Gold Nanoparticles in the Clinical Laboratory

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Abstract

In order to meet the challenges of effective healthcare, the clinical laboratory is constantly striving to improve testing sensitivity while reducing its time and cost. Gold nanoparticles (AuNPs) are proposed as one of the most promising 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 serves as a focused guide for preparation and utilization of AuNPs in the clinical laboratory.

Keywords: gold nanoparticles, diagnostics, immunoassay, molecular assay, biobar-code, nanomedicine.

Introduction

Gold nanoparticles (AuNPs), also referred to as colloidal gold, posses some astounding optical and physical properties that have earned them a prime spot among the new promising tools for medical applications. (1-3). Over the past couple decades, AuNPs have been the subject of intense research for use in biomolecule detection and imaging (4).Today, the unique properties of AuNPs offer to empower the clinical laboratory with more sensitive faster, and simpler assays, which are also cost-effective. AuNPs are also amenable for use in point-of-care testing and can be employed in novel testing strategies.

Structure and Properties of AuNPs

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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. They range in size from 0.8 to 250 nm and are characterized by high absorption coefficients (1, 5). 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 (6). A relatively small number (<300) of gold atoms in the nanoparticle is required for the manifestation of their distinct optical properties (7). This is in addition to a large molar extinction coefficients which 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 smaller than the diameter of the AuNPs, hits the AuNPs, it induces coherent, resonant oscillations of the metal 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 with 20 nm particles exhibits a SPR band with an absorption maximum of 520 nm, generating the solution's distinct red color (8-10). 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 less than 2 nm fail to exhibit SPR (11).The SPR band is affected by several factors including 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 (10, 13). As the core/shell ratio increases, the SPR band exhibits a red-shift (14).

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 (8, 10). The strong absorption of AuNPs can be used in colorimetric detection of analytes by measuring changes in the refractive index of AuNPs' environment caused by adsorption of the target analytes (10). Additionally, AuNPs posses some interesting catalytic properties, particularly in the size range 2-4 nm, allowing their utilization in electrochemical and chemiluminescent detections with enhanced sensitivity (7, 10, 15). For example, AuNPs were found to have a catalytic effect on the chemiluminescent reaction of the luminol-H2O2 system. AuNPs ranging in diameter between 6 and 99 nm were found to enhance the chemiluminescence (CL) signal of the luminol- H2O2 system, with maximum enhancement obtained using AuNPs with 38 nm diameter(16).

AuNPs also present some remarkable scattering properties which 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 (4, 14). For example, a 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 (4). The scattering signal intensity of AuNPs is superior to organic fluorescence dyes emission signals, where a 60 nm AuNP generates a scattering light intensity that is 4 to 5 orders of magnitude stronger than a fluorescein molecule (4, 17).

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One more feature of AuNPs that comes in handy in assays using AuNPs, 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 (18-19).

On another front, 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 (9, 20-21). The quenching occurs within the scope of two closely related phenomena; Förster/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 Förster distance, which is the distance between the donor and acceptor at which energy transfer efficiency is 50%. On the other hand, 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 (9, 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 (24).

Synthesis of AuNPs

There are several strategies for synthesis of AuNPs, in both aqueous and non-polar organic solvents (3, 25). 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 (26-27).

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 which binds to the surface of AuNPs (10, 27). Capping agents allow the control of the size of AuNPs by preventing particle growth and provide colloidal stability by inhibiting particle aggregation (10). Reduction of ionic gold can be achieved by chemical reduction e.g. using citric acid (28), sodium borohydride (29), or tetrakis(hydroxymethyl) phosphonium chloride (30). g-irradiation (31-32) or UV irradiation (33) can also induce reduction of chloroaurate solutions. Sonochemical reduction is also possible by ultrasound irradiation (34-35). Other more ecologically friendly biochemical reduction methods include the use of bacteria (36-37), fungi (38) and plant extracts (39-40), or with application of green synthesis photocatalytic methods (41).

The most common methods for the synthesis of AuNPs are citrate reduction, Brust-Schiffrin, and seeding growth methods (26, 42). This article will briefly discuss these synthesis methods while table 1 presents examples of the synthesis and functionalizations of AuNPs used in different applications. Figure 1 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, 43). 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 (44) and later modified by Frens in 1973 (28). 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 2). In this method, an aqueous solution of chloroaurate is reduced to metallic AuNPs by the addition of citrate which acts both as a reductant and surface capping agent that stabilizes the formed AuNPs (26, 28, 44). 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 (28, 42). Both the size and distribution of the particles are controlled by preparative conditions including concentrations, reducing/stabilizing agent to gold precursor ratio, pH and temperature (45-46). 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 (26, 47).

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Strength of the reducing and capping agent also affects the particle size. Citrate is a relatively weak reducing and capping agent compared to other agents like 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 (26). Although AuNPs with particle size ranging from 12 - 147 nm were produced by citrate reduction method, however, good quality particles can only be produced up to a size of 50 nm beyond which, particles are not spherical and are polydispersed (48).

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 to5.2 nm (42). These hydrophobic AuNPs are also sometimes called monolayer protected clusters and can be easily isolated, redissolved and functionalized. They are also extremely stable without any signs of decomposition such as particle growth or loss of solubility even after storage for several months at room temperature (42, 49-50).

In their first report, Brust and co-workers demonstrated the synthesis by reduction of Au(III) with sodium borohydride (NaBH4) in a two-phase system in the presence of thiol capping compounds (49). Brust-Schiffrin's method starts by transferring AuCl4- from aqueous solution to toluene using tetraoctylammonium bromide (TOBA) as the phase-transfer agent. AuCl4- is then reduced by aqueous solution of sodium borohydride in the presence of dodecanethiol (DDT); a thiol capping ligand (29, 49). Later, Brust and coworkers extended their method by the synthesis of p-mercaptophenol stabilized AuNPs in single phase system (50). The particle size is controlled by adjusting thiol to AuCl4- molar ratio, larger ratios give smaller average core size while cooled solution and fast addition of reducing agent gives more monodispersed small particles (42, 49).

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 (26, 51). Seeding growth is another common approach for controlling the size of nanoparticles, and is especially used when large particles are required (26, 48, 52-53). In seeding growth methods, small mono-dispersed particles are first prepared by common methods then used as seeds for the preparation of larger particles (26). 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 ion to seeds ratio (26, 48). Perrault et al. (48) reported the synthesis of AuNPs with sizes 50-200 nm. AuNP seeds were prepared by citrate reduction then used in growth reaction containing hydroquinone as reducing agent. Hydroquinone is a weak reducing agent that can only reduce gold in the presence of metallic gold seeds. Reducing amount of seeds resulted in larger particles (48).

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 (54). Methods used for the functionalization of AuNPs with biomolecules can be classified into electrostatic attraction, specific affinity interaction, and covalent binding (55).

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 (positively charged, at a pH below their isoelectric point) bind to the negatively charged citrate-capped AuNPs (55). Although it is a simple and direct approach, electrostatic attraction is a weak bond that cannot withstand harsh experimental conditions such as multiple washing steps, long incubation periods, or high salt concentrations (49).

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 (49, 55-56). For example, Gestwicki et al. (57) 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 (49). The most common approach to covalently bind biomolecule 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 (10, 49, 55) .

Assay Strategies Using AuNPs

AuNPs, both modified and unmodified, can be used for detection of biological targets such as nucleic acids and proteins, with enhanced sensitivity. Although imaging and immunoassays are currently the predominant applications for AuNPs, their use for nucleic acid detection is significantly expanding (10, 58-62). The coming sections will elaborate on the leading strategies for AuNPs-based diagnostics.

Immunological strategies

Liu et al. (4) developed a homogenous single step immunoassay model for protein detection utilizing the scattering properties of AuNPs. The direct assay detected mouse IgG with a detection limit of 0.5 ng/mL and had a dynamic range from 0.5 to 50 ng/mL. Anti-mouse antibodies were conjugated to AuNPs and incubated with the target IgG. In the presence of the target IgG, the AuNP-labeled antibodies bound to it, and the resulting complex caused formation of AuNP aggregates with increased size. This enhanced their light scattering, which was detected and quantified using a dynamic light scattering instrument.

An approach for multiplex detection of antibodies, utilizing a disposable chip for an electrochemical immunoassay was recently developed by Leng et al. (63). Capture antibodies; rabbit anti-goat IgG (RaH) and mouse anti-human IgG (MaH), were immobilized by passive adsorption onto two carbon electrodes on a chip. The model analytes, human IgG (H-IgG) and goat IgG (G-IgG), were subsequently added to the chip, followed by other RaH and MaH conjugated to AuNPs. The immobilized antibodies and the AuNP-labeled antibodies sandwiched the analytes and formed an immunocomplex. Electro-oxidation of the AuNP labels in HCl led to formation and immediate adsorption of AuCl4- on the carbon electrode surface. This resulted in a differential pulse voltammetric response which was measured and correlated with analyte concentration. This system seems particularly well suited for multiplex detection in a complex sample, as is the case of the detection of several biomarkers in plasma samples, because of the ease of introduction of additional working electrodes by screen printing in the disposable chip, and the good sensitivity obtained, namely a detection limit of 1.1 ng/mL and 1.6 ng/mL for HIgG and GIgG respectively (63).

In a utilization of the power, simplicity, and low cost of conductometric detection, Liu et al. (64) developed a proof-of-concept biochip for protein detection using AuNPs and silver enhancement. Antibodies were immobilized on the surface of a chip covered with an electrode array, which is manufactured using standard technologies. The target antigen was then added and captured by the immobilized antibody and is the sandwiched when another AuNP-labeled antibody was added. This was followed by the addition of a silver enhancement solution (contains silver ions and hydroquinone). The AuNPs catalyzed the reduction of silver ions into metallic silver by hydroquinone, and the metallic silver deposited onto the AuNPs, which increases the particle size. The deposits provided a shorter path for electron transfer between electrodes and resulted in an easily measureable change in electric conductance. The study illustrated the feasibility of this model biochip by detection of rabbit IgG as a model analyte, and had a detection limit below 240 pg/ml.

One of the most interesting nanoscale properties of AuNPs is their extremely large surface area. This feature imparts excellent catalytic properties to AuNPs and their conjugates, and also ensures high rates of surface functionalization. In an attempt to increase the available gold surface area for immune electrochemical detection, Lai et al. (65) utilized autocatalyzed deposition of Au3+ on the surface of AuNPs to obtain areas seven times larger than those prior to deposition. This was achieved by immobilizing rabbit IgG on a gold electrode, which was subsequently used to capture and immobilize anti-IgG tagged with AuNPs of small diameters (5 nm). Auto-catalyzed deposition of Au3+ on the surface was promoted by addition of gold chloride in formaldehyde.

Fluorescence quenching by AuNPs has also been used in immunosensors in a variety of formats such as, typical sandwich assays (66), and immunoassays involving the utilization of magnetic beads (67).

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), which can uncoil and expose their nitrogenous bases, allowing electrostatic attraction to the AuNPs' 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 DNA and its complementary unlabeled oligonucleotide sequence, AuNPs aggregate. This is because the oligonucleotide sequences bind to their complementary target, and this double-stranded DNA structure cannot adsorb on AuNPs due to the repulsion between its negatively-charged phosphate backbone and the negatively-charged coating of citrate ions on the surfaces of the AuNPs. In this situation, the oligonucleotide sequences are not free to 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 target aggregation of the AuNPs is prevented due to the presence of free oligonucleotide sequences that stabilize them, and the solution color remains red (54, 62, 68) .

Another useful property of AuNPs is their ability to catalyze the chemiluminescent reaction of the luminol-H2O2 system. This was utilized by Qi et al. (15) to enhance the obtained signal for development of a homogenous assay for detection of DNA hybridization. A target-specific oligonucleotide sequence is mixed with the target DNA, followed by denaturation, annealing, then cooling. AuNPs are then added to the mixture followed by a luminol-H2O2 solution, and the CL signal is measured. In case of presence of target DNA, the AuNPs aggregated (the oligonucleotide binds to the target and cannot stabilize the AuNPs), and a strong CL signal was observed. In the target's absence, the AuNPs remained dispersed (the oligonucleotide is free to stabilize them) and a weak CL signal was observed. This led to the conclusion that the change of surface properties of AuNPs upon aggregation, namely the altered negative charge density, is a determinant of their ability to catalyze the CL reaction of luminol-H2O2. The detection limit of this strategy was 1.1 fM of target DNA.

Modified AuNPs

AuNPs can be conjugated to oligonucleotides and be 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 then bind to biotinylated oligonucleotides (2, 49). The use of modified AuNPs for biological target detection is illustrated in the coming sections.

Multiple analyte assay platforms

Xia et al. (69) recently developed a potentially 'universal' AuNPs-based colorimetric biosensing platform. The approach, which demonstrated picomolar sensitivity for DNA detection, utilizes unmodified AuNPs and a conjugated cationic polyelectrolyte; poly [(9,9-bis (6′-N,N,N-trimethylammonium) hexyl) fluorene-alt-1,4- phenylene] bromide (PFP-Br). The basis of this approach is that at low salt concentration both ssDNA and dsDNA can stabilize AuNPs and prevent their aggregation. The polyelectrolyte favors binding ssDNA rather than dsDNA or folded DNA, under specific conditions. Therefore, upon addition of AuNPs to a solution containing ssDNA, followed by the polyelectrolyte, the AuNPs aggregate and solution turns blue since the ssDNA binds to the polyelectrolyte and cannot stabilize the AuNPs. On the other hand, in case of the presence of a complementary target, dsDNA is formed and the polyelectrolyte does not bind the dsDNA. This leaves the dsDNA free to stabilize the AuNPs and the solution remains red. This strategy proved effective in DNA detection also in spiked human serum. The assay is also adaptable to other targets such as proteins and small molecules, as the same difference in polyelectrolyte affinity was demonstrated in case of free aptamer and aptamer bound to its target (thrombin). The solution containing the aptamer alone turned blue as the AuNPs were free to aggregate with the aptamer being unavailable to stabilize them. Conversely, the solution containing both the thrombin aptamer and thrombin remained red. The adaptability of the strategy is dependent on the optimization of individual parameters, which makes this strategy rather flexible.

Another strategy is the bio-barcode assay (Figure 3), a new technique that employs AuNPs and has demonstrated zeptomolar sensitivity for DNA detection and attomolar sensitivity for protein detection (10). In this assay, the target antigens are isolated by a sandwich process involving oligonucleotide-modified (barcode DNA) 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 amplification process that occurs as a result of the large number of barcode DNA strands released corresponding to the antigen binding event. This barcode DNA can then be detected using a universal oligonucloetide 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 (9-10, 70-71). This technique has been adapted for chip-based analysis which allows for significant time, cost, and sample savings, in addition to amenability for use in point-of-care testing.

The power of electrochemiluminescence (ECL) was utilized in a bio-barcode recently developed by Duan et al. (72). The assay employs an ECL nanoprobe which consists of AuNPs modified with tris-(2,2′-bipyridyl) ruthenium (TBR) labeled cysteamine bound to a DNA probe specific to the target sequence. The ECL probe is mixed with the target and a second biotinylated DNA probe. In the presence of the target, it is sandwiched between the two specific DNA probes and the complex can be separated using streptavidin-coated magnetic beads, and drawn onto the surface of an electrode for signal measurement. The assays allowed quantitative DNA detection with 100 fM detection limit, and exhibited marked selectivity for single mismatches in human serum. The assay is cost effective compared to other bio-barcode assays, and probe preparation and assay application are simple.

Biological Target Detection

AuNPs-based assays present significant potential for simple, quick, and sensitive biomarker detection, including direct detection of pathogen nucleic acid or antigens, or their corresponding antibodies in the host. Different features of AuNPs were exploited for various diagnostic purposes, examples of which are presented below. Tables 3 and 4 highlight some immunoassays and molecular assays that employ AuNPs.

Viruses

Shawky et al. (54) developed a colorimetric assay for direct detection of unamplified HCV RNA from clinical specimens. The assay involves the extraction of HCV RNA from patient serum then mixing the RNA with a specific oligonucleotide sequence in a hybridization buffer containing salt. The mixture is denatured, annealed, and cooled to room temperature, and unmodified AuNPs are added. The solution color changed to blue (i.e. AuNPs aggregated) in HCV positive specimens, and remained red in the negative ones (Figure 4). The color change was observed within 1 minute of AuNPs' addition. 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 30 minutes. An additional advantage of this assay is that is circumvents the need for sophisticated expensive equipment such as thermal cyclers. Upon further optimization of sequence and annealing temperature this assay platform could be extended to the detection of various other infectious agents.

Griffin et al. (23) employed the exceptional fluorescence quenching properties of AuNPs and designed a NSET probe for the homogenous detection and quantification of a synthetic HCV RNA target (Figure 5). The probes consist of ssRNA labeled with fluorophores and adsorbed on the surface of AuNPs; the fluorophores in this state are quenched by the AuNPs, and the AuNPs are stabilized by the probes and remain dispersed in solution (red color). In the present of the target RNA, the probe binds to it and the dsRNA is released into the solution and 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 probes no longer stabilize them. The intensity of fluorescence and the target RNA concentration are directly proportional and therefore the method is used in HCV RNA quantification. The quenching efficiency improved by 3 orders of magnitude as the size of AuNPs increase from 5 to 70 nm, along with sensitivity. A detection limit of 300 fM of RNA was obtained with particle size of 110 nm.

Duan et al. (73) developed an assay for simultaneous detection of hepatitis B virus (HBV) and hepatitis C virus (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 from human sera. AuNP-labeled staphylococcal protein A was then added to detect captured antibodies, and silver staining was used to amplify the signal. This produced black spots on the assay chip that can be detected visually at the locations where the antibodies were found. The spot density was measured using a scanner. Turnaround time of the assay was under 40 minutes, and it was reported to detect down to 3 ng/mL of IgG.

Electrochemical detection is by far the most widely used technique in AuNP-based immunoassays. Shen et al. (24) developed an electrochemical immunoassay based on copper-enhanced AuNPs for the detection of hepatitis B surface antigen (HBsAg) using anodic stripping voltammetry (ASV). In this assay, two anti-HBsAg antibodies are utilized; one is conjugated to magnetic nanoparticles and the other to AuNPs. The two antibodies are mixed with the sample containing HBsAg and incubated at 37°C. The HBsAg is sandwiched between the two nanoparticle-conjugated antibodies and the resulting complex is then separated magnetically. Copper enhancer solution (composed of ascorbic acid and copper sulphate) is then added to the resuspended complex and incubated, which leads to copper deposition on the surface of AuNPs. Following another magnetic separation, the copper is then solubilized using nitric acid to release copper ions, whose concentration is then measured using anodic stripping voltammetry. The concentration of copper ions is 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 turn-around-time of 70 minutes, which is reasonably shorter than the currently used immunoassays for HBsAg detection. The assay has comparable performance to conventional enzyme-linked immunosorbent assay (ELISA).

Tang et al. (74) developed a bio-barcode assay for the detection of human immunodeficiency virus type 1 (HIV-1) with a detection limit 100-150 times lower than the corresponding ELISA assay. The assay was able to detect concentrations of the HIV-1 p24 antigen as low as 0.1 pg/mL versus 10-15 pg/mL detected by ELISA. Also, in patients with seroconversion, the assay detected infection 3 days earlier than the ELISA.

Bacteria

Soo et al. (75) developed a AuNP-based assay for detection of mycobacterium tuberculosis (MTB) and MTB complex (MTBC) in 600 patient sputum samples. AuNPs were conjugated to thiol-modified oligonucleotides to generate two probes each for detection of PCR-amplified MTB and MTBC DNA. The hybridization of the two probes to their target caused aggregation of AuNPs and a measurable red-shift in the absorbance of the solution. The assay had a sensitivity of 96.6% and 94.7% and a specificity of 98.9% and 99.6% for detection of MTBC and MTB, respectively, and the detection limit was 0.5 pmol of DNA.

Chlamydia trachomatis, a common causative agent of sexually transmitted diseases, was detected in urine using a colorimetric approach in the work of Jung et al. (76). This approach involves PCR amplification of the bacterial DNA, where one of the used primers is thiol-labeled, therefore, the amplicon yielded is thiolated at one end. When the amplicon is mixed with AuNPs, it adsorbs strongly on the surface of the AuNPs due to the strong affinity between AuNPs and thiols. Consequently, the AuNPs resist salt-induced aggregation and the solution color remains red. This resistance is attributed to the increase of negative surface charge density on the AuNPs (due to the negative charge of the phosphate sugar backbone of dsDNA) and the physical separation between AuNPs due to the adsorbed amplicons on their surfaces. In case of negative samples, no amplicon is produced by the PCR and subsequently when AuNPs are added, no thiolated dsDNA is available to stabilize them and they aggregated upon salt addition. This approach does not requires no modification of AuNPs and eliminates the need for extensive post-PCR processing, and can detect 100 copies of template DNA within about 1 hour of salt addition.

Biomarkers

Mayilo et al. (21) developed a prototype for a homogenous sandwich immunoassay based on using AuNPs as fluorescence quenchers. The assay detected cardiac troponin T (cTnT), a biomarker of myocardial infarction, with a limit of detection in the picomolar range, reportedly the lowest value published for this assay format for cTnT. The assay relies on two fragments of monoclonal antibodies for two different epitopes on the cTnT antigen. One of the fragments is conjugated with AuNPs while the other is labeled with a fluorescent dye (Cy3 or Cy3B), whose emission spectrum overlaps with the AuNP extinction band. When the cTnT antigen is present, it is sandwiched between the labeled antibody fragments. This brings the AuNP on one fragment close to the fluorescent dye on the other fragment. Consequently, the fluorescence of the dye is quenched by the AuNPs (with efficiency up to 95%). The detection was made using time-resolved fluorescence measurement, and the assay's detection limit was 0.02 nM (700 pg/mL) (21).

Anfossi et al. (77) developed another homogenous AuNP-based immunoassay model for the detection of human serum albumin (HSA) in urine. In this competitive assay the analyte; HSA, competes with AuNP-coated HSA for binding with the respective polyclonal antibody. The binding of the AuNP-HSA conjugate to the antibody resulted in a measurable a shift in the absorbance maximum of the solution, compared to the solutions lacking the analyte. 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 diagnosis of microalbuminuria.

A microfluidic system using surface immobilized bio-barcode assay was also developed for detection of prostate specific antigen (PSA), where concentrations as low as 40 fM were detected in serum samples. This value is two orders of magnitude lower than commercial ELISA and was achieved with a turnaround time of 80 minutes (71).

Georganopoulou et al. (78) have developed a bio-barcode for measuring amyloid-derived diffusible ligands, a potential soluble marker of Alzheimer's disease; amyloid-β-diffusable ligand, in cerebrospinal fluid specimens with attomolar sensitivity (78-79).

Single nucleotide polymorphisms

Another TB-related application was developed by Veigas et al. (80) who used AuNP-probes composed of AuNPs linked to thiol-modified oligonucleotides for the identification of multidrug resistant TB (MDRTB). The probes target single nucleotide polymorphisms (SNPs) in the beta subunit of the RNA polymerase gene of MTB (responsible for rifampicin resistance in 95% of resistant TB strains), after PCR amplification. In the presence of the target sequence, theAuNP-probe hybridizes to it, and the probe thus resists salt-induced aggregation, and the solution color remains red. This is due to formation of dsDNA providing charge stabilization and steric hindrance which prevents the aggregation of AuNP-probes. On the other hand, in the negative samples the AuNP-probes are free to aggregate in response to salt addition as they are not stabilized by the hybridization to their target, and the color of the solution turns blue. The assay had 81% concordance with the INNO-LiPA Rif TB (Innogenetics, Belgium) assay for detection of rifampicin-resistant TB strains. The assay is fairly simple to perform and requires just about 15 minutes after PCR amplification to obtain the results, as opposed to the complex INNO-LiPA which has a turnaround time of about 6 hours (80-81).

Small molecule detection

Zhang et al. (20) illustrated the potential utilizing the fluorescence quenching properties of AuNPs for multiplex molecular detection in a proof-of-concept experiment. They assembled 3' thiolated oligonucleotides at the surface of AuNPs (synthesized by citrate reduction), these oligonucleotides are specific to fluorescently-tagged aptamers targeting their respective molecules. In this probe structure, the fluorescence of the dye is markedly quenched due its close proximity to the AuNPs. When the target molecule is added, the aptamer binds to its target and is thus displaced and the dye's fluorescence is restored. Using this approach they designed a multicolor probe onto which aptamers specific for cocaine, potassium, and adenosine (labeled with different fluorescent dyes) were assembled. Upon mixing the targets with the probe and subsequent 1 hr incubation, the fluorescence of the different dyes was shown to increase in a concentration-dependent manner. This indicates the possible use of this approach as a simple and fast method for screening for target molecules.

Market 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 future for AuNPs in the marketplace. Additionally, the general nanotechnology investment trends, particularly in the healthcare sector, and growth of commercial suppliers back up this expectation. The 2009 estimate for the global nanomedicine market is $53 billion and is expected to reach $100 billion in 2014 (82). 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), on account of the potential in various medical applications. The NIH investment in 2001 was $40 million, and in 2011 the requested amount is $382 million from the budget of the National Nanotechnology Initiative (NNI), whose 2011 president's budget totals about $1.8 billion (83). Further downstream, 34 startup companies and 250 patent disclosure issuing or filings related to nanoparticle-based diagnostics and therapeutics were yielded by the industrial collaborations of the university-based National Cancer Institute (NCI) Alliance for Nanotechnology in Cancer Center, since its inception in 2005. It is also estimated that in 2014, billions of dollars of revenue will be attributed to the 16% of manufactured goods in the field of life science and health care, having a nanotechnology component (84).

On a more specific commercial market front, 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 more than triple by 2012, and 75.9% of this market is expected to belong to nanoparticles with biomedical applications (82). An example of current market successes is Nanosphere Inc. whose main focus area is nanotechnology-based molecular diagnostics. It made one of the two largest venture capitalist deals in nanotechnology in healthcare and life science in 2006, worth $57 million (runner up was worth $35 million), versus the average $10.4 million per deal for the remainder of the 12 deals that year (84). Nanosphere Inc. has obtained FDA clearance for two AuNP-based diagnostic tests. One test is for the determination of a patient's ability to metabolize Warfarin, the leading oral anticoagulant, via SNPs detection, and the other detection of influenza A & B and syncytial virus (85-86).

Conclusion

The known unique and versatile properties of AuNPs, along with the myriad of studies illustrating the potential for biomolecular detection, and the emerging clinical proofs-of-concept, are a testimony to the bright future of AuNPs in the field of in vitro diagnostics. At the same time, the increasing investments in research and emergence of successful startup companies drawing even more private funds, and already bringing approved commercial products to clinical laboratories, fortify the position of AuNPs in the future diagnostic market place. To top it off, as illustrated above, obtaining AuNPs via in-house synthesis or commercial sources (Table 5) is no obstacle, even to small laboratories with limited resources, and several AuNPs-based assays can be investigated with such resources. With ease of preparation and versatility of utilization options, the promise of cost-effectiveness, short turnaround times, enhanced sensitivity, and point-of-care testing, AuNPs are bound to have favorable winds in the their journey towards a key role in the arena in vitro diagnostics. All that is needed is some time and diligent thorough scientific work.

References

Tables and Figures

Table 1. Methods for Synthesis and Functionalization of AuNPs.

Synthesis Method

Size of AuNP (nm)

Surface cap

Conjugated Biomolecule

Bond Type

Application

Reference

Citrate reduction

12 - 13

Citrate

Thiol modified oligonucleotides

Covalent

SNP detection

(87)

Citrate reduction

13

Citrate

Thiol modified oligonucleotides with poly adenine spacer

Covalent

Detection of endonuclease activity

(88)

Citrate reduction

13

Citrate

Alkanethiol modified oligonucleotides

Covalent

SNP detection

(89)

Reduction by gallic acid

-

Gallic acid

-

-

Detection of lead ions

(90)

Citrate reduction

13

Citrate

Thiol modified DNA aptamers

Covalent

Detection of cocaine &adenosine

(91)

Citrate reduction

35

Citrate

Anti-EGFR

Electrostatic

Cancer cell imaging

(92)

EGFR: epithelial growth factor receptor

Table 2. Main characterization strategies of AuNPs.

Method

Utility

Reference(s)

TEM

Determination of nanoparticle morphology and size. It can also provide a picture of the AuNPs core (in case of core-shell structure).

(1, 43)

AFM

Size and morphology determination

(1)

SEM

Size and morphology determination

(54)

UV/visible spectroscopy

Determination of AuNPs concentration and estimated size.

(93)

Zeta potential measurement

Measurement of surface charge and stability of AuNPs and their conjugates

(3, 94)

AFM: atomic force microscopy; SEM: scanning electron microscopy; TEM: transmission electron microscopy.

Table 3. Examples of AuNPs-based immunoassays.

Target

Assay Description

Assay Performance

Reference(s)

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.

Detection limit was 0.17 nM.

(67)

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.

Detection limit was 10 ng/ml, an order of magnitude more sensitive than double-antibody sandwich ELISA.

(95)

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.

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

(19)

HBsAg

An electrochemical immunoassay based on copper-enhanced AuNPs for the detection of HBsAg using anodic stripping voltammetry.

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 minutes.

(24)

HBV and HCV IgG antibodies

A protein chip with immobilized HCV and HBV antigens is used to capture the antibodies. Staphylococcal protein A labeled with AuNPs detects the captured antibodies and silver staining is used to generate an amplified signal for visual detection. The silver enhancement results in black spots on the chip where the antibodies were captured.

Turnaround time was under 40 minutes, and the assay was reported to detect down to 3 ng/ml of IgG.

(73)

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 impedence spectroscopy.

Lower detection limit was 0.64 pg/mL and the linear dynamic range was from 3 to 100 pg/mL.

(96)

HSA

A competitive homogenous immunoassay where HSA, competes with AuNP-coated HSA for binding with the specific antibody. The binding of the AuNP-HSA conjugate to the antibody resulted in a measurable a shift in the absorbance maximum of the solution.

Detection limit of 3 mg/L in human urine.

(77)

PSA

Bio-barcode assay for PSA detection in patient serum with scanometric detection.

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.

(70)

AFP: Alpha-phetoprotein; AIV: Avian influenza virus; CA15-3: Cancer antigen 15-3; 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.

Table 4. Examples of AuNPs-based molecular assays.

Target

AuNPs Modification

Detection

Notes