Gold nanoparticles in the clinical laboratory: principles of preparation and applications

Transcription

1 Clin Chem Lab Med 2012;50(2): by Walter de Gruyter Berlin Boston. DOI /CCLM Review Gold nanoparticles in the clinical laboratory: principles of preparation and applications Hassan M.E. Azzazy1,2, *, Mai M.H. Mansour1, Tamer M. Samir1,3 and Ricardo Franco4 1 Yousef Jameel Science and Technology Research Center, The American University in Cairo, New Cairo, Egypt 2 Department of Chemistry, The American University in Cairo, New Cairo, Egypt 3 Faculty of Pharmacy, Misr University for Science and Technology, October City, Egypt 4 REQUIMTE, Departmento de Química, Faculdade de Ci ê ncias e Tecnologia da Universidade Nova de Lisboa, Campus de Caparica, Lisboa, Portugal Abstract In order to meet the challenges of effective healthcare, the clinical laboratory is constantly striving to improve testing sensitivity while reducing the required time and cost. Gold nanoparticles (AuNPs) are proposed as one of the most promis ing tools to meet such goals. They have unique optophysical properties which enable sensitive detection of biomarkers, and are easily amenable to modification for use in different assay formats including immunoassays and molecular assays. Additionally, their preparation is relatively simple and their detection methods are quite versatile. AuNPs are showing substantial promise for effective practical applications and commercial utilization is already underway. This article covers the principles of preparation of AuNPs and their use for development of different diagnostic platforms. Keywords: assay; bio-barcode; diagnostics; gold nanoparticles; immunoassay; molecular assay; nanomedicine. Introduction Gold nanoparticles (AuNPs) have been the subject of intense research for use in biomedicine over the past couple of decades. AuNPs, also referred to as colloidal gold, possess some astounding optical and physical properties that have *Corresponding author: Dr. Hassan M.E. Azzazy, The American University in Cairo, P.O. Box 74, New Cairo, 11835, Egypt Phone: , Fax: , hazzazy@aucegypt.edu Received January 23, 2011; accepted August 28, 2011; previously published online October 6, 2011 earned them a prime spot among the new promising tools for medical applications (1 3). Today, AuNPs offer to provide the clinical laboratory with more sensitive, faster, and simpler assays, which are also cost-effective. AuNPs can be used to develop point-of-care tests and novel testing strategies. Structure and properties of AuNPs The typical structure of AuNPs is spherical nano-sized gold particles, but they can also be composed of a thin gold shell surrounding a dielectric core, such as silica (gold nanoshells). They range in size from 0.8 to 250 nm and are characterized by high absorption coefficients (1, 4). AuNPs have some unique optical properties, such as enhanced absorption and scattering, where the absorption cross-section of AuNPs is 4 5 orders of magnitude greater than that of rhodamine 6G (5). A relatively small number ( < 300) of gold atoms in the nanoparticle are required for the manifestation of their distinct optical properties (6). This is in addition to large molar extinction coefficients that are proportional to particle size. Surface plasmon resonance (SPR) is the phenomenon behind the exquisite optical properties of AuNPs. When an electromagnetic radiation, of a wavelength much larger than the diameter of the AuNPs, hits the particles, it induces coherent, resonant oscillations of the metal free electrons across the nanoparticles. These oscillations are known as the SPR, which lie within visible frequencies and result in strong optical absorbance and scattering properties of the AuNPs. A colloidal gold solution of 20 nm particles exhibits a SPR band with an absorption maximum of 520 nm, generating the solution s distinct red color (Figure 1 ) (7 10). Typically, SPR absorption maxima between 517 and 575 nm are exhibited by AuNPs whose diameters range from 9 to 99 nm. However, AuNPs with core diameter < 2 nm fail to exhibit SPR (11). The SPR band is affected by several factors including the distance between AuNPs (12), the particle shape, and to a lesser extent the size of AuNPs and the refractive index of the medium. Also, in case of gold nanoshells the composition and core/shell ratio play a role (9, 13). As the core/shell ratio increases, the SPR band exhibits a red-shift (10). When AuNPs aggregate, interaction of locally adjacent AuNPs (plasmon-plasmon interaction) shifts their color to blue. Thus, the binding of AuNP-labeled entities to their respective target would lead to aggregation of the nanoparticles and a detectable shift in the optical signal (7, 9). The strong absorption of AuNPs can also be used in colorimetric

2 194 Azzazy et al.: Gold nanoparticles for in vitro clinical diagnostics Figure 1 Surface plasmon resonance (SPR) in dispersed and aggregated AuNPs. (A) Dispersed AuNPs absorb visible light at their SPR absorbance maximum (approx. 520 nm for AuNPs of approx. 15 nm) whereas the red color is not absorbed and solution color appears red. (B) When AuNPs undergo aggregation, their electronic clouds overlap and thus the aggregated particles behave as a larger particle with a SPR absorbance maximum at longer wavelength (referred to as red-shift). Thus, the solution color appears blue. detection of analytes by measuring changes in the refractive index of the AuNP s environment caused by adsorption of the target analytes (9). One more feature of AuNPs that comes in handy is their high surface-to-volume ratio. This allows signal enhancement both in assays where AuNPs are the signal generating particles, or when other signal generating molecules e.g., enzymes are immobilized on their surface (14, 15). AuNPs were reported to enhance electrochemical and chemiluminescent detections (6, 9, 16). AuNPs ranging in size from 6 to 99 nm, were reported to have a catalytic effect on the chemiluminescent reaction of the luminol-h O2 2 system with maximum signal enhancement obtained using 38 nm AuNPs (17). AuNPs also present some remarkable scattering properties that can also be utilized in bimolecular detection. The scattering properties are significantly enhanced and the scattering cross-sections of AuNPs are quite large as compared to other particles of similar size (10, 18). For example, an AuNP with a 30 nm diameter has a scattering cross-section that is 250 times larger than that of a polystyrene particle of the same size (18). The scattering signal intensity of AuNPs is superior to organic fluorescence dyes emission signals, where the scattering light intensity by a 60 nm AuNP is 4 to 5 orders of magnitude that of a fluorescein molecule (18, 19). AuNPs have an exceptional fluorescence quenching capability and with the overlap of fluorophore emission with the extinction band of AuNPs and the large molar extinction coefficients of AuNPs, they can quench virtually any fluorophore (8, 20, 21). The quenching occurs within the scope of two closely related phenomena; F ö rster/fluorescence resonance energy transfer (FRET) and nanometal surface energy transfer (NSET). FRET is the non-radiative energy transfer from an excited donor molecule to an acceptor molecule through near-field dipole dipole interaction. In addition to an overlap between the emission spectrum of the donor and the absorbance range of the acceptor, a typical distance of 2 8 nm between the donor and acceptor is needed for the energy transfer to occur. This is termed the F ö rster distance, which is

3 Azzazy et al.: Gold nanoparticles for in vitro clinical diagnostics 195 the distance between the donor and acceptor at which energy transfer efficiency is 50 %. However, NSET is more specific to metallic nanoparticles and involves more dipolar interactions on account of the presence of free conduction band electrons in the metallic nanoparticles (8, 20, 22, 23). In the case of NSET, the distance allowing effective energy transfer between the donor and acceptor is almost twice that of FRET due to the greater dipolar interactions, and the quenching effect correlates with the distance ( d ) between the AuNP and fluorophore as 1/ d 4 (in case of FRET 1/ d 6 ) (23 25). There is also a difference in the geometry of dipolar interactions between donor and acceptor in the case of NSET, where the isotropic dipole vector distribution of metallic acceptor increases the likelihood of energy transfer from the donor and thereby increases quenching efficiency (23, 26). Synthesis of AuNPs There are several strategies for synthesis of AuNPs, in both aqueous and non-polar organic solvents (3, 27). The synthesis methods are rather versatile, allowing production of AuNPs of different shapes, sizes, and dispersity, but only a few methods produce particles with uniform size and narrow size distribution (28, 29). AuNPs are generally synthesized by the reduction of a precursor with gold (III) ions, such as chloroaurate, to metallic gold in the presence of a capping agent that binds to the surface of AuNPs (9, 29). Capping agents control the size of AuNPs by preventing particle growth and provide colloidal stability by inhibiting particle aggregation (9). Reduction of ionic gold can be achieved by chemical reduction e.g., using citric acid (30), sodium borohydride (31), or tetrakis (hydroxymethyl) phosphonium chloride (32). γ-irradiation (33, 34) or UV irradiation (35) can also induce reduction of chloroaurate solutions. Sonochemical reduction is also possible by ultrasound irradiation (36, 37). Other more ecologically friendly biochemical reduction methods include the use of bacteria (38, 39), fungi (40) and plant extracts (41, 42), or with application of green synthesis photocatalytic methods (43). The most common methods for the synthesis of AuNPs are citrate reduction, Brust-Schiffrin, and seeding growth methods (1, 28). This article will briefly discuss these methods, while Table 1 presents examples of the synthesis and functionalization of AuNPs used in different applications. Figure 2 shows the sizes of AuNPs obtained by the different synthesis routes. The synthesized AuNPs can be characterized using a variety of tools, such as ultraviolet-visible (UV-vis) spectrophotometry, X-ray diffraction (XRD), transmission electron microscopy (TEM), and scanning electron microscopy (SEM) (1, 50). The main methods for characterization of AuNPs are presented in Table 2. Citrate reduction method Citrate reduction is one of the simplest and most popular methods of AuNPs synthesis, which was first reported by Turkevich et al. in 1954 (54) and later modified by Frens in 1973 (30). In addition to its simplicity, it has the advantages of being carried out in an aqueous phase and produces almost uniform spherical particles over a tunable size range (Figure 3 ). In this method, an aqueous solution of chloroaurate is reduced to metallic AuNPs by the addition of citrate that acts both as a reductant and surface capping agent that stabilizes the formed AuNPs (28, 30, 54). Controlling the size and monodispersity of AuNPs is crucial. AuNPs ranging in size typically between 16 and 147 nm can be prepared using this method and can be controlled via changing the reaction conditions (1, 30). Both the size and distribution of the particles are controlled by reaction parameters including concentrations, reducing/stabilizing agent to gold precursor ratio, ph and temperature (55, 56). In the citrate reduction method, citrate plays different roles, first it acts as a reducing agent that converts Au (III) to metallic gold nanoparticles, and second it passivates and stabilizes the formed nanoparticles, preventing particle growth. At high citrate concentrations, smaller particles are covered and stabilized by citrate leading to smaller particle size, while at low concentrations particle coverage by citrate is incomplete and particle growth continues leading to the formation of AuNPs with larger particle size (28, 57). Table 1 Methods for synthesis and functionalization of AuNPs. Synthesis method Size of AuNP, nm Surface cap Conjugated biomolecule Bond type Application References Citrate reduction Citrate Thiol modified Covalent SNP detection (44) oligonucleotides Citrate reduction 13 Citrate Thiol modified oligonucleotides Covalent Detection of endonuclease (45) with poly adenine spacer activity Citrate reduction 13 Citrate Alkanethiol modified Covalent SNP detection (46) oligonucleotides Reduction by Gallic acid Detection of lead ions (47) gallic acid Citrate reduction 13 Citrate Thiol modified DNA Covalent Detection of cocaine and (48) aptamers adenosine Citrate reduction 35 Citrate Anti-EGFR Electrostatic Cancer cell imaging (49) EGFR, epithelial growth factor receptor; SNP, single nucleotide polymorphism.

4 196 Azzazy et al.: Gold nanoparticles for in vitro clinical diagnostics Figure 2 Synthesis of AuNPs of different sizes. Strength of the reducing and capping agents also affects the particle size. Citrate is a relatively weak reducing and capping agent compared to other agents such as sodium borohydride for reduction and alkanethiol for capping as in the Brust-Schiffrin method. This accounts for the formation of larger particles ( > 10 nm) by the citrate method compared to < 5 nm by the Brust method (28). Although AuNPs with particle size ranging from 12 to 147 nm were produced by the citrate reduction method, good quality particles can however, only be produced up to a size of 50 nm. Beyond this size, particles are not spherical and are polydispersed (58). The Brust-Schiffrin method The Brust-Schiffrin method allows the synthesis of thermally and air stable particles with reduced dispersity and controlled size ranging from 1.5 to 5.2 nm (1). These hydrophobic AuNPs are also sometimes called monolayer protected clusters and can be easily isolated, redissolved and functionalized. They are also extremely stable and do not show signs of decomposition, such as particle growth or loss of solubility even after storage for several months at room temperature (1, 59, 60). In their first report, Brust and co-workers demonstrated the synthesis by reduction of Au (III) with sodium borohydride (NaBH 4 ) in a two-phase system in the presence of thiol capping compounds (59). Brust-Schiffrin s method starts by transferring AuCl 4 - from aqueous solution to toluene using tetraoctylammonium bromide (TOBA) as the phase-transfer agent. AuCl 4 - is then reduced by aqueous solution of sodium borohydride in the presence of dodecanethiol (DDT); a thiol capping ligand (31, 60). Later, Brust and co-workers extended their method by the synthesis of p-mercaptophenol stabilized AuNPs in a single phase system (59). The particle size is controlled by adjusting thiol to the AuCl 4 - molar ratio, larger ratios give smaller average core size, while cooled solution and fast addition of reducing agent give more monodispersed small particles (1, 60). Seed growth method Although controlling particle size is achieved by controlling nucleation and growth during particles growth by reducing and capping agents, respectively; it is difficult to control these intermediate steps during particle formation (28, 61). Seeding growth is another common approach for controlling the size of nano particles, and is especially used when large particles are required (28, 58, 62, 63). In seeding growth methods, small mono-dispersed particles are first prepared by common methods then used as seeds for the Table 2 Main characterization strategies of AuNPs. Method Utility References TEM Determination of nanoparticle morphology and size. It can also provide a (1, 50) picture of the AuNPs core (in case of core-shell structure) AFM Size and morphology determination (1) SEM Size and morphology determination (51) UV/visible spectroscopy Determination of AuNPs concentration and estimated size (52) Zeta potential measurement Measurement of surface charge and stability of AuNPs and their conjugates (3, 53) AFM, atomic force microscopy; SEM, scanning electron microscopy; TEM, transmission electron microscopy.

5 Azzazy et al.: Gold nanoparticles for in vitro clinical diagnostics 197 Figure 3 AuNP synthesis using citrate reduction method. preparation of larger particles (28). Reducing agents used in seed growth methods are usually weak; they are capable of reducing metal ions only in the presence of seeds. Particle size can be controlled by adjusting metal ions to seeds ratio (28, 58). Perrault et al. (58) reported the synthesis of AuNPs with sizes nm. AuNP seeds were prepared by citrate reduction then used in growth reaction containing hydro quinone as reducing agent. Hydroquinone is a weak reducing agent that can only reduce gold in the presence of metallic gold seeds. Reducing number of seeds resulted in larger particles (58). Functionalization of AuNPs In order to utilize AuNPs for clinical applications, their surface can be functionalized with appropriate recognition biomolecules, such as oligonucleotides or antibodies for specific biomarker detection in clinical specimens. It should be noted, however, that non-functionalized AuNPs can still be used for detection of biomolecular targets (51). Methods used for the functionalization of AuNPs with biomolecules make use of electrostatic attraction, specific affinity interaction, or covalent binding (64). Electrostatic attraction or physical adsorption binding methods are simple approaches that are based on electrostatic attraction between oppositely charged AuNPs surface and target biomolecules. For example, immunoglobulins can be conjugated to AuNPs where the negatively-charged citrate AuNPs bind to the positively charged amino acid side chains of the immunoglobulin. The conjugation is typically performed at a ph that is equal to or slightly higher than the isoelectric point of the immunoglobulin (64, 65). Although it is a simple and direct approach, electrostatic attraction is weak and cannot withstand harsh experimental conditions, such as multiple washing steps, long incubation periods, or high salt concentrations (60). Another approach of conjugating biomolecules to AuNPs is by utilizing biomolecule pairs with specific high binding affinities as linkers between AuNPs and other biomolecules. Such high affinity pairs include streptavidin/biotin, protein A/Fc immunoglobulin fragments, and concanavalin A/carbohydrate (60, 64, 65). For example, Gestwicki et al. (66) conjugated streptavidin-conjugated AuNPs to biotin-labeled concanavalin A tetramers. The resulting conjugates were used to visualize single multivalent receptor ligand complexes by TEM. Covalent binding of ligands to AuNPs surface provides high stability and can withstand harsh conditions, such as high salt concentration and elevated temperature (60). The most common approach to covalently bind biomolecules to the surface of AuNPs is based on Au-S covalent bond formation. Different biomolecules, such as thiolated oligonucleotides or proteins containing cysteine residues can be readily conjugated to AuNPs (9, 60, 64). Details of additional AuNPs functionalization strategies are provided elsewhere (3, 58, 64). Assay strategies using AuNPs AuNPs, both modified and unmodified, can be employed for the detection of target biomolecules, using various strategies, and by exploiting different features of AuNPs. The following sections will elaborate on notable strategies for AuNPs-based detection. Immunological strategies Liu et al. (18) developed a homogenous immunoassay for protein detection utilizing the scattering properties of AuNPs. The assay detected mouse IgG with a detection limit of 0.5

6 198 Azzazy et al.: Gold nanoparticles for in vitro clinical diagnostics ng/ml and had a dynamic range of 0.5 to 50 ng/ml. Binding of the target IgG to anti-mouse antibodies-aunps conjugates led to the formation of AuNP aggregates with increased size and enhanced light scattering properties. Leng et al. (67) recently developed an approach for multiplex detection of antibodies, by utilizing an electrochemical immunoassay. Capture antibodies; polyclonal rabbit anti-goat (RaG) and mouse anti-human IgG (MaH), were immobilized by passive adsorption onto two carbon electrodes on a disposable chip. The targets, human IgG (H-IgG) and goat IgG (G-IgG), were subsequently added to the chip, followed by detector antibodies, RaG and rabbit anti-human (RaH) conjugated to AuNPs. Electro-oxidation of the AuNP labels in presence of hydrochloric acid led to formation and immediate adsorption of AuCl 4 - on the carbon electrode surface. This resulted in a differential pulse voltammetric response that correlated with target concentration. The assay had a detection limit of 1.1 ng/ml and 1.6 ng/ml for HIgG and GIgG; respectively. This system seems particularly well suited for multiplex detection in a complex sample, such as plasma, because of the ease of introduction of additional working electrodes by screen printing on the chip and the good sensitivity obtained (67). In a utilization of the simplicity and low cost of conductometric detection, Liu et al. (68) developed a biochip for protein detection using AuNPs and silver enhancement. Antibodies were immobilized on the surface of a chip covered with an electrode array, which was manufactured using standard microelectromechanical systems (MEMS) technology. The target antigen was added and captured by the immobilized antibodies and then sandwiched by another AuNP-labeled antibody. Finally, a silver enhancement solution (silver ions and hydroquinone) was added. The AuNPs catalyzed the reduction of silver ions into metallic silver by hydroquinone, and the metallic silver deposited onto the AuNPs, which increased the particle size. The deposits provided a shorter path for electron transfer between electrodes and resulted in an easily measureable change in electric conductance. This biochip prototype had a detection limit of 240 pg/ml of rabbit IgG. The extremely large surface area of AuNPs imparts excellent catalytic properties on AuNPs and also ensures high rates of surface functionalization. Rabbit IgG were immobilized on a gold electrode, which was subsequently used to capture anti-igg tagged with AuNPs of small diameters (5 nm) (69). Auto-catalyzed deposition of Au on the surface was promoted by addition of gold chloride in formaldehyde. This strategy combined the efficient labeling of the IgG using small AuNPs and the improved electrochemical signal generated by the enlarged AuNPs (69). Fluorescence quenching by AuNPs has also been used in immunosensors in a variety of formats, such as typical sandwich assays (70) and immunoassays involving the utilization of magnetic beads (71). Examples of the utility of this property are illustrated in the Biological target detection section. Nucleic acid detection strategies Unmodified AuNPs The use of unmodified AuNPs can significantly contribute to the development of simpler, faster, and cheaper sensitive assays for nucleic acid detection. Citrate-coated AuNPs have a surface negative charge, which allows the adsorption of single-stranded DNA (ssdna) or RNA, which can uncoil and expose their nitrogenous bases, allowing electrostatic attraction to the AuNP s surface. Consequently, the negative charge on the AuNPs increases and so does the repulsion between the AuNPs, thus preventing their aggregation. Upon addition of AuNPs to a saline solution containing the target nucleic acid and its complementary unlabeled oligonucleotide sequence, AuNPs aggregate and the solution color changes from red to blue. This is because the oligonucleotide sequences bind to their complementary target, and this double-stranded structure cannot adsorb on AuNPs due to the repulsion between its negatively-charged phosphate backbone and the negativelycharged citrate ions on the surfaces of the AuNPs. In this situation, the oligonucleotides are not free to adsorb and stabilize the AuNPs, which can be forced to aggregate using salt and the solution color changes to blue. On the other hand, in the absence of the complementary target, aggregation of the AuNPs is prevented due to the presence of free oligonucleotide sequences that stabilize them, and the solution color remains red (51, 72, 73). Qi et al. (16) developed a homogenous chemiluminescent assay to detect DNA hybridization. A specific oligonucleotide sequence was mixed with the target DNA followed by denaturation, annealing, then cooling. AuNPs were then added to the mixture followed by a luminol-h 2 O 2 solution. In presence of target DNA, the AuNPs aggregated and a strong chemiluminescent signal was observed. The detection limit of this strategy was 1.1 fm of target DNA. In absence of the target, the AuNPs remained dispersed and a weak signal was observed. Based on zeta potential measurements, the surface negative charge density of aggregated AuNPs was much lower than that of dispersed nanoparticles. This allowed the AuNPs to effectively catalyze the luminol-h 2 O 2 reaction and led to an enhanced chemiluminescence signal. Modified AuNPs AuNPs can be conjugated to oligonucleotides and used as probes in various assay formats. The conjugation can be done in a number of ways, for example, the AuNPs have a high affinity to thiols and can thus readily bind to thiolated oligonucleotides. Also the AuNPs can be functionalized with streptavidin and bind to biotinylated oligonucleotides (2, 60). The use of modified AuNPs for biological target detection is illustrated later in this paper. Universal biosensors Xia et al. (74) recently developed a universal AuNPs-based colorimetric biosensing platform for detection of a wide range of targets including nucleic acids, proteins, small molecules, and ions. The approach, which demonstrated picomolar sensitivity for DNA detection with naked eye, utilizes ssdna probe, unmodified AuNPs and a conjugated water soluble cationic polyelectrolyte; poly [(9,9-bis (6 -N,N,Ntrimethylammonium) hexyl) fluorene-alt-1,4- phenylene]

7 Azzazy et al.: Gold nanoparticles for in vitro clinical diagnostics 199 bromide. The basis of this approach is that the conjugated polyelectrolyte specifically sequesters most of ssdna probe and prevents it from stabilizing AuNPs that aggregate and the solution color turns blue. However, in the presence of a complementary target, the probe-target dsdna is formed which weakly binds to the conjugated polyelectrolytes and becomes free to stabilize AuNPs against aggregation (solution color remains red). The absorbance ratio A520/A700 can be measured and used to calculate target concentration. This assay has also been adapted for detection of nonnucleic acid targets by employing specific aptamers which bind to specific mole cular targets. Upon target binding, aptamers change from single-stranded to a folded, compact structure that only weakly binds the conjugated polyelectrolyte. Anti-cocaine aptamer, thrombin aptamer, and a mercury-responsive sequence that folds in the presence of Hg (II) were employed in this assay to detect their respective targets. In the absence of targets and upon addition of 20 nm AuNPs, aptamers do not stabilize the AuNPs against aggregation in the presence of the polyelectrolyte. In the presence of targets, the aptamers fold and minimally bind to the polyelectrolyte and the solution color remains red as the AuNPs are stabilized. The reported detection limits for thrombin, cocaine, and Hg (II) were 10 nm, 10 μ M, and 50 μ M; respectively. Bio-barcode assay Another strategy is the bio-barcode assay (Figure 4 ), a new technique that employs AuNPs, and it has demonstrated zeptomolar sensitivity for DNA detection and attomolar sensitivity for protein detection (9). In this assay, the target antigens are isolated by a process involving oligonucleotide (barcode DNA)-modified AuNPs and magnetic microparticles. Both particles are functionalized with antibodies specific to the target antigen. The ultra-sensitivity of the bio-barcode assay is a result of effective sequestration of antigen and the signal amplification due to the large number of barcode DNA strands released corresponding to the antigen binding event. This barcode DNA can then be detected using a universal oligonucleotide probe conjugated to AuNPs and scanometric detection (utilizing silver enhancement). The bio-barcode approach allows detection of 30 copies of a target protein in a large mixture of other proteins. For DNA detection, the same principle of the assay applies, but the target is sequestered using specific oligonucleotides instead of antibodies (7 9, 75 77). Conventional bio-barcode assays are expensive and laborious and require sophisticated instruments. To overcome these disadvantages, modified bio-barcode methods have been developed that utilize electrochemical, colorimetric, fluorimetric, and chemiluminescent detection techniques. Duan Figure 4 Principle of bio-barcode assay. The antigen is sandwiched between two antibodies, one conjugated to AuNPs and the other to magnetic microparticles. The AuNPs are also conjugated to barcode DNA. The complex is separated magnetically and the barcode DNA is chemically released. The released barcode DNA is captured by immobilized oligonucleotides that are complementary to part of the barcode DNA and then AuNPs-labeled oligonucleotides complementary to another part of barcode DNA are added. This is followed by silver enhancement and scanometric detection [adapted with permission from (75)].

8 200 Azzazy et al.: Gold nanoparticles for in vitro clinical diagnostics et al. recently developed an electrochemiluminescent biobarcode assay (78). In this assay, AuNP were modified with Tris-(2,2 -bipyridyl) ruthenium labeled cysteamine, to boost electrochemiluminescence signal, and ssdna capture probe. The target DNA was sandwiched between a biotinylated capture probe and cysteamine-aunp-capture probe conjugate and the complex was finally captured by streptavidin-coated magnetic microparticles. The method had a reported detection limit of 100 fm and high selectivity for single-mismatched DNA detection. The bio-barcode technique has been adapted for chip-based analysis that allows for significant time, cost, and sample savings, in addition to amenability for use in point-of-care testing. Biological target detection AuNPs have been used for sensitive detection of viruses, bacteria, proteins and peptides, SNPs, and small molecules. Tables 3 and 4 highlight some immunoassays and molecular assays that employ AuNPs; respectively. Viruses Griffin et al. (23) employed the fluorescence quenching properties of AuNPs and designed a NSET probe for the homogenous detection and quantification of a synthetic hepatitis C virus (HCV) RNA target (Figure 5 ). The probe consists of ssrna labeled with a fluorophore and adsorbed on the surface of AuNPs; the fluorophore in this state is quenched by the AuNPs, and at the same time, the AuNPs are stabilized by the probe and remain dispersed in solution (red color). In the presence of the target RNA, the probe hybridizes with it and the dsrna is released into the solution. Therefore, the quenching effect is cancelled and the dye fluoresces. Additionally, the color of the solution changes from red to blue, due to aggregation of AuNPs as the probe no longer stabilizes them. The intensity of fluorescence is proportional to the concentration of the target RNA. The quenching efficiency increased by three orders of magnitude as the size of AuNPs increased from 5 to 70 nm. A detection limit of 300 fm of RNA was obtained with particle size of 110 nm (23). Shawky et al. (51) developed a colorimetric assay for direct detection of unamplified HCV RNA from clinical specimens. HCV RNA was extracted patient serum then mixed with a specific oligonucleotide sequence in a hybridization buffer containing salt. The mixture was denatured, annealed, and cooled to room temperature, then unmodified 15 nm AuNPs were added. The solution color changed to blue (i.e., AuNPs aggregated) in HCV positive specimens, and remained red in the negative ones (Figure 6 ). The assay had a detection limit of 50 copies/reaction and exhibited a sensitivity of 92 %, a specificity of 88.9 %, in addition to a short turnaround time of about 30 min. An additional advantage of this assay is that it circumvents the need for sophisticated expensive equipment, such as thermal cyclers. This assay platform could be developed to detect other infectious agents. Duan et al. (82) developed an assay for simultaneous detection of hepatitis B virus (HBV) and HCV antibodies using a protein chip and AuNP-based detection. Antigens from HBV and HCV were immobilized on a glass chip and used to capture HBV and HCV antibodies in serum. AuNP-labeled staphylococcal protein A was then used to detect captured anti bodies, and silver staining was used to amplify the signal. This produced black spots on the assay chip that can be detected visually and the spot density was measured using a scanner. The assay had a turnaround time under 40 min, and it detected down to 3 ng/ml of IgG. Electrochemical detection is a widely used technique in AuNP-based immunoassays. Shen et al. (81) developed an electrochemical immunoassay based on copper-enhanced AuNP tags for the detection of hepatitis B surface antigen (HBsAg) using anodic stripping voltammetry. In this assay, two anti- HBsAg antibodies were utilized; one conjugated to magnetic nanoparticles and the other to AuNPs. The two antibodies were incubated at 37 C with the sample and the HBsAg was sandwiched between the two nanoparticle-conjugated antibodies and the resulting complex was then separated magnetically. Copper enhancer solution (ascorbic acid and copper sulfate) was then added to the resuspended complex leading to copper deposition on the surface of AuNPs. Following another magnetic separation, the copper was solubilized using nitric acid to release copper ions, whose concentration was then measured using anodic stripping voltammetry. The concentration of copper ions was proportional to the concentration of the HBsAg present in the sample. The assay had a linear range from 0.1 to 1500 ng/ml, a detection limit of 87 pg/ml, and a turnaround time of 70 min. The assay had a comparable performance to HBsAg enzyme-linked immunosorbent assay (ELISA) but with a considerable reduction in turnaround time. Tang et al. (93) developed a bio-barcode assay for the detection of human immunodeficiency virus type 1 (HIV-1) with a detection limit times lower than the corresponding ELISA. The assay was able to detect concentrations of the HIV-1 p24 antigen as low as 0.1 pg/ml vs pg/ml detected by ELISA. Also, in patients with seroconversion, the assay detected infection 3 days earlier than the ELISA. Bacteria Soo et al. (88) developed a colorimetric AuNP-based assay for detection of Mycobacterium tuberculosis (MTB) and MTB complex (MTBC) in 600 sputum samples. AuNPs were conjugated to thiol-modified oligonucleotides to generate two probe pairs for detection of nested PCR-amplified DNA targets of MTB and MTBC. The hybridization of the two probes to their target caused aggregation of AuNPs and a change in color from red to purple/blue and a decrease in absorbance at 525 nm. The assay gave a positive reaction by spectrophotometry 2 h after addition of probes in presence of 0.5 pmol of DNA. The assay had a sensitivity of 96.6 % and specificity of 98.9 % for detection of MTBC. The reported sensitivity and specificity for MTB detection were 94.7 % and 99.6 %, respectively. Chlamydia trachomatis was detected in urine using unmodified AuNPs (85). This approach involved PCR amplification

9 Azzazy et al.: Gold nanoparticles for in vitro clinical diagnostics 201 Table 3 Examples of AuNPs-based immunoassays. Target Assay description Assay performance References AFP AFP was sandwich between two monoclonal antibodies; one conjugated to AuNPs and the other to the magnetic nanoparticle. The complex was separated magnetically. The supernatant, containing the excess AuNP-antibody conjugates was then added to a known solution of the fluorophore fluorescein isothiocyanate. The fluorescence of the fluorophore was quenched by the AuNPs in the conjugate and the fluorescence intensity of the fluorophore correlated with the concentration of AFP. AIV AIV was sandwiched between a specific pentabody conjugated to magnetic nanoparticles and a monoclonal antibody conjugated to AuNPs. A magnetic field was then used to separate the complex and hydroquinone is added, which was reduced by the AuNPs to quinine, whose optical density is measured. This allowed quantitative detection of AIV. CA15-3 An enhanced ELISA, where the AuNPs were used as carriers for the detector antibody (conjugated to horseradish peroxidase) to concentrate the enzyme to enhance the generated optical signal. ENO1 An electrochemical sandwich immunoassay for ENO1, a marker associated with small lung carcinoma. HBsAg An electrochemical immunoassay based on copper-enhanced AuNPs for the detection of HBsAg using anodic stripping voltammetry. HBV and HCV IgG antibodies A protein chip with immobilized HCV and HBV antigens was used to capture the antibodies. Staphylococcal protein A labeled with AuNPs detects the captured antibodies and silver staining was used to generate an amplified signal for visual detection. The silver enhancement resulted in black spots on the chip where the antibodies were captured. hgh A monoclonal anti-hgh antibody was immobilized on a gold electrode coated with AuNP. When the immobilized antibody bound to the hgh in the sample, the charge transfer properties of the electrode changed. The change correlated with hgh concentration. The change was measured using electrochemical impedance spectroscopy. HSA A competitive homogenous immunoassay where HSA, competes with AuNP-coated HSA for binding to the specific antibody. The binding of the AuNP-HSA conjugate to the antibody resulted in a measurable shift in the absorbance maximum of the solution. PSA Bio-barcode assay for PSA detection in patient serum with scanometric detection. Detection limit was 0.17 nm. (71) Detection limit was 10 ng/ml, an order of magnitude more sensitive than double-antibody sandwich ELISA. Sensitivity of the ELISA assay doubled after the use of AuNPs as carriers for the signal generating enzyme. Linear range was 0 60 U/mL. The assay had a detection limit of 11.9 fg (5 μ l of 2.38 pg/ml solution), and linear dynamic working ranging from to 10-8 g/ml (twice as broad as that of commercial ELISA). The assay turnaround time was almost half that of commercial ELISA and required 1/20 of the sample volume. The assay had a linear range from 0.1 to 1500 ng/ml, a detection limit of 87 pg/ml, and a turnaround time of 70 min. Turnaround time was under 40 min, and the assay was reported to detect down to 3 ng/ml of IgG. Lower detection limit was 0.64 pg/ml and the linear dynamic range was from 3 to 100 pg/ml. Detection limit of 3 mg/l in human urine. (84) Detection limit of 330 fg/ml. The assay is 300 times more sensitive than current PSA assays, thus allowing redefinition of undetectable PSA levels, a critical factor for patients undergoing radical prostatectomy in detecting recurrence. AFP, alpha-phetoprotein; AIV, avian influenza virus; CA15 3, cancer antigen 15 3; ELISA, enzyme-linked immunosorbent assay; ENO1, alpha-enolase; HBsAg, hepatitis B surface antigen; HBV, hepatitis B virus; HCV, hepatitis C virus; hgh, human growth hormone; HSA, human serum albumin; PSA, prostate-specific antigen. (79) (15) (80) (81) (82) (83) (76)

10 202 Azzazy et al.: Gold nanoparticles for in vitro clinical diagnostics Table 4 Examples of AuNPs-based molecular assays. Target AuNPs modification Detection Notes References Chlamydia trachomatis None Colorimetric Pathogen detected in human urine samples after PCR. (85) Detection limit: 100 template copies. HCV None Colorimetric Direct detection of HCV RNA. (51) Turnaround time: 30 min. Sensitivity of 92 %. Specificity of 88.9 %. Detection limit: 50 RNA copies/reaction. Influenza A (H1N1) Streptavidin Impedimetric Detection limit: 577 pmol/l. (86) MTB Thiol-oligonucleotides Colorimetric Detection limit 0.75 μ g total DNA. (87) Turnaround time: 2 h. 100 % concordance with commercial INNOLiPA-Rif-TB. Cost: $0.35 per sample. MTB Thiol-oligonucleotides Spectrophotometry/visual Sensitivity: 94.7 %. (88) Specificity: 99.6 %. Detection limit: 0.5 pmol of DNA. Mutations in EGFR None Colorimetric Assay detected mutations in unamplified genomic DNA from (89) non-small cell lung cancer patient specimens. SNPs Hairpin oligonucleotides Visual Detection limit: 10 pm. (90) Turnaround time of 25 min. SNPs in DMD gene Thiol-oligonucleotides Colorimetric Specific discrimination between wild-type and mutant genotype for SNPs in exon 30 of the DMD gene. Telomere DNA None Colorimetric Picomolar detection limit. (92) Linear range to mol/l. DMD, Duchenne muscular dystrophy; EGFR, epidermal growth factor receptor; HCV, hepatitis C virus; MTB, Mycobacterium tuberculosis ; SNPs, single nucleotide polymorphisms. (91)

11 Azzazy et al.: Gold nanoparticles for in vitro clinical diagnostics 203 Figure 5 Principle of RNA detection assay based on fluorescence quenching by unmodified AuNPs. (A) In absence of complementary target sequence, the fluorescent dye-labeled oligonucleotides remain adsorbed onto the AuNPs and the fluorescence is quenched, and the AuNPs remain dispersed (red). (B) Upon hybridization with the complementary target, the labeled oligonucleotide moves away from the AuNPs, thereby restoring the dye s fluorescence and allowing aggregation of AuNPs (blue). of the bacterial DNA, where one of the used primers was thiol-labeled, therefore, the resulting amplicon was thiolated at one end. Upon mixing with AuNPs, the amplicon adsorbed strongly on the surface of the AuNPs due to the strong affinity between AuNPs and thiols. Consequently, the AuNPs resisted salt-induced aggregation and the solution color remained red (positive sample). This resistance was attributed to electrostatic repulsion caused by increased of negative charge density on the surface of AuNPs on account of the adsorbed amplicons. Presence of the adsorbed amplicons imparts a physical barrier that impedes particle aggregation. In case of negative samples, AuNPs aggregated upon salt addition (0.37 M) and solution color changes to blue. This approach does not require modification of AuNPs and can detect 100 copies of template DNA. Proteins and peptides Mayilo et al. (21) developed a homogenous sandwich immunoassay employing AuNPs as fluorescence quenchers. The assay detected cardiac troponin T (ctnt), a biomarker of Figure 6 Principle of AuNPs-based assay for detection of unamplified HCV RNA. myocardial infarction, with a limit of detection in the picomolar range. The assay relies on bivalent antibody fragments that bind two different epitopes of ctnt. One of the antibody fragments was conjugated to AuNPs while the other was labeled with a fluorescent dye (Cy3 or Cy3B), whose emission spectrum overlapped with the AuNP extinction spectrum. When the ctnt was present, it was sandwiched between the labeled antibody fragments. This brought the AuNP and the fluorescent dye to a close proxi mity leading to quenching of the fluorescence by AuNPs (efficiency up to 95 % ). The detection was achieved using time-resolved fluorescence measurement, and the assay s detection limit was 0.02 nm (21). Anfossi et al. (84) developed another homogenous AuNP-based immunoassay for the detection of human serum albumin in urine. In this competitive assay the albumin in sample competed with albumin-coated AuNP for binding to a specific polyclonal antibody. The binding of the AuNP-albumin conjugate to the antibody resulted in a measurable shift in the absorbance maximum of the solution. The concentration of albumin in the sample correlated to the extent of inhibition of antibody binding to albumincoated AuNP. The assay showed good correlation with the reference nephelometric method and had a 3 mg/l limit of detection in human urine, thus making it suitable for detection of microalbuminuria.

12 204 Azzazy et al.: Gold nanoparticles for in vitro clinical diagnostics A microfluidic system using surface immobilized biobarcode assay was developed for detection of prostate speci fic antigen (PSA). PSA concentrations as low as 40 fm were detected in serum samples (77). This value makes the assay more sensitive that commercial ELISA with an improvement of two orders of magnitude, and the reported assay turnaround time was 80 min (77). In another study, Georganopoulou et al. (94) developed a bio-barcode for measuring amyloid-β diffusable ligand; a potential soluble marker of Alzheimer s disease, in cerebrospinal fluid of 30 subjects with attomolar sensitivity (94, 95). Single nucleotide polymorphisms (SNPs) Another MTB-related assay was developed by Veigas et al. (96) who used AuNPs linked to thiol-modified oligonucleotides for the identification of SNPs in the β subunit of the MTB RNA polymerase gene that are associated with multidrug resistance. SNPs in the β subunit of the RNA polymerase gene are responsible for rifampicin resistance in 95 % of resistant MTB strains. In the presence of the complementary target sequence the AuNPs resisted saltinduced aggregation, and the solution color remained red. In the negative samples, the AuNP-probes were free to aggregate in response to salt addition and the color of the solution turned blue. Absorbance at 526 nm (contribution of the nonaggregated fraction of AuNP-probes) and absorbance at 600 nm (aggregated AuNP-probes) were measured. Absorbance ratio of 526 nm/600 nm of 1.0 was considered the threshold to discriminate between positive and negative samples. For the direct identification of MTBC samples, the assay had a sensitivity of 84.7 %, a specificity of 100 %, a PPV of 100 %, and a NPV of 36.4 %. The assay generated positive results for 35 out of 42 MTBC samples (83 % ) that were positive by the INNO-LiPA Rif TB assay (Innogenetics, Belgium) (96). biosensor (97). The assay uses two nanoparticle conjugates; concanavalin A conjugated to fluorescent CdTe nanocrystals (quantum dots; QDs), and thiolated β-cyclodextrins conjugated to AuNPs. In absence of glucose, the two conjugates can come in a close proximity and FRET occurs between the QDs (energy donor) and the AuNPs (energy acceptor), and the fluorescence of the QDs is quenched. When glucose is present, it displaces the β -cyclodextrin conjugate and binds to the concanavalin A. As a result, the AuNPs and the QDs move farther apart and the AuNPs cannot quench the QD s fluorescence (Figure 7 ). The intensity of the QDs fluorescence signal was proportional to glucose concentration. The assay had a detection limit of 50 nm and a high selectivity for glucose over other sugars and most biomolecules present in human serum. Radhakumary et al. (98) developed a method for visual detection of glucose based on AuNPs functionalized with glucose oxidase (GO). AuNPs synthesized using citrate reduction were modified with alkanethiol groups and functionalized with GO. The functionalized AuNPs were mixed with urine samples spiked with different concentrations of glucose, and Small molecules Zhang et al. (20) utilized the quenching property of AuNPs to develop a novel aptamer-based multicolor nanoprobe for simultaneous detection of adenosine, potassium ions, and cocaine in a homogenous solution. First, three aptamers (anti-adenosine aptamer, potassium-specific G-quartet, and anti-cocaine aptamer) were synthesized and labeled with different fluorophores at their 5 end. Second, 3 thiolated DNA strands with complementary sequences for the three apta mers were assembled at the surface of AuNPs. Third, the three aptamers were mixed and hybridized with their complementary sequences on the surface of AuNPs (multicolor nanoprobe). Due to the close proximity of the fluorophores to AuNPs, significant fluorescence quenching took place due to NSET effect. The presence of one target in the solution caused the corresponding aptamer to unhybridize from its complementary sequence on the AuNP surface and the generation of a fluorescent signal that correlated to the analyte concentration. The fluorescence quenching properties of AuNPs were also employed in the development of a FRET-based glucose Figure 7 Principle of a glucose sensor based on AuNPs quenching of fluorescence of quantum dots (QDs). (A) In absence of glucose the QD-concanavalin A conjugate and the AuNP-β-cyclodextrin conjugates are in close proximity and the QDs fluorescence is quenched by the AuNPs via FRET. (B) When glucose is present, it displaces the β-cyclodextrin conjugate and binds to concanavalin A, thereby restoring the QDs fluorescence.

13 Azzazy et al.: Gold nanoparticles for in vitro clinical diagnostics 205 Table 5 Examples of commercially available AuNPs and their suppliers. Vendor Product examples Website BB International (Cardiff, UK) AuNPs reference materials for biomedical research. Certified by the National Institute of Standards and Technology (NIST). BioAssay Works (Ijamsville, Maryland, USA) Nanopartz (Loveland, Colorado, USA) Sigma-Aldrich (St. Louis, Missouri, USA) Strem Chemicals (Newburyport, Massachusetts, USA) Colloidal gold, streptavidin-conjugated AuNPs, protein and antibody-coated AuNPs. AuNPs (5 100 nm) and gold nanorods. AuNPs coated with methyl, amine, carboxyl, biotin, and neutravidin terminal groups, AuNPs conjugation kit. Colloidal gold, AuNPs functionalized with different thiol compounds and organic compounds. AuNPs (3 90 nm) and sugar-coated AuNPs the color of the solution changed immediately from red to blue at glucose concentrations 100 μ g/ml. This rapid noninvasive assay also utilized UV/visible measurement and had a detection limit of 5 µg/ml; the shift in SPR peak correlated directly with glucose concentration up to 90 μg/ml. Commercial potential of AuNPs The known exquisite properties of AuNPs along with the multitude of studies illustrating the benefit and viability of their clinical use, herald a bright commercial future for AuNPs. Additionally, the general nanotechnology investment trends, particularly in the healthcare sector, and growth of commercial suppliers of nanoparticles back up this expectation. The 2009 estimate for the global nanomedicine market is $53 billion and is expected to reach $100 billion in 2014 (99). At the same time, the US National Institutes of Health (NIH) investment in nanotechnology is growing at an impressive rate (12.5 % between 2008 and 2009) (100). Further downstream, 34 startup companies and 250 filed or granted patents related to nanoparticle-based diagnostics and therapeutics were yielded by the industrial collaborations of the university-based National Cancer Institute Alliance for Nanotechnology in Cancer Centers, since its inception in 2005 (100). The market for nanoparticles with applications in biomedicine, pharmaceuticals, and cosmetics was estimated to be worth $204.6 million in 2007, and this figure is expected to triple by 2012, with 76 % of this market expected to belong to nanoparticles with biomedical applications (99). It is to be noted that AuNP-based tests for detection of SNPs (related to warfarin metabolism) and influenza virus detection have already obtained FDA clearance (101, 102). Table 6 Comparison of performance parameters of AuNPs-based assays and conventional ones for selected pathogens. Pathogen Point of comparison Conventional assay AuNPs-based assay References HCV Assay/target Real-time PCR (COBAS AmpliPrep/COBAS TaqMan HCV) HCV RNA (51, 103) Detection limit 15 IU/mL a 50 RNA copies/reaction Turnaround time 2 3 h 0.5 h Estimated cost $100 $5 Complexity level 3 1 AIV Assay/target Sandwich ELISA Whole virus (79) Detection limit Approx. 100 ng/ml 10 ng/ml Turnaround time < 2 h Approx. 1 h Complexity level 2 2 MTB Assay/target Bacterial culture PCR-amplified MTB DNA (88, 104) Sensitivity 80 % 94.7 % Specificity 98 % 99.6 % Turnaround time 2 8 Weeks Hours Complexity level 2 2 a 1 IU typically equals 3 5 RNA copies. AIV, avian influenza virus; HCV, hepatitis C virus; MTB, Mycobacterium tuberculosis. Complexity level (trained personnel, number of steps): simple (1), intermediate (2), complex (3).

14 206 Azzazy et al.: Gold nanoparticles for in vitro clinical diagnostics Conclusions AuNPs have been employed in a multitude of assay prototypes for detection of various biomarkers. As illustrated above, obtaining AuNPs via in-house synthesis or commercial sources (Table 5 ) is no obstacle, even to small laboratories, and relatively simple AuNPs-based assays may be developed with limited resources. The surface chemistry of AuNPs can be easily adjusted to allow functionalization with different molecules making them easily adaptable for detection of disease biomarkers. The unique optophysical properties of AuNPs have allowed their detection using a wide range of techniques, such as colorimetry, scanometry and fluorometry. AuNP-based assays are showing promise for improved clinical performance as compared to current assays (Table 6 ). AuNPs can be employed in classical diagnostic formats (e.g., as reporter molecules in ELISA and detection of nucleic acid targets after amplification). 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17 Azzazy et al.: Gold nanoparticles for in vitro clinical diagnostics 209 Hassan Azzazy received his BSc and graduate diploma in Biochemistry from Alexandria University, Egypt. In 1994 he received his PhD in Biochemistry and Molecular Biology from the University of North Texas Health Science Center in Fort Worth, TX, USA. He was a postdoctoral fellow and then an assistant professor in the Pathology and Medical Technology departments at the University of Maryland School of Medicine in Baltimore, MD. He is an adjunct professor with the Graduate School of Management and Technology, University of Maryland University College, Adelphi, MD. He has board certifications in Clinical Chemistry and Molecular Diagnostics from the American Board in Clinical Chemistry, Washington, DC. Dr. Azzazy currently serves as professor of Chemistry at the American University in Cairo (AUC). In 2009, he founded the Novel Diagnostics and Therapeutics group at Yousef Jameel Science and Technology Research Center, AUC. He is interested in developing low tech novel diagnostics and identifying therapeutic candidates using biotechnology and nanotechnology tools. He actively collaborates with researchers in Japan, The Netherlands, Qatar, Portugal, and USA. Dr. Azzazy has received Excellence in Research and Teaching Awards from AUC and the State Prize in advanced technological sciences from the National Academy for Scientific Research & Technology. Mai Mansour graduated in 2006 from the American University in Cairo (AUC), Egypt with a BS in Chemistry with a specialization in clinical analysis. She is currently enrolled in the MS Biotechnology program at AUC, investigating rapid detection of multi-drug resistant tuberculosis. She has completed a year-long medical technologist training program. Since graduation, she has worked as a research assistant with the Novel Diagnostics and Therapeutics group at the Yousef Jameel Science and Technology Research Center. Mai also trained in clinical diagnostics at Oregon Health and Sciences University, Portland, OR, USA. Mai has seven papers in peer-reviewed journals on nanodiagnostics, and detection of gene doping in sports. Tamer Samir graduated from Cairo University, School of Pharmacy in He received his MSc in Microbiology from Cairo University in He is currently finishing his PhD at Cairo University investigating isocitrate lyase as a potential drug target for Mycobacterium tuberculosis. He is an assistant lecturer in Misr University for Science and Technology. In 2009, he joined the Novel Diagnostic and Therapeutics group at the American University in Cairo as a research assistant with Professor Azzazy. He is working to develop nanogold assays for detection of Mycobacterium tuberculosis. Ricardo Franco obtained a degree in Applied Chemistry, a Masters in Biotechnology, and a PhD in Bioinorganic Chemistry (1995) at the Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Portugal, where he is currently a tenured Assistant Professor in Biochemistry. He has been a recipient of a Humboldt Foundation post-doctoral fellowship (1996). He has been a visiting Assistant Professor to the Sandia National Laboratories, Albuquerque, NM, USA (2001), and to the College of Medicine, University of South Florida, Tampa, FL, USA (2000). His research interests are in the bionanotechnology field, namely in the development of gold nanoparticle-based immuno- and enzymatic-assays for environmental applications and for clinical diagnosis. He actively collaborates with several (bio)nanotechnology laboratories in Portugal, Spain, Germany, Egypt and the USA.