Optical Assays Based on Colloidal Inorganic Nanoparticles

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, TiO₂ and ZnS as well as insulator NPs including SiO₂ 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.

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 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.

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 HAuCl₄ 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. 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) 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].

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, 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 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 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 × 10¹⁰ PFU/mL (18 pM) T7 [111] (Fig. 2A).

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 Hg²⁺ based on chelation reaction between Hg²⁺ 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 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 Pb²⁺, 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 Pb²⁺detection. In this method, the presence of Pb²⁺ 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 Cd²⁺, Ni²⁺ and Co²⁺ 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 Hg²⁺. 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 Al³⁺ 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 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.

Table 1.

AuNP-based colorimetric detection using interparticle-crosslinking mediated aggregation

Target	Examples	Target of Nucleic Acids	Shape of AuNPs	Functionalization	Linear Range	LOD	Size of AuNPs	Ref
Pathogens	Mycobacterium tuberculosis (MTB) and Mycobacterium tuberculosis complex (MTBC)	Amplified target DNA.	NR	Thiol DNA-probe	20-0.5 pmol	1 pmol	13.7 ± 0.8 nm	[119]
	E,coli O157:H7	NR	Spherical	MEA-AuNPs	2.91×108−16×108 CFU/mL−1	108 CFU/mL−1	13 nm	[120]
	Pseudomonas syringae	Amplified target DNA.	Spherical	Thiol DNA-probe	200-2 ng/μl	15 ng/μL	13 nm	[69]
	Salmonella Typhimurium	Amplified target DNA.	Spherical	Thiol DNA-probe	NR	21.78 ng/μL	20 nm	[108]
	Helicobacter pylori	Amplified target DNA	Spherical	Thiol DNA-probe	NR	10 CFU/mL−1	13 nm	[92]
Viruses	Influenza A	Viral RNA	Spherical	mAb-AuNPs	NR	7.8 HAU	13 nm	[110]
	Tomato leaf curl New Delhi virus (TolCNDV)	Amplified DNA target	Spherical	Thiol DNA-probe	NR	7.2 ng	19 nm	[114]
	papillomavirus type 16 and type 18	amplified DNA target	Spherical	Thiol DNAprobe	1.4×100-1.4×10−5 μM	1.4×10−4 μM	13 nm	[109]
Cancer	Cancerous Cells	-	Spherical	Ap-AuNPs	0-40000 cell	1000 cell	20, 50 and 100 nm	[63]
	MCF-7	-	Spherical	ssDNA-AuNP probes	101-105 cell	10 cell	25 nm	[84]
	Breast Cancer Cells	-	Oval-shape	AuNPs- monoclonal anti-HER2/c-erb-2 antibody AuNPs- RNA aptamer	NR	100 cells/mL	14 nm length and 18 nm width	[121]
Metal Ions	Cd2+, Ni2 + and Co2 +	-	Spherical	peptide- AuNPs	0-3 μM Cd2+, 0-5 μM Ni2+, 0-10 μM Co2+	0.05 μM Cd2+, 0.03 μM Ni2+, 2μM Co2+	NR	[122]
	pb2+	-	Spherical	GSH-AuNPs	NR	100 nM	5-8 nm	[115]
	Hg2+	-	Spherical	Chitosan-AuNPs	9-50 μM	1.35 μM	16 nm	[112]
	Al3+	-	Spherical	MMT-AuNPs	1-10 μM	0.53 μM	12 nm	[117]
	Melamin, Hg2+		Spherical	Cysteamine-AuNPs (CA-AuNPs)	0.05-3 μM Hg2+, 0.08-1.6 μM Melamin	Hg2+=30 nM, Melamin=80 nM	13± 2 nm	[123]
	Mg2+		Spherical	Tryptophan-AuNPs	0.1-2.0 μmol L−1	0.2 μmol L−1	15 nm	[124]
Enzymes and Protein	Protein kinase activity		Spherical	STV-AuNPs	0.0005-0.05 U μL−1.	0.0005-0.02 U μL−1	13 nm	[81]
	Flt-1 protein	-	Spherical	p-AuNPs	0.2-10 nM	0.2 nM	13± 2nm	[118]
Others	Botulinum neurotoxin serotype A light chain (BoLcA)	-	Spherical	PEG –AuNPs, Eutravidin- AuNPs	NR	5-0.1 nM	20 nm	[88]
	H2S	-	Spherical	AE-AuNPs	3–10 μM	0.2 μM	13 nm	[125]
	Spermine	-	Spherical	ctDNA-AuNPs assembly	0.1–2 μM	11.6 nM	13 nm	[126]
	Clenbuterol		Spherical	Cysteamine AuNPs	50 nM-1 μM	50 nM	10 nm	[127]

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].

Fig. 3.

A schematic illustration of non-crosslinking AuNP and AgNP aggregation-based colorimetric detection

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

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 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-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.

Table 2.

AuNP-based colorimetric detection based on non-crosslinking aggregation mechanism

Types	Examples	Target of nucleic acids	Shape of AuNPs	Functionalization	Linear Range	LOD	Size of AuNPs	Ref
Bacteria	E. coli	unamplified genomic DNA	spherical	Thiolated DNA probe	215-27 ng	54 ng	20 nm	[131]
	Salmonella spp	Amplified by NASBA	_	Thiolated probe-AuNPs	100-0.5×104 CFUs/mL	5 CFUs	17-23 nm	[137]
	Salmonella typhimurium DT104	mAb-AuNPs	“Popcorn” shaped	mAb-AuNPs	NR	103 CFU/g	30 nm	[138]
Viruses	Dengue virus (DENV)	genomic RNA	-	DDZ-AuNP	1×101-1×106 TCID50 units	1×101 TCID50 units	15 nm	[139]
	Shrimp Taura syndrome virus (TSV)	RT LAMP Products	spherical	Thiolated-TSV oligonucleotide- AuNPs	100 pg–1 fg	1 pg total RNA	10-20 nm	[140]
Cancer biomarkerss	Breast cancer	-	spherical	peptide substrate	0-0.2 μg/mL	0.05 μg/mL	20 nm	[133]
Metal Ions	Hg2+	-	spherical	Ds-DNA	. 25 nM to 1μM,	0.4 nM	12 nm	[141]
	Pb2+	-	Anisotropic AuNPs and spherical AuNPs	DTT-AuNPs	1-1000 nM	∼9 nM	∼47nm	[142]
Fungus	Paracoccidioides brasiliensis	complementary DNA (cDNA)	spherical	Thiolated probe-AuNPs	NR	20 ng/μL−1	23 nm	[143]
Others	SNPs and DNA mutations	complementary DNA	Nanospheres nanorods, nanotriangles	ssDNA- nanoparticles	NR	NR	AuNS: 40 (d) nm, AuNR: 20 (d)×42 nm, AuNR: 15 (d)×45 nm, AuNR: 13 (d)×51 nm, AuNT: 20 (th)×50nm	[134]
	Nitrite	-	nanorods	4-Amino thiophenol-AuNPs (4-ATP)	0.25-5 ppm	<1 ppm	NR	[144]

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 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 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 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.

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/μl−1	0.8125 ng/μL	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 productو 40 ng for genomic DNA	14±2 nm	[153]
Viruses	Maize chlorotic mottle virus	RT-PCR target products	Spherical	2.4 ng/μL-1.875 pg/μL	<30 pg/μL 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]
Biomarkers	Plasmodium vivax lactate dehydrogenase (PvLDH), Plasmodium falciparum LDH (PfLDH)	-	Spherical	0 pM–1 μM	1.25 pM and 2.94 pM	NR	[155]
	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 μM	0.1μM	13 nm	[163]
	Thrombin	-	Spherical	1 pM-5 μM	1 pM	15 nm	[151]
	Protein kinase	-	Spherical	0-1 U/μL	0.232 mU/μL	13 nm	[164]
	Caspase-3-activity	-	Spherical	0- 0.3 μg mL_1	0.01 and 0.005μg/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 protein RNA	-	Spherical	NR	2.4 nmol/L	15nm	[166]
Drugs	Ramoplanin	-	Spherical	0.30-1.30 ppm	0.01 ppm	13nm and 23nm	[158]
	Methomphetamin	-	Spherical	2-10 μM	0.82μM	13 nm	[156]
	Steptomycin	-	Spherical	0-4000 nM	73.1 nM	15 nm	[157]
Metals Ions	Hg2+		Spherical	NR	30 nM	13 nm	[167]
	Cd2+	-	Spherical	0-50 μM	5 μM	∼13 nm	[168]
	Cu2+	-	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]
	Cu2+	-	Spherical	0.5-10 μM	250 nM	13nm	[171]
	Hg2+	-	Spherical	50-300 nM	15 nM	13 nm	[172]
	Hg2	-	Spherical	0.5-10 nM	0.26 nM	15 nm	[173]
Others	Sulfide	-	Spherical	0-10 μM	80 nM	8.1± 1.1 nm	[174]
	Thiocyanate	-	Spherical	0-2 μM	1 μM	13 nm	[175]
	Hypochlorite	-	Spherical	NR	1.5μM	13 nm	[176]
	Urea	-	NR	20-150 mM	20 mM	NR	[177]
	17β-estradiol	-	-	1-105 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]

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 NaBH₄ 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].

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.

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]
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]
Ag Nano sphere, triangular	SPR	Ascorbic acid quantitative detection	[186]
Ag Nano sphere 2.5-28 nm	SPR	Phosphate, glutathione, Cu2+ 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]

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

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 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 μM. 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 μM 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.

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 μM	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 μM	13 nM	[231]
GSH	Pb(II)	0.5-4 μM	0.5 μM	[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 μM	0.23 μM	[246]
Alizarin Red S	Al3+	1-5.3 μM	0.12 μM	[247]
Dextrin	Cu(II)	50–200μM	50 μM	[213]
Sucrose	Endrin	0.05–5.00μg mL−1	0.015 μg/mL	[248]
-	Melamine	0.002-0.25mM	2.32 μM	[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 μM	1.8 μM	[252]
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	< 5μM	[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]

An interesting colorimetric assay was developed based on the morphology transition of Ag nanoprisms for sensing of Hg²⁺ over a linear range from 10 to 500 nM with a LOD of 3.3 nM. Ag nanoprisms capped with 1-dodecanethiol (C₁₂H₂₅SH), 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 Hg²⁺ 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 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 × 10⁻⁸M 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].

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 permission

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].

The oxidation of some biomolecules, such as glucose and cholesterol, catalyzed by specific enzymes, is accompanied by the generation of hydrogen peroxide. H₂O₂ 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 H₂O₂, glucose, cholesterol and other H₂O₂ 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].

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 SiO₂ coating, and labeled with biotin. Next AuNPs were prepared and their surfaces were also coated with SiO₂ 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.

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]
Au@Ag core-shell	H2S	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]

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 H₂O₂ 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

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 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 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.

The magnetic properties of any material depend on the orbital and spin motions of its electrons [294]. There are five fundamental types of magnetic materials: diamagnetic, ferromagnetic, paramagnetic, antiferromagnetic, and ferromagnetic. The magnetic susceptibility is the main property by which a material is classified as a magnetic material. Magnetic properties can be measured using several methods, including SQUID (superconducting quantum interference device) [295] magnetometry and vibrating sample magnetometry (VSM)[296].

The most recognized technique for the study of the MNPs surface is X-ray photoelectron spectroscopy (XPS). Stabilization of MNPs with different coatings affects the surface charge of the MNPs. To study this effect, zeta potential analysis can be performed on MNPs with various coatings, It is clear that by modifying the NPs with various coated layers, the zeta potential changes can be controlled in order to achieve the optimum condition for the specific assay with regard to, pH, temperature, functional and surface groups (Figure 11) [283].

Fig. 11.

Zeta-potential of Fe₃O₄ NPs (a), Fe₃O₄/SiO₂ NPs (b) and native silica (c) [283]. Reprinted with permission

In each step of functionalization, FT-IR spectroscopy can be carried out to detect which functional group is attached to MNPs. To estimate the functionalization efficiency, the thermal gravimetric analysis (TGA) procedure can be performed on the coated MNPs [284].

A brief survey on the applications of MNPs showed that they were mostly used in industrial, biological, medical, and environmental sciences [297].

Hu, et al. developed a technique for the removal of hexavalent chromium present in the effluents of a metal-processing plant using magnetic separation based on maghemite [298]. In medical sciences, MNPs can be employed in various applications such as targeted drug delivery, biosensors, and bioimaging. MNPs are also widely used in more specialized areas such as radiotherapy, virus detection, gene therapy, enzyme catalysis, and magnetic cell separation[299]. Cole,et al reported that MNPs modified with PEG could be deployed for targeting brain tumor via external magnetic fields. They concluded that enhanced magnetic brain tumor targeting could be achieved because of the relatively long circulation lifetime of the modified NPs [300]. Hua, et al demonstrated that iron oxide nanoparticles coated with polymeric poly-[aniline-co-N-(1-one-butyric acid) aniline] (SPAnH) enhanced the therapeutic capacity and thermal stability of 1, 3-bis (2-chloroethyl)-1-nitrosourea (BCNU), which is an important chemotherapeutic agent used for the treatment of several types of brain cancer, including glioma, glioblastoma multiforme, medulloblastoma and astrocytoma [301].

7- Colorimetric Detection Based on MNPs

MNPs, due to their non-toxicity, biocompatibility and ability for remote manipulation, are interesting candidates for assays based on colorimetric detection. Liang, et al developed a technique based on the detection of organophosphorus pesticides and nerve agents using Fe₃O₄ magnetic nanoparticles as a peroxidase mimetic. The MNPs were mixed with two different enzymes acetylcholinesterase (AChE) and choline oxidase (CHO) as shown in Figure 12 [302]. The AChE and CHO enzymes catalyze the formation of H₂O_2, which in turn causes the MNP-catalyzed oxidation of a substrate, 3,3´,5,5´-tetramethylbenzidine (TMB) leading to a color reaction [302]. In this way a screening tool could be developed for ultra-sensitive detection of organophosphate compounds such as pesticides and neurotoxic nerve agents.

$$\text{Acetylcholine}+{\mathrm{H}}_2\mathrm{O}\stackrel{\text{Ache}}{\to }\text{choline}$$ (1)

$$\text{Choline}+{\mathrm{O}}_2\stackrel{\text{CHO}}{\to }{\mathrm{H}}_2{\mathrm{O}}_2$$ (2)

$${\mathrm{H}}_2{\mathrm{O}}_2+\text{TMB}\stackrel{\text{MNP}}{\to }\text{Oxidized}\text{TMB}$$ (3)

Fig. 12.

MNPs catalyze the oxidation of a colorimetric substrate, TMB, to generate a color reaction in the presence of AChE and CHO producing hydrogen peroxide after incubation in acetylcholine chloride solution. [302]. Reprinted with permission

Zhang, et al studied a similar assay system employing the catalytic activity of MNPs to oxidize TMB in the presence of H2O2. They employed a sandwich assay using two different aptamers that recognized thrombin. Biotinylated thrombin aptamer 1 was loaded into a 96-well plate by streptavidin binding. Next amino-terminal thrombin aptamer 2 was assembled onto the chitosan modified Fe₃O₄ MNPs (prepared via a solvothermal method) through glutaraldehyde coupling. When the thrombin sample and aptamer 2 modified MNPs were successively added the MNPs became linked to the 96-well plate and were detected by a color change ([303].

7-1 Colorimetric detection based on combinations of AuNPs and MNPs

MNPs such as nickel, cobalt, and iron can be used in various biological applications; these materials, however, rapidly oxidize in the presence of water and oxygen. One of the most effective methods to prevent oxidation relies on the generation of a protective layer of materials such as silica and gold to form a core-shell structure. Superparamagnetic iron oxide nanoparticles (SPIONs) have a pronounced tendency to accumulate and agglomerate and hence coating the nanoparticles can be an effective solution to prevent their agglomeration. Moreover, coatings also enhances properties such as biocompatibility and stability. Among the different materials used to generate a layer on the iron oxide nanoparticles (IONPs), metals such AuNPs have attracted attention due to their biocompatibility, stability and facile synthesis methods [304]. Nanoparticles coated with AuNPs show good biocompatibility and can easily react with bio-molecules such as polypeptides, nucleic acids, and polysaccharides. The combination of IONPs and AuNPs with a core-shell structure (IONPs-AuNPs), has resulted in many applications in the biotechnology and medical fields and has proven particularly relevant for the identification of analytes, using formats such as immune-electrochemical sensors [304]. Pang,et al developed a detection method for DNA point mutations that was based on a ligase reaction between DNA sequences and Fe₃O₄/Au core/shell nanoparticle probes [305]. Using MRI and core-shell structured IONPs- AuNPs, He, et al successfully carried out in vivo tumor detection using dual mode contrast agents [306]. They reported covalent conjugation of lectin to Fe₂O₃@Au core@shell NPs(lectin-Fe₂O₃@AuNP) for T2-weighted magnetic resonance and X-ray computed tomography dual-modality imaging in vitro and in vivo (Figure 13) [306].

Fig. 13.

Lectin conjugated to core-shell IONPs-GNPs. Reprinted (adapted) with permission from [306]. Copyright (2014) American Chemical Society

Core-shell structured IONPs-AuNPs can be covalently modified by fluorescent quantum dots. These fluorescent quantum dots need to be functionalized in order to allow better coupling of biomolecules or other probes (Figure 14) [307].

Fig. 14.

Magnetic-fluorescent composite particles consisting of a magnetic core, silica spacer, and fluorescent quantum dots covalently bonded to the silica surface.

Xie, et al used Fe₃O₄/Au core-shell NPs to separate histidine-tagged (His-Tag) maltose-binding protein (MBP)[308]. The intact gold shell enabled these NPs to be modified and bio-functionalized for various biodetection and biosensing purposes. The authors concluded that Fe₃O₄/Au−NTA−Ni2+ can be used for sensitive, effective, and specific separation of His-Tag targets from a mixture of lysed cells (Figure 15) [309].

Fig. 15.

Surface modified Fe₃O₄ with mercaptopropionic acid, followed by conjugation of nitrilotriacetic acid (NTA) and subsequently chelating Ni²⁺ chelation. Reprinted (adapted) with permission from [309] Copyright (2010) American Chemical Society

7-2 Colorimetric detection based on combinations of AgNPs and MNPs

Silver nanoparticles have been extensively used in various fields because of their antibacterial properties. The use of these particles in biosensors for the detection and treatment of diseases like cancer is also notable. AgNPs are considered to be non-toxic, non-stimulant, non-allergenic, stable in various conditions, hydrophilic, and heat-resistant [310]. Due to the simplicity of the process needed for their generation and because of the ability to control their size and shape, chemical reduction is one of the most commonly used methods to synthesize this type of NPs [311]. When comparing Ag with Au nanoparticles, Tang, et al reported the following advantages, AgNPs show a surface plasmon band in the range of 390-420 nm that is significantly different from that of Au and the extinction coefficient of the surface plasmon band for an Ag NP is almost 4 times larger than that for Au NPs of the same size [312]. The authors also described a new method to develop more advanced immunosensors to detect carcinoembryonic antigen in a clinical immunoassay using chemically functionalized core-shell Fe₃O₄@AgNPs [312]. Ranc, et al developed a method for rapid analysis of dopamine levels in artificial samples of cerebrospinal fluid [313]. Their method used a nanocomposite that consisted of magnetite and AgNPs connected by carboxymethyl chitosan (Figure 16) [313]. This method successfully showed an improvement in the detection of dopamine levels at low concentrations [313].

Fig. 16.

Determination of dopamine in an artificial sample of cerebrospinal fluid and in mouse striatum brain tissue using a Fe₃O₄/Ag nanocomposite. Reprinted (adapted) with permission from [313] Copyright (2014) American Chemical Society

8- Colorimetric detection based on other inorganic nanoparticles

In addition to the noble metal and magnetic nanoparticles that have been discussed above, other mineral nanoparticles, such as semiconductor nanoparticles including ZnO, TiO₂, and ZnS, as well as insulator nanoparticles including SiO₂, have also been used in colorimetric assays. SiO₂, due to their optical and fluorescent properties, are widely used in rapid diagnostic processes that require high sensitivity [314, 315]. These NPs can be used as coatings for other NPs due to their stability and catalytic properties [316–318]. Ag@SiO₂ core shell NPs were used in a study to detect pyrethroid insecticides (including lambda-cyhalothrin, LC) based on a colorimetric technique [317]. Pyrethroid insecticides can be toxic to the nervous system. Ag@SiO₂ core shell nanoparticles modified with amines (NH2), interacted with LC through hydrogen bond formation causing a color change from yellow to pink. The detection limit of this technique was reported to be 1.0×10⁻⁶ M of LC [317] (Fig. 17).

Fig. 17.

Schematic illustration of Ag@SiO₂-NH2 NPs detection of lambda-cyhalothrin (LC) [317]. Reprinted with permission

For detection of H₂O₂ and glucose, γ-Fe₂O₃/SiO₂ nanoparticles (JFSNs) were developed by Sun et al. These NPs show a peroxidase-like catalytic activity and are stable over time. JFSNs modified with glucose oxidase GOx were used to detect glucose. GOx initially catalyzes the oxidation of glucose to H₂O₂ and gluconic acid at pH 7.0. Subsequently, H₂O₂ is adsorbed on the surface of JFSNs and catalytically oxidizes TMB producing a blue color [319] (Fig. 18).

Fig. 18.

Schematic illustration of colorimetric detection of H₂O₂ and glucose by glucose oxidase and peroxidase-like activity of JFSNs. In presence of glucose, GOx catalyzes oxidation of glucose. Subsequently, glucoronic acid and H₂O₂ are produced. H₂O₂ decomposition is catalyzed by JFSNs, and if TMB is present a blue color is formed. Reprinted (adapted) with permission from [319] Copyright (2015) American Chemical Society.

Zinc oxide (ZnO) is a non-toxic, biocompatible material with high catalytic activity. ZnO nanoparticles have been widely used in diagnostics due to their optical, chemical, and electrical properties [320–323]. The catalytic and optical properties of these ZnO-NPs have been exploited in colorimetric assays [324, 325]. In a study conducted by Hayat et al, ZnO-NPs incorporated into carbon nanotubes were synthesized for the detection of cholesterol. These NPs displayed peroxidase like activity. In the presence of cholesterol oxidase, H₂O₂ is produced as the product of cholesterol oxidation. Because of the presence of H₂O₂, catalyzed by ZnO-NPs/CNTs, a chromogenic substrate, 2,2′-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid (ABTS) is oxidized and the color changes to green. The authors suggested that the use of composite nanoparticles with peroxidase-like activity is a valuable alternative to conventional enzyme assays in colorimetric procedures [324].

Another type of inorganic NPs are composed of titanium dioxide, TiO₂. TiO₂-NPs function as photocatalysts, with a high surface area, and high absorption coefficient [326–328]. A colorimetric technique based on the use of dithizone (DZ) anchored on mesoporous TiO₂-NPs was proposed for the detection and removal of Bi(III) ions. Bi(III) ions have high bacteriostatic properties and can be very dangerous to aquatic organisms. In this approach, spherical shaped TiO₂ mesoporous NPs were synthesized. Bi(III) binds with high affinity to DZ to form the [(DZ) 3-Bi] complex, with a consequent color change. In the presence of mesoporous TiO₂-NPs, a stable complex (TiO₂-[(DZ) 3-Bi]) was formed. This led to the complete disappearance of color to generate a transparent solution. This technique was used for the detection and removal of Bi(III) ions in solution [326].

Quantum dots (QDs) are semiconductor nanoparticles with tunable sizes that are widely used in optics, due to their quantum properties. In a recent study NPs composed of ZnS deposited on montmorillonite (ZnS-MMT) were produced in vacuum with high peroxidase-like activity for the detection of H₂O₂ according to the scheme in Fig. 19. In the presence of H₂O₂, ZnS-MMT composite NPs oxidize TMB due to their peroxidase-like activity producing a color change to blue. This study reported the a H₂O₂ detection limit of 1.04838×10⁻⁵ M [329] (Fig. 19).

Fig. 19.

Schematic illustration of colorimetric detection of H₂O₂ by catalytic activity of ZnS-MMT nanocomposites. ZnS-MMT catalyzes the decomposition of H₂O₂ to OH radicals to oxidize TMB to form blue-colored oxTMB [329]. Reprinted with permission

Some studies using colorimetric detection based on other inorganic nanoparticle are summarized in Table 8.

Table 8.

Colorimetric detection based on other inorganic NPs

Type of Inorganic NPs	NPs	Targets	Functionalization	Linear Range	LOD	Size of NPs	Ref
SiO2NPs	SiO2	Cd2+	NR	10−1-10−9 M	1×10−8M	-	[330]
	Ag@SiO2 core shell	Pyrethroids	Amine (NH2)	1.0×10−6-2.0×10−3	1×10−6M	Ag@SiO2 =30 nm	[317]
	SiO2	2,4,6-trinitrotoluene (TNT)	3-aminopropyl-triethoxysilane (APTES)	500 nM-3.0 mM	100 μM	240 ± 10 nm	[331]
	Au@SiO2 core shell	Hg2+	Porphyrin	5-10 ppb	1.2 ppb for Hg2+	Au@SiO2 core/shell = 40 nm	[332]
	Fe3O4@SiO2 core shell	Hg2+	Porphyrin	NR	NR	core shell = 500 nm	[333]
	Ag@SiO2 NPs	Hg2+	Amine-SiO2	NR	5 μM	AgNPs = 14 nm SiO2 NP s = 250nm	[334]
	γ-Fe2O3/SiO2	H2O2 and Glucose	GOX	1-100 μM of H2O2, 0-20 μM of glucose	10.6 nM of H2O2, 3.2 μM of glucose	γ-Fe2O3/SiO 2= ~20-120 nm	[319]
Zinc oxide and ZnS NPs	ZnO NPs/CNTs	Cholesterol	NR	0.5-500 nM	0.2 nM	NR	[324]
	ZnO/ZnS core shell nanoparticles	Cu2+	NR	15-1500 μM	15 μM (~0.96 ppm)	NR	[323]
	ZnS	H2O2	ZnS-MMT nanocomposites	0.07-0.6 mM	1.04838×10−5 M	30-40 nm	[335]
Titanumoxid NPs	mesoporous TiO2	Bi(III) ions	NR	0.001-1 ppb	1 ppb.	10 nm	[326]
	SiO2 TiO2	Hg2+	Azobenzene-coupled receptor SiO2 and TiO2 (AR-SiO2@ AR-TiO2)	NR	28 nM	mesoporous silica and the AR-SiO2 = 2.24 and 2.15 nm TiO2 and AR-TiO2 = 30-60	[336]

9- Conclusion and Future Outlook

In this review, the properties and application of a wide range of inorganic nanoparticles that have been used optical sensors has been discussed. Each type of nanoparticle has its own unique properties that can be used in optical diagnostic techniques, including colorimetric assays. Noble metal NPs exhibit local surface plasmon resonance and metal oxide NPs also possess extremely small size and useful potential. Quantum dots and nanocomposites exhibit fluorescence and other useful optical properties; their significant fluorescence intensity changes make them a promising choice for optical assays. Nanocomposites are made of two or more different component materials. The nanoparticles discussed in the paper have many applications in detection of a wide range of molecules and analytes due to their unique properties.

Colorimetric sensors are attractive because of their simplicity, high sensitivity and specificity and low cost. Most of them produce color changes that can simply be detected by the naked eye. Because of their properties, colorimetric sensors have attracted considerable attention. Early and timely detection of infectious and biological agents, food, and environmental contaminants are important in preventing and treating diseases. Another advantage of nanoparticles relates to their efficiency for detection of dangerous microorganisms and biomarkers of cancerous tissues both in vivo and in vitro.

AuNPs have high stability and biocompatibility and together with other molecules, they can serve as an effective sensor to detect target molecules, AgNPs are also useful for optical sensors since the nanoparticles have a higher extinction coefficient than AuNPs. Thus AgNPs can be used with DNA molecules and other recognition systems to create selective colorimetric biosensors that are used to detect important analytes.

Metal nanoparticles possessing magnetic properties including iron oxide NPs are widely used in colorimetric techniques due to their superparamagnetic properties as well as their small size and large surface area. However, these NPs tend to have high reactivity and low stability compared to metal oxide nanoparticles. They tend to agglomerate irreversibly. Therefore, it is usually necessary to protect them against the surrounding environment by coating them with a protective shell. The use of different surface coatings can improve stability and increase biocompatibility by reducing their interactions with cells or proteins.

In addition to noble metal and magnetic nanoparticles, the properties and applications of ceramic and quantum nanoparticles were also discussed. According to their unique features, such as high surface area to volume ratio, photocatalytic activity and high absorption, ceramic and quantum nanoparticles can be also deployed for removal of hazardous metal ions and a variety of toxic compounds.

SiO₂ NPs act as insulating nanoparticles with easy synthesis and good biocompatibility, and act as a coating can be used to stabilize other NPs. These NPs are very important for the formation of core-shell and other nanocomposite structures. Therefore, they can play an important role in colorimetric assays. The use of nanocomposites or multi-purpose nanoparticles allows several different analytes to be detected simultaneously. This results in increased versatility for the diagnostic techniques.

The surface plasmon resonance wavelength and absorption spectrum of the nano-shells depends on the size ratio of the core to the shell. Unlike metal NPs, the optical absorption of which remains constant at a certain wavelength, the ability to tailor the size of NPs and intensify the SPR is among their advantages as optical sensors.

Despite the importance of inorganic NPs in diagnostic sensors, there are still some problems surrounding their synthesis methods, their tendency to precipitate, and the ability to finely control the shape and size of the NPs, as well as their sustainability in environmental conditions. In order to use NPs in the development of medical and clinical diagnostic assays, efforts to optimize these parameters are essential in order to detect biomolecules and biomarkers in biological fluids and clinical samples. In order to overcome these problems, it is necessary to focus on controlling the basic parameters such as the size and shape of the NPs to make4 them suitable for applications in optics, catalysts, photonics, electronics and related fields. It is also possible to increase their biocompatibility and reduce their toxicity to individuals and also to the environment. However, due to the high performance of inorganic NPs in optical sensors, it is becoming possible to incorporate them into microarray designs, microfluidic devices and lab-on-a-chip set ups to simultaneously detect multiple pathogens or analytes. Micro-arrays and chips will be designed for the simultaneous detection of bacteria, viruses and other pathogens. Using metal NPs is much easier than other technologies for these microscale devices, and their ability to provide a range of optical read-outs including in SPR and SERS means that applications of colloidal AuNPs will continue to grow.

Fig. 8.

Shell etching according to the concentration of copper ions [275]. Reprinted with permission