidic pH, Cyt c undergoes conformational changes, inducing
aggregation of NPs, and a consequent shift in the color of the solution towards blue [239].
Environmental protection is one of the most critical challenges present in the modern era. In this
regard, detection of hazardous chemicals is of paramount importance. Table 5 summarizes the
detection of several analytes relevant for biology and health using AgNP-based colorimetric
assays.
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Table 5. Colorimetric assays based on AgNPs
AgNP Surface modification or
aggregation/dispersion agent
Analyte Linear range LOD Ref.
Lysine Hg(II) 1 nM-30 M 1 nM [227]
Gelatin Hg(II) 0.5-800 nM 0.125 nM [240]
Starch Hg(II) 10 ppb - 1 ppm - [241]
Iminodiacetic
Acid
Pb(II) 0.4-8 M 13 nM [231]
GSH Pb(II) 0.5-4 M 0.5 M [242]
GSH Pb(II) 10
-
9
-10
-
3
M 10
-
9
M [243]
- Cr (VI) 10
-
9
-10
-
3
M 1 nM [244]
Isonicotinic acid hydrazide Cr(III) 10
-
6
-5×10
-
5
M 4.5×10
-
7
M [245]
N-acetyl-L-cysteine Ni(II) 2-48 M 0.23 M [246]
Alizarin Red S Al
3+
1-5.3 M 0.12 M [247]
Dextrin Cu(II) 50–200M 50 M [213]
Sucrose Endrin 0.05–5.00g mL
-
1
0.015 g/mL [248]
- Melamine 0.002-0.25mM 2.32 M [249]
Sulfanilic acid Melamine 0.1-3.1 µM 10.6 nM [250]
Melamine Cyanuric acid 1) 1.0–6.5 mg/L
2) 1-25 mg/L
1) 0.6 mg/L
2) 0.25 mg/L
[251]
SDS Cyanide 16.7–133.3 M 1.8 M [252]
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Chitosan Thiocyanate 1-14 ppm 1 ppm [253]
p-aminobenzenesulfonic acid Pymetrozine 0.02 to 0.09 mg/L 0.01 mg/L [254]
- Enterobacter cloacae P99 b-lactamase 5-600 pM 5 pM [255]
Methylcellulose Aerosol oxidative activity 5-25 ng 10 ng [256]
Citrate Quaternary ammonium surfactants 10
–
7
-5×10
–
5
M < 5M [257]
ssDNA Biological thiol nM nM [258]
- Calf intestine alkaline phosphatase
Protein kinase A
- 1 unit/mL
0.022 unit/mL
[259]
Chitosan Glucose 5.0 ×10
-
6
-2.0 ×10
-
4
M
100 nM [260]
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An interesting colorimetric assay was developed based on the morphology transition of Ag
nanoprisms for sensing of Hg
2+
over a linear range from 10 to 500 nM with a LOD of 3.3 nM.
Ag nanoprisms capped with 1-dodecanethiol (C
12
H
25
SH), when exposed to Hg mixed with I
ions, lost the organic protective layer. The silver atoms reacted with I
-
ions to form AgI. These
interactions were accompanied by transformation of nanoprisms and a color change [261].
Glucose induced alteration of the nanoprism morphology in the presence of glucose oxidase, has
been used for a glucose assay in the linear range 2.0 × 10
-7
- 1.0 × 10
-4
M [262].
An electron transfer reaction between Ag atoms and Hg ions has been used for sensing mercury.
Upon this redox interaction and the conversion of AgNPs to a Ag-Hg nanoalloy, the color of
solution changed according to the Hg
2+
concentration [263, 264].
Various amino-acids and metabolites, involved in biological interactions, are chiral molecules
and are present as L or D enantiomers. Differentiation of enantiomers is important especially in
the drug manufacturing industry. L-cys and D-cys can be distinguished by a colorimetric assay
because of their different effects on aggregation/dispersion of nucleotide capped AgNPs [265].
Aromatic ortho-trihydroxy phenols, attached by hydrogen bonding to the surface of chitosan
modified AgNPs, were oxidized by reduction of Ag ions on the surface. The resulting changes in
UV-Vis absorption spectra of NPs were used for quantitative analysis of gallic acid, pyrogallol,
and tannic acid [266].
5-1-Colorimetric detection based on combinations of AuNPs and AgNPs
While the most important property of AuNPs is the ability for surface functionalization, AgNPs
show higher extinction coefficients. The combination of these two properties can lead to a
sensing system characterized by good selectivity and sensitivity. To prepare these hybrid NPs,
Ag@Au and Au@Ag core-shell nanostructures, as well as NPs made of Ag-Au alloys have been
developed [267-269]. Colorimetric assays based on these structures have been devised by using
the changes in UV-Vis spectra caused by aggregation/dispersion of NPs or by alteration of the
shell by etching. The interaction of Ag ions with organic molecules containing carbonyl groups
leads to Ag
+
reduction to silver atoms. Based on this fact, both Ag
+
and organic molecules
containing a carbonyl group have been detected by the formation of a Ag shell on the Au core
[267, 270, 271]. In the presence of thiol terminated hyper branched polyethylenimine (HPEI), the
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formation of a Ag shell onto the Au core induced a color change from red to brown to green.
This resulted in a significant improvement in the selectivity and sensitivity of the Ag ion assay.
The graph of absorbance vs. Ag
+
concentration was linear in the range of 8.76×10
-9
-1.27×10
-4
M
with a LOD of 8.76×10
-9
M by UV-Vis spectra and 8.76 × 10
−8
M by eye [270]. Formaldehyde
and glucose, due to their carbonyl group, reduce Ag
+
, this resulted in formation of a Ag shell
onto the AuNP core (Fig. 7). The resultant color change was proportional to the concentration of
these organic analytes [267, 271].
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Fig. 7 Color change induced by Ag shell formation on AuNP core. The shell thickens and the
resultant color change is proportional to the glucose concentration [271]. Reprinted with
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Anions and cations, in the presence of appropriate reagents, can etch or change the chemical
composition of the shell of mixed Au-AgNPs. Variations in thickness or texture according to the
concentration of analytes have been detected by colorimetry [269, 272-275].
Fig. 8 Shell etching according to the concentration of copper ions [275]. Reprinted with
permission
The oxidation of some biomolecules, such as glucose and cholesterol, catalyzed by specific
enzymes, is accompanied by the generation of hydrogen peroxide. H
2
O
2
oxidizes and etches the
Ag shell of the Au@AgNPs. The color change due to the shell etching can be used in the
colorimetric assay of H
2
O
2
, glucose, cholesterol and other H
2
O
2
producing biomolecules [276,
277].
In addition to core-shell NPs, AuAg-alloy nanoprobes have also been used in colorimetric
assays. In addition to the advantages of core-shell nanostructures, these NPs are easily
synthesized and show a single absorption band [268].
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Tamada and co-workers designed a colorimetric biosensor composed of triple-layered AgNPs
and AuNPs. AgNPs (5-nm diameter) were coated with myristates, followed by a SiO
2
coating,
and labeled with biotin. Next AuNPs were prepared and their surfaces were also coated with
SiO
2
and labeled with biotin. The hybrid AgNPs were attached to a gold substrate. In the
presence of streptavidin (model analyte), a sandwich interaction occurred between streptavidin
and biotin binding on both sides, leading to the adsorption of AuNPs on the top of the biosensor,
inducing color and absorption spectral changes [278]. A marked color change could be observed
at less than 30% surface coverage. The color change was attributed not only to the LSPR effect,
but also to the multiple light trapping effect derived from the stratified Au and Ag NPs, as
predicted by a finite-difference time-domain simulation. Table 6 summarizes some assays
mediated by these hybrid NPs.
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Table 6.Linear ranges and LODs of Ag-Au hybrid nanoprobes
NPs/modification Analyte Linear range LOD Detection base Ref.
Au@Ag core-shell HCOH 0.1–40 mM 50 nM Shell formation [267]
Au@Ag core-shell Glucose 0.04 - 1 mM 10 nM Shell formation [271]
Ag@Au core-shell Cyanide 0.4-32 µM 0.16 µM Shell etching [269]
Au@Ag core-shell Cyanide 0.4–100 mM 0.4 mM Shell etching [274]
Ag@Au core-shell Nitrite 1.0 - 20.0 µM 0.1 µM Shell etching [272]
Au@Agnanorod core-shell Cu2+ 3–1,000 nM 3 nM Shell etching [275]
Au@Ag core-shell Iodide 0.5-80 mM 0.5 mM Shell composition change [273]
DNA-embedded core–shell
Au@Ag
Glucose 0.00–0.20 and 1.00–
100 µM
10 nM Shell etching [276]
Au@Ag core-shell Glucose,
cholesterol
0.5 - 400 mM 0.3-300
mM
0.24 mM
0.15 mM
Shell etching [277]
Thiolated oligonucleotide
functionalized Ag@Au core-shell
Target DNA - - Aggregation [279]
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Au@Ag core-shell H
2
S 50 nM - 100 µM 50 nM Shell composition
changing
[280]
Ag-Au alloy NP Hg 0.02–100 ppb 0.01 ppb Shell composition
changing
[281]
Oligonucleotide functionalized Ag-
Au alloy NP
Target DNA - - Aggregation [268]
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Table 6 shows that colorimetric assays based on Au-Ag hybrid nanoprobes often possess
lower LODs compared with other colorimetric assays. These nanoprobes can be immobilized
on a solid matrix to construct portable and user-friendly sensors and chips [278, 281].
Jin and co-workers utilized poly L-histidine modified hybrid nanoshells for colorimetric
detection. They immobilized glucose oxidase enzyme on the surface of hollow Ag-Au
nanoshells. Within a range of glucose concentration the enzyme produced H
2
O
2
and the Ag
atoms dissolved leading to shell removal. Changes of absorbance, caused by the generation of
porosity, constituted the basis of this glucose analysis [282].
6- Magnetic Nanoparticles (MNPs): Synthesis, Functionalization, Properties and
Applications
In recent years, many studies have been conducted with the aim of developing magnetic
nanoparticles (MNPs) [283]. These MNPs have been widely used in various applications in
the context of biomedicine, biotechnology, engineering, and material sciences [284]. MNPs
can be synthesized using either chemical, physical or biological methods. Several of these
methods are summarized in Table 7 [285].
Table 7. Methods for the synthesis of MNPs [285]
Method
Physical Gas-phase deposition
Electron beam lithography
Chemical
Sol-gel synthesis
Oxidation Method
Chemical co-precipitation
Hydrothermal reaction
Flow injection synthesis
Electrochemical method
Aerosol/vapor phase method
Sonochemical
decomposition reactions
Supercritical fluid method
Synthesis using nanoreactors
Biological Microbial incubation
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The difficulty of controlling the particle size at the nano-scale is one of the main
disadvantages of physical methods. Chemical methods (wet chemistry) are simpler, more
flexible, and offer more effective control over size, composition, and in some cases, the shape
of the NPs [286] . In some chemical methods such as the sonochemical decomposition
method, the supercritical fluid method and the sol–gel method, the particle size can be well-
controlled by adjustment of the process parameters. Functionalization is one of the more
important processes that are performed on the MNPs.
MNPs have a high chemical reactivity and a large specific surface area which make them
sensitive to oxidation and agglomeration [283]. Oxidation of the MNPs occurs on the
surfaces, causing dramatic changes in their functional and structural properties.
Agglomeration of MNPs also hinders their processing [287]. Hence, protective methods are
normally used to preserve the NP-specific magnetic properties. Protection of MNPs can be
accomplished using different organic and inorganic materials such as carbon and metal
oxides [283].
Recently, protection methods that rely on the use of organic and inorganic coatings have been
developed. The most common organic coatings are surfactants (stearic acid, elaidic acid,
oleic acid, trioctylphosphonic acid and lauric acid etc.) and polymers (dextran, chitosan,
starch, arabic gum and gelatin etc.) [283]. Amongst inorganic coatings, metal oxides (cobalt,
titanium oxide and aluminum oxide), precious metals (gold and platinum), silica, and carbon
are the most commonly used [283].
After this step, functionalization can be performed according to the specific application of
MNPs. Usually, MNPs are required to be chemically stable, well dispersed in liquid media,
and uniform in size [287]. Intermolecular interactions such as electrostatic chemo-adsorption
and covalent conjugation are often used for the functionalization of MNPs [283]. Several
studies have reported the synthesis of iron oxide nanoparticles, coated with a silica shell that
can be subsequently functionalized via attachment to gold NPs[285]. Riva, et al used silica
coating to prevent MNPs aggregation in aqueous environment and biological media [288] .In
another study Muzio, et al. coated the MNPs by a silica shell, that were subsequently
functionalized with a multi-layered conjugated linoleic acid coating [289].
One technique which is widely used for surface functionalization is silanization, which
results in high stability in acidic conditions, inertness to redox reactions, and low
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cytotoxicity. Treccani et al used silane precursors to functionalize the surface of MNPs and
then successfully used them for protein immobilization (Figure 9) [290].
Fig. 9. (A) Silane molecular structures. (B) Schematic of the silane condensation reaction
[290]. Reprinted with permission
One of the methods most commonly deployed for MNP surface functionalization relies on
the use of thiol groups. Using this method, Huixia, et al successfully functionalized MNPs to
immobilize and separate bovine serum albumin (BSA) in solution [291]. Another type of
surface functionalization method that can be used for MNPs is based on the presence of
amine groups (Amine-Functionalized Core-Shell MNPs). Chen, et al demonstrated that by
conjugating silica to amine groups, fluorescein molecules can quickly and quantitatively
associate with MNPs via electrostatic binding, leading to a significant increase in the
absorption per nanoparticle [292].
An additional method to functionalize MNP surfaces relies on the use of organic and
organometallic catalysts. Hu, et al reported functionalization using chiral catalysts
immobilized on MNPs [293]. The immobilized catalysts were readily recycled via magnetic
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decantation and could be re-used up to fourteen times without any decline in activity (Figure
10)[293].
Fig. 10. Immobilization of Chiral Ru Catalyst on MNPs.
Reprinted (adapted) with
permission from
[293]
. Copyright (2005) American Chemical Society.
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