Gold Nanoparticles and Nanocomposites in Clinical Diagnostics Using Electrochemical Methods

1 Amity Institute of Biotechnology, Amity University Uttar Pradesh, Sector 125, Noida 201303, India 2Department of Chemistry, Pusan National University, Busan 609-735, Republic of Korea 3 Institute of BioPhysio Sensor Technology, Pusan National University, Busan 609-735, Republic of Korea 4 Faculty of Nanotechnology and Advanced Materials, HMC, and Graphene Research Institute, Sejong University, Seoul 143-747, Republic of Korea 5 Department of Chemistry, DDU Gorakhpur University, Gorakhpur 273009, India 6Department of Chemistry, Indian Institute of Technology Roorkee, Roorkee 247667, India


Introduction
The last decade has witnessed an exponential progress of activities in the field of nanoscience and nanotechnology worldwide, motivated both by the anticipation of considerate new science and by the impending trust for applications and financially feasible impacts. The prime action in this field has been in the production and characterization of new materials consisting of particles with dimensions in the order of a few nanometers, purported nanocrystalline materials. These nanosized materials have properties that are often significantly different from their counterparts with the bulk size [1,2].
Inorganic, organic, and biological nanomaterials may have existed in nature since the evolution of life started on earth [3]. Some evident examples are microorganism and fine

Synthetic Routes for the Preparation of AuNPs
Inorganic nanomaterials have received remarkable attention in the past few years due to their optical and well-defined size related to electronic properties [29][30][31]. Development of new materials in the nanometer scale called nanoparticles (NPs) is expected to form the basis of many of the technological and biological innovations of this century [32][33][34][35]. Particularly, AuNPs are being considered in chemistry, biological, and medical due to their oxide-free surface, bioconjugation properties, good biocompatibility, and unique optical properties. Specifically, because of optical activity, AuNPs are extensively used in colorimetric biosensors [36,37], drug delivery [38][39][40], cancer imaging [41][42][43][44], and cancer therapies [45,46]. They also have the potential to help establish specific beneficial processes and achieve selectivity within biological settings. These applications have sparked great interest in the development of synthetic methods for preparing different gold-based nanostructures. The general synthesis methods of AuNPs are briefly described in this section.

General Synthetic Routes.
AuNPs can be manufactured into a variety of shapes including Au nanospheres, nanorods, nanoshells, nanocubes, nanoclusters, and nanostars [46][47][48]. The development of simple and versatile methods for the preparation of NPs in a size-or shape-selected and controlled manner has been a challenging but intellectually rewarding task ( Figure 2). Most commonly, a Au(III) salt is reduced to Au(0) to form an activated species, either in a single step or via a Au(I) intermediate followed by reduction to Au(0). These activated Au(0) species are thermodynamically unstable and rapidly aggregate to form nuclei and eventually the desired gold nanoparticle, as presented in Figure 1. This is typically achieved not only by altering the relative growth rates of different facets by the selective localization of surface-modifying or capping agents, but also by the modulation of nucleation and reaction parameters such as time, temperature, reagent concentration, and pH [49][50][51][52]. On the basis of reported literature,  the following methods have been widely used in the synthesis of nanoparticles.

Physical
Methods. Many of the physical methods have been involved to the synthesis of Au nanoparticles [53][54][55]. In these methods, the size of the NP is controlled by temporarily inactivating the source of evaporation or by slowing the rate by introducing gas molecules to collide with the particles. The growth of nanoparticles generally occurs rapidly, from milliseconds to seconds, requiring precise control over experimental parameters.

Photochemistry (UV, Near-IR), Sonochemistry, and Radiolysis.
Photochemical synthesis of nanoparticles is carried out by the light-induced decomposition or the reduction of a metal salt by photogenerated reducing agents such as solvated electrons (radiolysis). UV-irradiation is another method that can improve the quality of the AuNPs especially when it is used in synergy with micelles or seeds [55]. Near-IR laser irradiation facilitates an enormous size growth of thiol-stabilized AuNPs [56]. Sonochemistry can also be used for the synthesis of AuNPs within the pores of silica [57]. Radiolysis has been used to control the AuNPs size or to synthesize them in the presence of specific radicals [58], and the mechanism of AuNPs formation upon -irradiation has been carefully examined [59]. Laser photolysis has been used to produce AuNPs in block copolymer micelles [60]. Laser ablation is another technique for AuNPs synthesis that has been used under various conditions, whereby size control can be induced by a laser.

Chemical Methods.
Chemical methods have emerged to be indispensable for synthesizing nanocrystals of various types of materials. These methods are generally carried out under mild conditions and are relatively straightforward. One of the important factors that determine the quality of a synthetic procedure is the monodispersity of the nanocrystals obtained. It is desirable to have nanoparticles of nearly the same size, in order to be able to relate the size and the property under study. A variety of reducing agents, such as sodium borohydride, presence of sodium hydroxide, ascorbic acid, sodium citrate, trisodium citrate, and hydrogen peroxide have been used in the reduction of Au(III) ions to Au(0) [61][62][63]. Such reduction takes place in the presence of one or more water-soluble polymers, surfactants, or capping agents AuNPs are considered for use as conductors from printable inks to electronic chips. As the world of electronics becomes smaller in size, nanoparticles are important components in the electronic chip design. AuNPs are being used to connect resistors, conductors, and other elements of electronic devices [20].

Photodynamic therapy
Photodynamic therapy that is a mode of treatment by laser light at a particular wavelength is combined with exogenous chromophores to accomplish a therapeutic effect. Near-IR absorbing AuNPs (including Au nanoshells and nanorods) produce heat when excited by light at wavelengths from 700 to 800 nm. This enables these nanoparticles to destroy targeted tumors. When light is applied to a tumor-containing AuNPs, the particles rapidly heat up, killing tumor cells in a treatment also known as hyperthermia therapy [21].

Therapeutic agent delivery
Therapeutic agents can be covered with AuNPs. Whereas the NIR-resonant Au/silica nanoshells used in therapeutic and imaging applications have an average diameter of 120-140 nm [22].

S e n s o r s
AuNPs can be used in different sensors. For example, a colorimetric sensor based on AuNPs can identify if foods are suitable for consumption [23]. Other methods, such as surface-enhanced Raman spectroscopy, exploit AuNPs as substrates to enable the measurement of vibrational energies of chemical bonds. This strategy could also be used for the detection of proteins, pollutants, and other molecules label-free [24].

P r o b e s
AuNPs also scatter light and can create an array of interesting colors under dark-field microscopy. The scattered colors of AuNPs are currently used for biological imaging. Also, AuNPs are relatively dense, allowing them helpful as probes for transmission electron microscopy [25].

D i a g n o s t i c s
AuNPs are also used to detect biomarkers in clinical and medical diagnosis. They are also useful in the lateral flow immunoassays, a common household example being the home pregnancy test [26].

C a t a l y s i s
AuNPs are used as catalysts in a number of chemical reactions [27]. The surface of AuNPs can be used for oxidation or reduction of certain chemicals. AuNPs are being developed for fuel cell applications. These technologies would be useful in the automotive and display industry.

Fluorescence properties
Fluorescence-based detection techniques are among the most highly sensitive and popular biological tests in the clinical analysis. AuNPs show outstanding behavior of antiphoto bleaching under the presence of powerful light [28].

Reducing agent
Capping agent    Journal of Nanoparticles and with introducing external supplied energy such as photo irradiation, ultrasound irradiation, or heating. These methods allow for adequate control of the size and concentration of the dispersed particles. Moreover, the surface-modifying or capping agents help to make colloidal stabilization and prevent NPs aggregation [62]. The most common approach to achieve colloidal stability proceeds via chemical binding of ligands at the surface of the NPs and a covalent linkage between the ligand. It may change the properties of the NPs through a modification of their electronic density and the dielectric constant of the surrounding medium. A strategy based on the physical adsorption of ligands on the surface of the nanoparticles may be preferable, in order to maintain the intended properties of the nanomaterials. The two reduction methods are outlined as follows.

Borohydride Reduction.
The preparation of metal NPs in solution is most commonly based on the chemical reduction of metallic ions to be metalized in organic/aqueous solvent. The reduction by borohydride has been in existence for a number of years [64,65]. Although, various metal nanocrystals have been synthesized via the borohydride reduction method, but this method is widely used in the synthesis of AuNPs. Superhydride and hexadecylaniline have been used as alternative reagents to NaBH 4 for the reduction of Au(III) in the synthesis of thiol-stabilized AuNPs [66]. Problems associated with borohydride reduction include its irreproducibility, especially in the aqueous medium and the incorporation of boron in the product [67].

Citrate Reduction.
The most common method of synthesizing gold nanoparticles is through the use of citrate. Jana et al. reported that adding sodium citrate to a boiling solution of chlorauric acid led to the formation of gold nanoparticles [68]. Later work showed that variations in temperature and the ratio of reactants allowed for control over the core size from 10 to 100 nm [69]. More recent work has shown that Au/citrate solutions reduced with NaBH 4 allow the synthesis of nanopartic1es <10 nm in average core size. Despite being the most common method of producing gold nanoparticles, citrate-stabilized nanoparticles are disadvantageous for several reasons. First, they cannot be isolated from solution, making it difficult to store or study them in the solid state. Second, their stability with changes in pH or ionic strength is minimal. Finally, and most importantly, their functionalization, either through ligand exchange derivatization of carboxylic acids in the ligand shell, is extremely limited.

Seeding-Growth Method.
The seeding-growth procedure is another popular technique that has been used for a number of years. Recent studies have successfully led to control of the size distribution in the range 5-40 nm, whereas the sizes can be manipulated by varying the ratio of seed to metal salt [70]. The step-by-step particle enlargement is more effective than a one-step-seeding method to avoid secondary nucleation. Gold nanorods have been conveniently fabricated using the seeding-growth method [71]. In a typical seeded growth reaction, a previously synthesized "seed" nanoparticle is introduced to a growth solution containing an Au(III) salt, a weak reducing agent, and a directing agent (most commonly a surfactant). Initially, the seed particles grow through the slow diffusion of gold atoms onto their surface.

Biosynthesis Methods.
As far as the synthesis of nanoparticles is concerned there is a great and growing need to develop clean, nontoxic, and environmentally friendly synthetic procedures. Biological methods have recently been considered as possible environmentally friendly nanofactories. Both living and dead microorganisms are gaining importance by virtue of their facile assembly of nanoparticles. Prokaryotic bacteria have primarily attracted the most attention in the area of biosynthesis of metal nanoparticles [72]. Different research groups continued to accomplish some excellent work based on uni-and multicellular organisms, Algae, and Fungi to synthesize inorganic materials, both intra-and extracellularly. However, the use of plants and their extracts for NP synthesis is a comparatively new and underresearched technique. The majority of research on the use of plants to synthesize AuNPs has been investigated without the need for their removal from the plant biomatrix [73]. More recently synthesized mesoporous material SBA-16 are used synthesis of AuNPs for preventing the aggregation or growth of metal NPs [74].

Overview of Synthesized AuNPs.
Despite the progress achieved, concerns, and problems with the preparation of metal NPs remain, such as the byproducts from the reducing agent, the multiple steps often required, and the high concentration of protective agents. In addition, a simple modification of the surface functionalization of the citratestabilized AuNPs with a variety of chemical and biochemical vectors is also another important factor to be considered. The ability to synthesize smaller AuNPs while preserving their biocompatibility and derivability would present new alternatives for these areas of research. To date, isotopic replacement of the solvent has solely been explored in the case of Au nanorods seeding-growth. Finally, the modification of the reducing and reduced species due to isotope replacement offers unique insights into the AuNP formation mechanism [75]. Recently, AuNPs are synthesized using monosodium glutamate as a reducing agent. However, the particles possess the tendency to aggregate in high molar ratios of gold capped with glutamates and are therefore unstable. Consequently, PMMA is introduced as a polymer-capping agent to stabilize the AuNPs [76]. Thus, nature of the particle stabilizer, solvent, reaction condition, namely, pH, temperature, and so forth, plays crucial role in determining the final size of the particles. It is also important to note that the citrate/aqueous synthesis of AuNPs is the most employed recipe in today nanotechnology explosion. All these novel procedures offer an attractive alternative to the existing ones and open a full range of possibilities for biological studies.

Nanosensors Based on Nano-AuNPs in Clinical Chemistry
AuNPs are one of the highly used particles in sensors used for clinical chemistry. In this section, the detection of some important clinical molecules has been summarized using AuNPs or AuNPs-composite sensor. A review on the use of AuNPs and AgNPs for clinical diagnosis has also appeared in the literature [77]. It has been demonstrated that the use of nanoparticles not only improves the performance of sensors but also permits arrays of sensors to be constructed for parallel sensing [78]. The chemical methods have also been found to produce AuNPs of different shapes such as spherical, nanoplates, and flower shapes [79,80]. A comparison of spherical AuNPs and nanogold plates-based indium tin oxide (ITO) sensor has been reported for the determination of dopamine and ascorbic acid [81]. The nanoplates based sensor has been found to exhibit higher electrocatalytic activity for both compounds due to their (1 1 1) lattice as the basal plane and due to more available surface edges. In addition the increased surface area of nanoplates, due to their large dimension (400-500 nm), also appears to play a significant role. A review on the biopharmaceutical applications of AuNPs has also appeared in the literature describing the detection of diseases biomarker for the diagnosis of patients with germ cell tumors and hepatocellular carcinoma [82]. The biosensing and therapeutic applications of AuNPs have been found due to their catalytic properties. An electrochemical immunoassay method has also been reported for the detection of tumor markers based on electrochemical stripping of AuNPs, which were deposited by immunosensor array with a reduction reaction catalyzed by AuNPs [83]. The method was sensitive, and a detection limit of 3.5 pg/mL was observed with acceptable reproducibility. Significant methods for the determination of brain neurotransmitters have been developed in the past few years using AuNPs. One commonly known neurotransmitter is dopamine (DA), chemically known as 4-(2-aminoethyl) benzene-1,2-diol, widely present in the mammalian brain tissues and plays a pivotal role in the function of the cardiovascular, hormonal, renal, and central nervous systems. Abnormal levels of DA have been linked with Parkinson's disease, Tourette's syndrome, Schizophrenia, attention deficit hyperactive disorder, and generation of pituitary tumours. Similarly, serotonin (5-hydroxytryptamine or 5-HT) is a monoamine neurotransmitter widely distributed in the brain tissues and plays a significant role in temperature regulation, muscle contraction, liver regeneration, endocrine regulation, depression, and so forth. It has also been found that DA and 5-HT influence each other in their respective releasing, and hence simultaneous determination of DA and 5-HT has been carried out using AuNPs-based nanosensors. These nanosensors have been found rapid and convenient for the simultaneous determination of DA and 5-HT in human serum and urine with detection limit of 0.5 and 3.0 nM, respectively [84]. The large excess of ascorbic acid (AA) in biological fluids did not interfere in the determination. Norepinephrine (NE) is also a significant neurotransmitter present in many parts of the central nervous system, where it is engrossed in emotional arousal, blood pressure regulation, and mood disorders. The determination of NE in biological fluids has also been carried out at AuNPs nanosensor using square wave voltammetry. A stable layer of AuNPs was deposited on the surface of ITO, and the nanosensor was used for the detection of NE. The reduction of NE was observed in two pH dependent peaks, and the linear dynamic range was found between 100 nM and 25 M with a detection limit of 87 nM [85]. Attempts have also been made to determine DA in human urine using AuNPs-coated poly(3,4ethylene-dioxythiophene) nanosensor using sodium dodecyl sulfate [86]. The DA concentration versus peak current plot showed two linear regions: 0.5-20 ML −1 and 25-140 ML −1 . The method was found highly sensitive and reproducible with good selectivity and long term stability. AuNPs have also been used in the composites for the determination of neurotransmitters. One of such examples is the fabrication of nano-Au/cysteamine/GC sensor for the determination of DA in presence of high concentration of ascorbic acid (AA). The cysteamine was bound at the surface of glassy carbon (GC) using cyclic voltammetry [87]. The oxidation of DA occurred at 0.175 V, and linear dynamic range for the determination was observed between 1.0 × 10 −8 and 2.5 × 10 −5 ML −1 with a detection limit of 4 nML −1 . The method was simple, and satisfactory results were observed. The electrodeposition of AuNPs clusters over insulating overoxidized polypyrrole film modified GC has been used by Guo et al. [88] for the determination of DA and 5-HT in presence of 100-fold concentration of AA in human blood serum. The oxidation peaks of the three compounds were observed at 0.37, 0.20 and 0.01 V versus SCE for 5-HT, DA, and AA, respectively. The method was sensitive, and a detection limit of 1 nM for 5-HT and 15 nM for DA was observed. Another important clinical target is tryptophan (2amino-3-(1H-indol-3-yl)-propionic acid, Trp), an important and essential amino acid for humans and herbivores. It is also a potent precursor of several metabolites such as 5-HT, melatonin, and niacin. It is an indispensable ingredient of various types of proteins, therefore, must be added in human nutrition for establishing and maintaining positive balance of nitrogen. A nanosensor based on the electrodeposition of AuNPs onto carbon nanotube film has been used for the amperometric determination of Trp at the physiological pH [89]. The linear dynamic range was found between 30 nM and 2.5 M, and a detection limit of 10 nM was observed. The method was successfully used to determine tryptophan in pharmaceutical samples. In another attempt, AuNPs attached multiwall carbon nanotubes (MWCNT) on ITO have been used for the determination of Trp [90]. The use of AuNPsdoped MWCNT shifted the peak potential of Trp to 0.669 V as compared to 0.760 V in absence of AuNPs, which clearly indicated the high electrocatalytic activity of the fabricated nanosensor. The determination of Trp was carried out in human urine and blood serum using standard addition method, and good reproducibility was observed. AuNPs have also seen their applications in clinical analysis of glucose.
A glucose nanosensor based on AuNPs decorated MWCNT at GC was used by Lim et al. [91]. The sensor was further modified with a layer of glucose oxidase and chitosan and used as electrochemiluminescence nanosensor. The sensor showed excellent performance for glucose detection and a wide linear range of 1-1000 M was observed with a detection limit of 0.5 M. A colorimetric bioassay for the glucose determination in human serum has been developed using self-assembled AuNPs with a thiol derivatized mannose [92]. The change in simple surface plasmon absorption band resulting from this method provides a selective method to quantify glucose. A linear concentration range of 500-4000 g/mL with a detection limit of 363 g/mL was observed. The practical utility of the method was examined by applying the colorimetric assay for the determination of glucose in human serum samples. The reported method is simple, and results can be achieved in 10 min and can be applied for the determination of human blood glucose levels without the use of enzymes.
Morphine (MO), a well-known alkaloid, is frequently used to relieve severe pain for patients, especially for those who undergo a surgical procedure. However, when overdosed or abused, MO is toxic and can cause disruption in the central nervous system. Therefore, to prevent overdose-induced toxication, it is necessary to sensitively determine the concentrations of MO in patient's blood or urine. An easy-to-use approach for directly electrodepositing AuNPs onto carbon paste electrode (CPE) to construct AuNPs modified carbon paste electrode (AuNPCPE) is performed [93]. The electrochemistry of MO is investigated by cyclic voltammetry, differential pulse voltammetry, and electrochemical impedance measurements. The results indicate that this strategy provides a promising approach for highly sensitive morphine sensing that offers an excellent response for morphine in the concentration range of 4.0 × 10 −7 − 2.0 × 10 −4 mol L −1 , with a detection limit of 4.21 × 10 −9 mol L −1 . AuNPCPE has also been successfully applied to the determination of morphine in urine samples with a low detection limit and satisfactory recovery. The good results indicate that AuNPCPE holds great promise in practical applications. Apart from the direct application of AuNPs in clinical chemistry of electrochemically active compounds, AuNPs have also attracted attention in the immunoassay of proteins and hormones. For example, an enzyme-catalyzed Ag deposition on irregular shaped AuNPs has been used for the electrochemical immunoassay of alpha-fetoprotein [94]. The assay was carried out using a sandwich-type protocol, and a wide dynamic linear range from 0.1 to 200 ng mL −1 was observed. The interesting feature of the nanobiosensor was its disposable nature and acceptable reproducibility, as no significant difference was observed in the analysis of 10 clinical serum samples. An electrochemical assay was also developed by using AuNPspolyaniline-nanogold microspheres (GPGs) for detecting thyroid stimulating hormone [95]. The GPGs were first synthesized and functionalized with horseradish peroxidaseconjugated thyroid-stimulating hormone antibody (HRP-Ab2), and then thyroid-stimulating hormone (TSH) was detected. The strong attachment of HRP-Ab2 was observed on GPGs and resulted in good reproducibility, and a detection limit of 0.005 IU mL −1 TSH was observed. The analysis of 15 spiked serum samples exhibited no significant difference between the developed method and the commercially available enzyme-linked immunosorbent assay (ELISA).

Biosensors Based on AuNPs and Conjugates
Biosensors are analytical devices that are based on the coupling of an immobilized biologically active compound called a receptor with a signal transducer and an electronic amplifier [96]. After the discovery of nanomaterials, various interdisciplinary strategies have been developed for the sensitive detection of various analytes present in biological and environmental matrix. Among all nanomaterials, Au nanoparticles (AuNPs) have widely been used because of their sizedependent electrical properties, high surface area-to-volume ratios, high electrocatalytic activities, and ease of chemical modification. Various biorecognision elements (antibodies, aptamers, peptides, DNA, etc.) have been attached in various ways to achieve a low detection limits. In this section some of the important fabrications strategies have been discussed based on various types of biorecognision elements.
The general working principle of the immunosensors relying on the specific immunochemical identification of antibodies antigens immobilized on a transducer to antigens in the sample media can produce analytical signals dynamically varying with the concentrations of analytes of interest. In the recent years construction of immunosensors using AuNPs have received significant attention. Numerous immunosensors are developed so far where antibodies are directly attached with the AuNPs. The ability of AuNPs to provide a stable immobilization of biomolecules, biocompatibility, and ease of surface functionalization provides a major advantage of AuNPs in the development of immunosensors. Various approaches like layer by layer selfassembly using nafion [97], dithiothreitol 3, cysteamine [98], 4-aminothiophenol [99], polyvinyl butyral [100] have been used in the fabrication of immunosensors. In one study, a spacer arm antibody was covalently attached onto the AuNPs and applied for the electrochemical immunoassay [101]. AuNPs have also been widely applied for the detection of cancer biomarkers. This study described that AuNPs were efficient in preserving the action and orientation of the antibody, and it can form a key platform in several medical immunodiagnostics. Very recently, inkjet-printed AuNPs arrays coupled with electrochemical detection cancer biomarker interleukin-6 (IL-6) in serum have been developed. Biorecognision antibodies for IL-6 were linked onto the eight-electrode array and used in sandwich immunoassays. A biotinylated secondary antibody with 16-18 horseradish peroxidase labels was used, and detection was achieved by hydroquinone-mediated amperometry with a clinically applicable detection limit of 20 pg mL −1 [102]. Apart of cancer biomarker detection, strategies using AuNPs have also been developed to understand the cancer immunology. In this regard, a quick and sensitive protocol for the detection of alpha-enolase (ENO1) is developed using an electrochemical sandwich immunosensor. Since ENO1 has been correlated with small cell lung cancer, nonsmall cell lung cancer, and head and neck cancer, it has been used as a potential diagnostic marker for lung cancer. The anti-ENO1 monoclonal antibody was adsorb on polyethylene glycol-modified disposable screen-printed electrode as the detection platform. The polyclonal secondary anti-ENO1-tagged AuNPs congregates as electrochemical signal probes. This AuNPs congregatebased assay provided an amplification approach for detecting ENO1 at trace levels, leading to a detection limit as low as 11.9 fg (equivalent to 5 microL of a 2.38 pg/mL solution [103]. Another example of AuNPs for signal amplification was shown by Zhu et al. [104]. In this case, an amperometric immunosensor with an enhanced sensitivity for the detection of neomycin (Neo) was prepared by covalently immobilizing a monoclonal Neo antibody onto a new nanoconducting surface, as a sensor probe. The probe was used to detect Neo in a sandwich-type approach, where the secondary antibody was attached to AuNPs-decorated multiwall carbon nanotubes labeled with hydrazine. Hydrazine on the conjugate served as a catalyst for the reduction of hydrogen peroxide, and the catalytic current was monitored at −0.45 V versus Ag/AgCl. The performance of the immunosensor with and without AuNPs on the probe and the conjugate was compared. A linear dynamic range for Neo analysis was obtained between 10 ng/mL and 250 ng/mL with a detection limit of 6.76 ± 0.17 ng/mL. In another signal amplification strategy, multifunctionalized AuNPs (MFAuNPs) was developed and applied for the electrochemical impedance immunosensor. The sensor was utilized for the detection of protein A, through the stepwise immobilization of 1, 6-hexanedithiol, AuNPs, and IgG on the Au electrode via self-assembling technique. The interfacial properties of the modified electrodes were evaluated in the presence of Fe(CN) 6 4−/3− couple redox as a probe by cyclic voltammetry and electrochemical impedance spectroscopy. The electrochemical behavior of the redox probe was affected by the accumulation of treated substances on the electrode surface. It was interesting to note that there is no significant increase in the electron transfer resistance after the binding of protein A to IgG on the electrode surface without amplification. The changes in the electron transfer resistance on the IgG-modified electrodes became more sensitive after the MFAuNPs were introduced for signal amplification. The increments of amplified impedance showed good correlation of the detection of protein A in the range of 5-1000 pg/mL with a detection limit of 1 pg/mL. In this strategy, the MFAuNPs were successfully applied for the highly sensitive and selective amplification of biomolecules in the picogram range [105]. Very recently, a new electrochemical detection methodology has been developed describing the practical application and integration of nanoparticle-enhanced detection into electrochemical biosensing technologies [106]. Immunoglobulin E (IgE) was used as a model protein biomarker, which possesses two distinct epitopes for antibody (anti-IgE) and DNA aptamer binding. A surface sandwich assay format was utilized involving the specific adsorption of IgE onto a gold electrode surface that was premodified with a monolayer of aptamernanoparticle conjugates followed by the specific interaction of alkaline phosphatase-(ALP-) conjugated anti-IgE. A femtomolar detection of IgE has been achieved. The signal enhancement associated with the nanoparticle was clearly demonstrated using analytical methods and control experiments. Another focus in recent time is to detect important cardiac biomarker such as troponin-I using AuNPs. In one study, troponin-I has been detected using an immunoassay where a specific monoclonal antibody against troponin-I was modified on the GNP-coated ITO electrode surface by selfassembly and characterized by electrochemical techniques. The immunoreaction was monitored by measuring open circuit potential (OCP). This method was fairly simple and stable. A linear dependence of OCP changes according to troponin-I concentrations is observed in the range of concentration from 1 to 100 ng/mL [107]. AuNPs have also been used for the evaluation of protein phosphorylation using a simple electrochemical method. This approach of protein phosphorylation is expected to be much simpler and sensitive compared to the conventional methods using mass spectrometry. The method relies on the labeling of a specific phosphorylation event with Au nanoparticles, followed by electrochemical detection [108]. This study demonstrates the use of AuNPs as electroactive indicators for kinase activity and inhibition has been demonstrated. Furthermore, the electrochemical protein phosphorylation assay will be helpful for the development of cost-effective biosensors for signal transduction studies. Apart from these approaches, the effect of the AuNPs size, ranging from 5 nm to 80 nm, on the electrochemical response of screen-printed carbon electrodes (SPCEs) used as electrochemical transducers is investigated for the first time recently [109]. To confirm this size effect, a simple hydrodynamic modelling and calculation at the nanoscale level is applied so as to find the effect of the size of AuNPs upon the electrochemical response. The results show that the greatest signal for AuNPs suspension for the same concentration of total Au is obtained for the 20 nm sized nanoparticles. The size effect is also studied for AuNPs acting as electroactive labels in an immunosensor that employs magnetic beads as platforms of the bioreactions. The best response for the 5 nm AuNPs in this case is due to the fact that in the immunosensing conditions the Brownian motions are minimized because the AuNPs contact with the electrotransducer surface is induced by the immunoreaction, and the fast magnetic collection of the nanoparticles used as antibody labels upon application of a magnetic field. These findings can be extended towards the tuning sensor fabrication for other clinical molecule detection [110]. The successful interaction between Au and SH has been utilized by Shim's laboratory for the development of electropolymerized self-assembled layer on AuNPs for the detection of inducible nitric oxide synthase (i-NOS) in neuronal cell culture [111]. The results show that AuNPencapsulated conductive polymer are good nanostructured materials as biosensor probes and have a potential application in cell biosensors. The self-assembled layer modified electrode was found to have excellent properties in terms of better conductivity and lower charge transfer resistance. The main advantage of this sensor probe is its simplicity compared to the classical immunoassay methods or optical 8 Journal of Nanoparticles detection-based immunosensing systems. This method was also extended for the toxicity analysis of endocrine disruptors through the in vitro monitoring of i-NOS concentrations using an AuNP-encapsulated conductive polymer-modified immunosensor [112].
AuNPS have also been well utilized in the detection of various kinds of cells such as bacteria and cancer cells. For instance, a disposable immunosensor for Salmonella-enterica subsp. enterica serovar Typhimurium LT2 (S) detection using a magneto-immunoassay and AuNPs as label for electrochemical detection is developed [113]. The immunosensor relies on the application of a screen-printed carbon electrode that incorporates a stable magnet underneath. Salmonella containing samples (i.e., skimmed milk) were examined by using anti-Salmonella magnetic beads (MBs-pSAb) as detector and sandwiching afterwards with AuNPs-modified antibodies (sSAb-AuNPs) detected using differential pulse voltammetry with the detection limit of 143 cells mL −1 . This AuNPs detection tools together with magnetic field application report a limit of detection lower than the usual commercial method carried out for comparison purposes in skimmed milk samples.
A fast cancer cell detection and quantification method, based on the electrocatalytic properties of AuNPs towards the hydrogen evolution reaction has been reported recently [114]. This method effectively detects the rare circulating tumor cells (CTC) through the epithelial cell adhesion molecule (EpCAM), a 30-40 kDa type I glycosylated membrane protein expressed at low levels in a variety of human epithelial tissues and overexpressed in most solid cancers [115]. The method establishes a selective CTC assay capable of detecting 4 × 10 3 cancer cells in suspension that can be extended to several other cells detection scenarios. In another study, a novel strategy for the rapid identification and high sensitive detection of different kinds of cancer cells by means of electrochemical and contact angle measurements has been performed. A simple, label-free method based on the functionalized AuNPs-modified interface has been utilized to distinguish the different cancer cells and drug resistant blood cancer cells. The detection limit was ∼10 3 cells mL −1 which is highly applicable for clinical applications of cells [116]. Conducting polymer nanofibers-AuNPs nanocomposite has also been applied for the detection of cancer cells. In this regard, polyaniline nanofibers-(PANI-NF-) AuNPs composites were prepared and based on the simple electrostatic reaction between the positive charges on the surface of PANI-NF and the negatively charged AuNPs [117]. This nanocomposite was utilized to develop an electrochemical cytosensor based on the self-assembly of foliate on PANI-NF/AuNP nanocomposite. This cytosensor was applied for the in vitro detection of cancer cells based on the selective binding between the folate receptor on cancer cell surface and a folate immobilized on PANI-NF/AuNPs nanocomposite. This cytosensor has several advantages, such as easy construction, short response time, high sensitivity, easy regeneration, good stability, and freedom of additional labeling, which can be thus used for a highly sensitive detection of cancer cells.

Conclusions
AuNPs such fascinating features as ease of synthesis and surface functionalization that are highly biocompatibility, sensitive, and decent optical properties make AuNPs still the most attractive nanomaterial and one of the most studied in the bioanalytical field. AuNPs have emerged as promising nanoplatforms for efficient diagnostics and therapeutics by merging the characteristic properties they possess at the nanometric scale with the practical immobilization of specific ligands on the surface. Therefore, they have become ideal candidates for molecularly sensitive detection, highly efficient contrast agents for molecular imaging, and carriers for targeted drug and gene delivery. Nonetheless, a better fundamental understanding of AuNPs behavior in biological systems needs to be addressed in future, as well as the engineering of novel AuNPs, which can overcome the drawbacks related to currently developed AuNPs. In future AuNPs possessing enhance biodistribution, and enhanced electrical and optical characteristics will provide better way towards clinical diagnostics.