key: cord-0036944-unvsfyr9
authors: Franco, Ricardo; Pedrosa, Pedro; Carlos, Fábio Ferreira; Veigas, Bruno; Baptista, Pedro V.
title: Gold Nanoparticles for DNA/RNA-Based Diagnostics
date: 2015-08-07
journal: Handbook of Nanoparticles
DOI: 10.1007/978-3-319-15338-4_31
sha: ab774b72751d8d151e4da5e48f0faa1ac7f165bc
doc_id: 36944
cord_uid: unvsfyr9

The remarkable physicochemical properties of gold nanoparticles (AuNPs) have prompted development in exploring biomolecular interactions with AuNPs-containing systems, pursuing biomedical applications in diagnostics. Among these applications, AuNPs have been remarkably useful for the development of DNA/RNA detection and characterization systems for diagnostics, including systems suitable for point of need. Here, emphasis will be on available molecular detection schemes of relevant pathogens and their molecular characterization, genomic sequences associated with medical conditions (including cancer), mutation and polymorphism identification, and the quantification of gene expression.

Gold nanoparticle ARMS Amplification- Examples of consecutive steps of nanoparticle synthesis, ligand functionalization, and optional bio-conjugation. AuNPs of different morphologies (rod-shaped, triangular plates, but mostly spherical) can be linked to thiolated bifunctional ligands (11-mecarcaptoundecanoic acid (MUA); thiolated nickel(II) nitrilotriacetate (Ni-NTA); or singlestranded thiolated nucleic acid oligomers). Optional additional bio-conjugation can include antibodies (e.g., chemically cross-linked to MUA) or a His-tagged protein (binds to Ni-NTA) electromagnetic field of light, the AuNPs' free electrons collectively oscillate with respect to the positive metallic lattice [2] . This process is resonant at a particular frequency and is termed localized surface plasmon resonance (LSPR). Considering AuNPs, both the electric field intensity and the scattering and absorption cross sections are strongly enhanced at the LSPR frequency, which therefore occur in the visible (Fig. 2) . Because of this, optical cross sections of metal nanoparticles (10-100 nm) are roughly five orders of magnitude larger than those of traditional organic dyes and fluorophores [2] . LSPR may be easily tuned by changing the nanostructure size, shape, composition, or environment [2, 3] (Fig. 2) . Nanodiagnostic methodologies have the ability to make molecular biological tests become faster, more sensitive, and flexible at reduced costs [4] . These new approaches can be integrated with conventional standard analytical methods, improving current techniques, and have been shown to be more sensitive and specific than conventional commercial molecular diagnostics. However, the great majority of these new systems still need further evaluation and validation with clinical samples and targets to transpose these tools from laboratory to clinic.

Despite the variety of nanoscale systems for biomolecular assays, AuNP-based systems have been mostly explored due to their unique physicochemical properties and are becoming a key element of nanotechnology-based detection of pathogens [5] . The development of schemes that can improve the sensitivity and specificity of molecular diagnosis methods and consequently to decrease the impact of a certain phenotype/ disease is an easier task since the completion in 2003 of the Human Genome Project. In fact, targets have been revealed and their genetic sequence is well known [6] .

This chapter presents several examples of applications involving AuNPs that can be translated into clinical applications. DNA/RNA detection schemes are based on the remarkable optical properties of AuNPs supplementary to the easiness of chemical functionalization through thiol ligands (e.g., thiol-modified oligonucleotides, antibodies, and other biomolecules). Recognition events occur at the nanoscale, i. e., in a one-to-one interaction between analytes and the nanoscale structures that act as signal transducers, allowing for increased sensitivity at lower costs. Each section encompasses a set of approaches based on the nanoscale properties involved in detection, namely: (i) colorimetric sensing depending on interparticle distance, which represents the most developed approach, especially for nucleic acid detection; (ii) fluorescence quenching/enhancement properties of AuNPs; (iii) plasmonics and light-scattering properties, including detection based on surface-enhanced Raman (SERS) and LSPR spectroscopies; (iv) piezoelectric sensors using AuNPs to increase sensitivity of detection based on mass increase; and (v) electrochemical detection methods based on electrical signal enhancement or generation provided by AuNPs (summarized in Table 1 ). The final section will present the latest advances in the utilization of AuNPs for diagnostics at point of care, using lateral flow devices.

Among detection methods based on AuNPs, colorimetric approaches are the most frequent, allowing for simplicity and portability, making them ideal for diagnostics Fig. 2 Size, shape, and composition tunability of the plasmon resonance of gold nanostructures, (a) tuning the LSPR frequency of the gold nanorod long-axis mode by synthetically controlling aspect ratio, (b) silica core-gold nanoshells show plasmon resonance frequency tunable from the visible to the NIR by changing the shell thickness relative to the core size, (c) increase in the plasmon scattering to absorption ratio by increase in particle volume in spherical AuNPs (Reprinted with permission from Jain et al. [3] Copyright 2008 American Chemical Society) for point of care (POC). These methods rely on the colorimetric changes of an AuNP colloidal solution upon aggregation. The aggregation process can be either mediated by changes to the dielectric medium or upon recognition and binding to a specific target. Colloidal solutions of spherical AuNPs present an LSPR band that strongly depends on interparticle distance. These systems rely on the ability of complementary targets to modulate and control interparticle interactions (e.g., attractive and repulsive forces), which define whether AuNPs are dispersed or aggregated.

Functionalized AuNPs: Cross-Linking Approach AuNPs modified with thiolated oligonucleotides led to the first application of AuNPs in nucleic acid detection. In their approach, Mirkin and coworkers [7] functionalized AuNPs with oligonucleotides modified with a thiol group at their 3 0 and 5 0 ends, whose sequences were contiguous and complementary to a target in a tail-to-tail (or head-to-tail) conformation. The hybridization of the two Au-nanoprobes with the target resulted in the formation of a polymeric network (cross-linking mechanism), which brought AuNPs in close vicinity to cause a red to blue color change, which can readily be detected visually, or by UV-vis spectroscopy (Fig. 3b) .

The first application of this approach, for pathogen detection, was described by Storhoff and coworkers [8] . They developed a "spot-and-read" colorimetric detection method for the identification of DNA sequences. In this assay, the color change of DNA-modified gold probes was detected after spotting onto a glass surface illuminated by a light beam. This light-scattering method allowed detection of DNA in zeptomole quantities without signal or target DNA amplification. In comparison to the absorbance-based methods, this approach allowed a 4 order of magnitude increase in sensitivity. This way, it was possible to detect the mecA sequence gene in methicillin-resistant Staphylococcus aureus directly from genomic DNA samples. The system showed high sensitivity with a limit of detection (LOD) of 66 ng/μL of DNA. Following the same approach, several groups were able to develop methods for human immunodeficiency virus type 1 [9] and Cryptosporidium parvum [10] with direct detection capabilities, allowing to circumvent expensive enzymatic DNA amplification reactions. Recently, Weigum et al. developed an amplification-free molecular assay for the detection of Cryptosporidium parvum oocysts [11] . The assay targeted the C. parvum 18 s rRNA, with an LOD of 4 Â 10 5 copies of RNA per μL per reaction mix. The ability to detect the C. parvum oocysts without the need for complex amplification is of utmost relevance in resource-limited settings where protozoan detection is needed the most.

Toward the development of naked eye scheme for detection and characterization of pathogen's RNA, Gill et al. integrated a nucleic acid sequence-based amplification (NASBA) and AuNP probes for the specific detection of Mycobacterium tuberculosis [12] . The 16S rRNA of mycobacterial RNA was amplified via isothermal NASBA and then hybridized with specific probes. This method showed an LOD of 10 CFU ml À1 with a sensitivity and specificity of 94.7 % and 96 %, respectively. Following the same detection approach, Soo et al. designed a set of 

Yang et al. [115] Au-nanoprobes for M. tuberculosis complex strains DNA identification [13] , showing a detection limit of 0.5 pmol of DNA. The 2-h assay comprises two main steps: Fig. 3 Gold nanoparticle-based colorimetric assays, (a) colorimetric assay based on naked AuNPs, (b) cross-linking hybridization assay, (c) non-cross-linking hybridization assay (Reproduced from Larguinho and Baptista [92] with permission from Elsevier) target DNA amplification via single or nested PCR, followed by detection using the specific nanoprobes.

During the last few years, several advances have been aimed at reducing the gap between light-scattering imaging and naked eye sensitivity. Earlier methods used enzymatic amplification techniques, such as PCR, asymmetric PCR, and NASBA [14] [15] [16] [17] [18] [19] . Recently, Zagorovsky and Chan reported on the integration of a multicomponent DNA-responsive DNAzyme with colorimetric detection using AuNPs, allowing for nonenzymatic signal amplification [20] . This constitutes a simple and fast colorimetric approach for the detection of DNA targets of bacteria, viruses, and parasites (LOD of 50 pM). This method is also able to detect multiple sequences in parallel. The color readout discards the use of complex equipment that makes it suitable for POC assays. More recently, this same approach was used for the naked eye and direct (amplification-free) detection of Kaposi's sarcomaassociated herpes virus [17] and Salmonella sp. [16] . Kalidasan Functionalized AuNPs: Non-Cross-Linking Approach Baptista and coworkers developed an inexpensive approach for the colorimetric detection of DNA sequences [21] . This method uses just one Au-nanoprobe, instead of the two required in the cross-linking method (see previous section). Detection is achieved after comparison of the solutions' color following salt addition. The presence of a complementary target does not allow nanoprobe aggregation, and the solution retains the original red color; non-complementary/mismatched targets allow gold nanoprobe aggregation, and the solution turns blue. The first clinical application of this strategy was in the rapid and sensitive detection of Mycobacterium tuberculosis [22] (Fig. 3c) . The gold nanoprobes were functionalized with thiol-modified oligonucleotides harboring a sequence derived from the pathogen's RNA polymerase β-subunit gene sequence. This methodology had been previously tested in clinical samples and, if associated to PCR, shows great sensitivity [21] . This protocol was further used for the rapid detection of M. tuberculosis complex (MTBC) members and simultaneous identification of mutations linked to antibiotic resistance [23] . LOD was determined at 75 nM. Using a set of three Au-nanoprobes based on the gyrB locus, specific identification of MTBC, M. bovis, and M. tuberculosis was easily achieved [24] . Considering application at POC, Baptista and colleagues integrated this non-cross-linking method in an optoelectronic platform (an amorphous/nanocrystalline biosensor and a light emission source) that assesses the colorimetric changes. This low-cost simple platform may be of use once integrated in a microfluidic test device for improved TB diagnostics with a tenfold reduction of reagents [25] [26] [27] . Recently, in an effort to increase sensitivity and ease of use, this detection strategy was integrated onto a paper-based platform [28] . Differential color scrutiny is captured and analyzed with a generic "smartphone" device to evaluate assay results and perform RGB analysis. The possibility to send the acquired information to a central lab is also considered via 3G technology. GPS location may be added to each test image, which would provide actual epidemiologic data on MTBC (Fig. 4) .

Following the same trend as the cross-linking method, several optimizations have been made toward higher sensitivity with the integration of NASBA or other RNA amplification methods [29, 30] . Liandris et al. optimized the non-crosslinking approach described above to the detection of TB without the need for target amplification [31] . The detection is based on the fact that double-and singlestranded oligonucleotides show distinct electrostatic behaviors. Hybridization leads to double-stranded DNA formation, which cannot uncoil sufficiently to expose its bases toward the Au-nanoprobe. Therefore, the Au-nanoprobe aggregates under the acidic conditions of the test. More recently, Padmavathy et al. reported on the visual direct detection of Escherichia coli without the need for nucleic acid amplification. This approach is able to detect~54 ng for unamplified genomic DNA, while reducing the overall time for detection to less than 30 min [32] . The non-cross-linking method was further applied to the identification and quantitation of RNA associated with human disease [33] . This system was successfully applied in the detection of BCR-ABL fusion gene mRNA, a hallmark for chronic myeloid leukemia [34] . This approach was capable to detect less than 100 fmol/μl of the specific RNA target (less than 10 ng/μl of total RNA). Also, the non-cross-linking approach could distinguish common-base mismatch (SNPs) within the β-globin gene [35] . The authors could detect three different individual mutations using only one Au-nanoprobe.

Functionalized AuNPs: Sandwich Assays DNA sandwich methods are very common in various POC systems. Microarrays, streptavidin-biotin stripes, and lateral flow cytometer systems are usually based on this method [36, 37] . Although each method varies in the building block, all share the same principle: a DNA probe is fixed on a support in the form of stripes or spots, followed by sample DNA hybridization to the fixed probe [38] [39] [40] [41] . The second probe, consisting of AuNPs functionalized with 5 0 -thiol-modified oligonucleotides (the Au-nanoprobe), is then hybridized to a second region of the target sample DNA. Total complementarity with both probes prevents the Au-nanoprobes from being removed by washing and yields the final assay result after signal enhancement by silver deposition (Fig. 5) . Sandwich-based assays are being developed in a lateral flow strip scheme with increased sensitivity and are today a suitable alternative for the detection of PCR amplicons [37, [42] [43] [44] [45] [46] [47] .

Storhoff and coworkers introduced the first application of the DNA sandwich approach for pathogen and human detection, applying it to methicillin resistance S. aureus, and human MTHFR gene [48] . Later, Cao and coworkers developed a similar method using a visual DNA microarray using Au and Ag stain together with 

A B C D Fig. 5 Microarray DNA detection via AuNPs. DNA hybridization to microarrays and detection using silver-amplified gold nanoparticle probes. Following target hybridization to a capture probe immobilized on the array surface, a secondary Au-nanoprobe is used for detection. DNA target and Au-nanoprobe hybridization can be performed in a single step. An additional signal amplification step may be introduced via silver deposition (Reproduced from Storhoff et al. [48] with permission from Elsevier) multiplex asymmetrical PCR. This approach allowed for the simultaneous detection of Ureaplasma urealyticum and Chlamydia trachomatis. The surface immobilized 5 0 -end-amino-modified oligonucleotides, acted as capturing probes to bind the complementary biotinylated targets. Au-streptavidin conjugates specifically bind to biotin, and agglomeration was enhanced with silver for enhanced colorimetric signal. The sensitivity of this assay has been much improved by combination with silver/gold deposition to enhance light-scattering properties and further decrease detection limits, allowing visual or optical detection (e.g., image scanner). Using a similar approach, Zhao and coworkers developed a microarray platform using AuNP-based genomic microarray assay for specific identification of avian influenza virus H5N1 RNA [41] . Viral RNA was detected within 2.5 h using capture-targetintermediate hybridization, and AuNP triggered silver staining, without target amplification or any enzymatic reactions (LOD of 100 f. for purified PCR amplicons and TCID 50 units for H5N1 RNA).

Unmodified AuNPs have also been used for detection of DNA/RNA biomarkers [49] . These systems rely on the unspecific adsorption of biomolecules to non-functionalized AuNPs, usually profiting from the differential affinity of ssDNA and dsDNA to the AuNPs' surface. ssDNA nucleobases electrostatically interact with the citrate capped AuNPs' surface, thus stabilizing the AuNPs against higher ionic strengths. dsDNA molecules do not adsorb the same way to the AuNPs and, as such, aggregation occurs (Fig. 3a) . Griffin et al. used unmodified AuNPs for the detection and quantification of hepatitis C virus (HCV) RNA. Their approach consists on the adsorption of a synthetic ssDNA probe complementary to the target [50] . Whenever the target is present, the ssDNA probes and target hybridize and stop being available to stabilize the AuNPs. Despite a clear colorimetric visual detection, the use of hyper-Rayleigh scattering measurement allowed for an increase in sensitivity of two orders of magnitude. Shawky et al. used the same method using clinical samples to detect the presence of HCV RNA [51] . The isolated RNA was added to a solution containing the complementary oligonucleotide probe, and after a denaturing and annealing cycle, unmodified AuNPs were added. This extremely simple and inexpensive assay, which does not include an RT-PCR step, presented a detection limit of 50 copies/reactions and exhibited a sensitivity of 92 % and a specificity of 89 %. A similar approach using an S1 nuclease to discriminate single mismatches in DNA with unmodified AuNPs was proposed, in which dNMPs adsorb to AuNPs stabilizing them further in relation to ssDNA [52] . In the presence of a mismatch between the synthetic oligonucleotide and the DNA sample, the S1 nuclease degrades ssDNA-ssRNA hybrid into its monomers (dNMPs), which increase the stability of AuNPs in solution. Also using an enzyme-mediated process, [53] tested the activity of ribonuclease H (RNase H) activity of HIV-1 reverse transcriptase. In the presence of RNase H activity RNA-DNA hybrids are formed, otherwise ssRNA remains in solution adsorbing to the AuNPs. [54] took advantage of asymmetric PCR to generate long ssDNA amplicons that stabilize AuNPs in solution after salt addition. However, if no amplification occurs, no long ssDNA amplicons are present, leading to AuNPs aggregation due to the increasing ionic strength (LOD set at 10 ng of target DNA).

Fluorescence is frequently used for biosensing due to sensitivity and easiness of implementation. The extraordinary quenching of fluorophores by noble metal NPs prompted the development of several new approaches with remarkable sensitivity and specificity. Gold nanoprobes can take advantage of the interesting luminescent properties of AuNPs for molecular diagnostics. In fact, AuNPs can cause fluorescence enhancement or quenching of chromophores in their vicinity. Chromophores within ca. 5 nm of the surface of the AuNP have their fluorescence quenched by non-radiative pathways, while chromophores at larger distances (>10 nm) show a fluorescence enhancement of up to 100-fold [55] [56] [57] [58] . The quenching properties of AuNPs have been applied in two approaches: molecular beacons, which rely on NPs functionalized with fluorescent-labeled hairpin structures, and AuNPs nanoprobes with ssDNA that hybridize to another fluorescent-labeled ssDNA probe [59] . Molecular beacons, i.e., AuNPs functionalized with fluorescent-labeled ssDNA where the NP act as a fluorescence quencher via the NP surface energy transfer occurring between the dye (donor molecule) and the NP's surface (acceptor). On the other hand, noble metal nanoprobes rely on NPs functionalized with ssDNA hybridized to a complementary fluorescent-labeled ssDNA probe. Since ssDNA naturally adsorbs to the AuNPs, the proximity of the fluorophore with the nanoparticle is close enough for a 98 % of quenching efficiency. In the presence of a complementary target, dsDNA is formed resulting in a spatial separation of the fluorophore from the AuNPs, thus restoring the fluorescence signal (Fig. 6) .

The combination of AuNPs and semiconductor quantum dots (QDs) as a Förster resonance energy transfer (FRET) system has been used to develop fluorescence competition assays for nucleic acid, protein, and antibody/antigen detection where the dye is replaced by QDs Fluorescence modulation by AuNPs has been used to monitor specific nucleic acid hybridizations. Two different research groups have used this approach to detect synthetic DNA of Campylobacter and HCV synthetic RNA, respectively, with single-base mismatches detection capability [50, 60] . Deng Zhang et al. used a biobarcode-type assay to obtain a fluorescence signal of PCR-amplified Salmonella enteritidis DNA [61] . In this type of assay, two types of nanoparticles are functionalized. Firstly, magnetic sulfo-SMCC-modified nanoparticles are functionalized with oligonucleotide sequence complementary to the sample DNA. Secondly, AuNPs are bi-functionalized with an oligonucleotide sequence specific for the target (in lower percentage), and another sequence that contains a nucleotide sequence 3 0 modified with a fluorophore (in higher percentage). Both nanoparticles are mixed with the sample DNA and, in case of total complementarity, the sample DNA serves has linker of both nanoparticles. After the hybridization step, a magnetic field is generated to isolate hybridized NPs from non-hybridized ones, and the thiol bonds are broken releasing the fluorophore-labeled oligonucleotides (LOD of 21.5 fM). Ganbold et al. describe a similar method, but the synthetic probe is functionalized with a fluorophore whose fluorescence is quenched if the ssDNA is adsorbed to the particle but not if there is complementarity to the target [62] . Standard fluorescence signal is measured but there is also the possibility of using sub-aggregation conditions for Raman spectroscopic analysis. The method was tested with influenza A (H1N1) synthetic DNA and was able to discriminate single-base mismatches.

In another application, Dubertret and coworkers used Au-nanobeacons to detect single-base mismatches with 100Â more sensitivity than that of conventional molecular beacons [63] . Similarly, Beni et al. used Au-nanobeacons to successfully detect a mutation that occurs in 70 % of cystic fibrosis patients using nM concentrations of DNA target [64, 65] . In the second case, AuNPs can be combined with dye-labeled ssDNA probe and is used to detect specific DNA targets mediated by energy transfer mechanisms (FRET). The method consists in designing probes that identify complementary and contiguous sequences on the target, where hybridization forces the dye into close vicinity of the AuNPs' surface and the fluorescence signal decreases [66] . More recently, Wang et al. proved that the fluorescence quenching/enhancement conferred by AuNP could be used as an SNP genotyping system suitable for point of care [67] .

Plasmonic sensing here refers in the broader sense to detection based on lightscattering techniques, namely, SERS and LSPR spectroscopies. These techniques take advantage of the different size and shapes of AuNPs to detect the biding of molecules and alterations to conformation. Plasmonic nanoparticles act as signal transducers capable of converting minute changes to the local refractive index into sharp spectral shifts [68] (Fig. 2) .

Raman scattering originates from the inelastic scattering of photons that interact with the analyte molecule, changing its vibrational states [59] . This interaction generates unique narrow spectrum bands that may be enhanced by metal nanostructures, allowing multiplex detection assays. Metallic nanosurfaces associated to SERS allow detection of specific biomolecules and is usually performed by means of a molecule with an intense and characteristic Raman signature (e.g., dye). SERS detection methods show great similarities to those previously described methods in colorimetric and fluorescent assays.

The first proof of concept of this approach for pathogen detection was introduced by Cao et al., with a multiplexed detection of oligonucleotide targets with AuNPs labeled with oligonucleotides and Raman-active dyes [69] . AuNPs help the formation of the silver coating, i.e., SERS promoter for the dye-labeled particles captured by the target molecules. This may be employed in a microarray format. Six DNA targets with six Raman-labeled NP probes were easily distinguished together with two RNA targets with single nucleotide polymorphism (LOD of 20 fM). This allowed for the multiplexed direct detection of hepatitis A virus, hepatitis B virus, HIV, Ebola virus, Variola virus, and Bacillus anthracis.

Hu et al. developed a sensitive DNA-SERS biosensor based on multilayer metal-molecule-metal nanojunctions [70] . The sensor could detect as little as 0.1 atomolar of a HIV-1 DNA sequence. High sensitivity may be attained via a bio-barcode approach combined with a silver-enhanced spot test. Isola and coworkers explored a similar approach for HIV detection using Raman-active dye-labeled DNA on Ag or AuNPs [71] . Upon target recognition, the cross-linking cages the Raman reporter in "hot spots" between NPs and, thus, enhances the SERS signal.

Quartz crystal microbalances (QCM) have been extensively investigated as transducers for hybridization-based DNA sensors, such as for the detection of gene mutations associated with disease and foodborne pathogens. Improving the sensitivity of QCM sensors has been based on probe immobilization and signal amplification strategies that include nanoparticles [72] . Nanoparticles are effective amplifiers for QCM DNA detection because they have a relatively large mass compared to that of DNA targets [73] . The use of AuNPs coupled to the DNA targets acts as "mass enhancers," i.e., signal amplification, thus extending the limits of QCM DNA detection (Fig. 7) .

Chen and coworkers introduced the first nanoparticle-amplified QCM DNA sensor for foodborne pathogens [72] , using the sandwich hybridization of two specific probes: one specific to E. coli O157:H7 immobilized onto the piezoelectric surface and a second conjugated to the AuNPs as "mass enhancer" and "sequence verifier" by amplifying the frequency change of the piezoelectric part.

The oscillation frequency of the piezoelectric sensor decreased with increasing weight at the sensor's surface (i.e., sandwich hybridization involving the target oligo, the sensor's probe, and the circulating DNA-functionalized AuNP). Thus, PCR products amplified from concentrations of 1.2 Â 10 2 CFU/mL of E. coli O157: H7 were easily detectable. Wang et al. also used AuNPs to functionalize QCM for E. coli DNA detection, employing two sizes of AuNPs to increase sensitivity [73] . First, 18-nm AuNPs were immobilized onto the QCM surface to support the ssDNA probes that will bind specifically to the biotinylated DNA of target bacteria. The gold layer binds a higher number of ssDNA molecules to the QCM, thus increasing sensitivity. The biotinylated DNA from the target organism binds to the sensor, which is recognized by avidin- [72] with permission from Elsevier) fragment of the Ba813 gene and the 340-bp fragment of the pag gene in plasmid pXO1 [74] . A thiol DNA probe was immobilized onto the QCM gold surface to hybridize to the target ssDNA obtained by asymmetric PCR. The DNA-functionalized QCM biosensor could specifically recognize B. anthracis (and distinguish from its closest species, Bacillus thuringiensis) -LOD of 3.5 Â 10 2 CFU/mL for B. anthracis vegetative cells without culture enrichment.

Other physicochemical properties of AuNPs have also been used in detection protocols, such as electrochemical activity. AuNPs are also extremely useful in electrochemical bioassays, to bind enzymes to electrodes, mediate electrochemical reactions as redox catalysts, and amplify recognition signals of biological processes [1, 75, 76] . Examples of applications in DNA detection include direct detection of AuNPs anchored onto the surface of the genosensor, conductimetric detection, and AuNPs as carriers of other AuNPs or of other electroactive labels [77] . In general, electrochemical biosensors employ potentiometric, amperometric, or impedimetric transducers. Zhang et al. described an approach that makes use of the bio-barcode method to detect DNA from Salmonella enteritidis and Bacillus anthracis in a multiplex assay where the characteristic molecular signature sequences are labeled with cadmium and lead ions, respectively [78] . Following magnetic separation, the ions are cleaved from the oligonucleotides and the square-wave anodic stripping voltammetry analyzed on screen-printed carbon electrode (SPCE) chips. The differential signal of both ions allows the parallel read of either DNA signature. Further signal augmentation was attained via introduction of PCR amplification and the detection limit set at 0.5 ng/mL for Cd 2+ and 50 pg/mL for Pb 2+ .

Also using an electrochemical approach, it was possible to directly detect M. tuberculosis DNA with a detection limit of 1.25 ng/mL [79] . Firstly, AuNP are dual-labeled with a complementary sequence of target DNA and an enzyme alkaline phosphatase. Secondly, Au-nanoprobes are fixed onto indium tin oxidecoated glass plates that work as electrodes, and the extracted DNA followed by a second mix of dual-labeled AuNPs are then added and let to hybridize. Then, in the presence of paranitrophenyl phosphate, if both probes hybridize, the substrate is converted in paranitrophenol generating a signal that can be measured by differential pulse voltammetry.

Vetrone et al. describe a bio-barcode-based assay in which, instead of using a signature DNA sequence, the amount of gold separated via the magnetic nanoparticles is measured [80] . Hydrochloric acid promotes dissolution on an SPCE's plate that measures Au 3+ ions. The method was capable to detect unamplified DNA specific of Salmonella enterica serovar Enteritidis (S. enteritidis) at an LOD of 7 ng/uL.

Multiplex approaches have also been described. Li and colleagues used DNA arrays on gold surface combined with reporting silver nanoprobes to detect herpes simplex virus, Epstein-Barr virus, and cytomegalovirus by differential pulse voltammetry. The silver tag is allowed for the detection of as little as 5 aM of target DNA. Zhang et al. explored anodic stripping voltammetry using a screenprinted carbon electrode chip together with bio-barcoded AuNPs and magnetic NPs, to detect B. anthracis and S. enteritidis [78] .

The lateral flow assay (LFA) or lateral flow immunochromatographic assay was introduced in 1988 by Unipath and is the most common commercially available POC diagnostic platform [81] . Most of the success of LFAs is due to the low cost and simplicity of operation. In fact, these devices are currently used in resourcepoor or non-laboratory environments [37] . Generally, LFAs consist of a porous white membrane striped with a line of antibodies or antigens that interact with AuNP-antibody nanoprobes visible by the naked eye (Fig. 8) . These types of platforms can support competitive or noncompetitive immunoassays. Competitive immunoassays are used for detection of low molecular weight target molecules like pesticides, hormones, and drugs, whereas competitive formats are used to detect high molecular weight targets with at least two binding sites [82] . With a suitably configured system, LODs in the picomolar range may be easily obtained.

Many LFAs have been developed on the capillary test strip platform during the past 30 years. LFAs are easy to use, disposable, fast to perform, and relatively cheap [83] . Integration of such approaches with gold labels introduce several advantages in lateral flow designs, since they have been shown to be stable in liquid and dry without loss of signal [84] . Today, these platforms are "everywhere," from pregnancy, heart attack, blood glucose, and metabolic disorders to small-molecule detection (e.g., narcotics, drugs, toxins, antibiotics, etc.) and even pathogens, such as anthrax, salmonella, and some viruses. LFAs have been applied to immunodiagnostics, RNA detection, and even identification of whole bacteria. Some of the more recent designs and publications show the detection of DNA without the need of amplification by PCR opening yet another vast field of new applications. In fact, Wilson and coworkers demonstrated how unlabeled PCR products can be detected with an antibody-free lateral flow device at room temperature [85] . Trials are being conducted for massive multi-parallel screening together with LFAs microarrays [86] .

Recently several lateral flow strip-based devices have been developed. Chua et al. developed a typical design where the detector reagent recognizes the fluorescein haptens of PCR-amplified DNA target and produces visual red lines with a recorded LOD of 5 ng of DNA [87] . Rastogi et al. optimized this approach via the integration of locked nucleic acid conjugated Au-nanoprobes together with signal amplification protocols for as little as 0.4-nM DNA [40] . More recently, Rohrman et al. presented a lateral flow assay coupling NASBA RNA amplification with Au-nanoprobes and gold enhancement solution to quantitate amplified HIV RNA [88] . These results suggest that the lateral flow assay can be integrated with amplification and sample preparation technologies, allowing viral load testing to monitor therapy response in limited resource setting.

Also, quantification of microRNA was demonstrated by Shao-Yi Hou et al. using of LFA nucleic acid test strips and AuNPs to detect miRNAs as low as 1 fmol [89] .

LFAs evolved from a simple device into increasingly sophisticated platforms with internal calibrations and quantitative readouts. Nevertheless, single-use POC test devices are often affected by critical issues associated to dispersion of dried reagents into the sample, sample mixing with reagents, and effective control of incubation time and conditions. Several enabling technologies such as printing and laminating of components and microfluidic technologies are contributing to advances in LFAs. For a deeper perception on this technological perspective, including recent innovations in LFA technology, see the excellent review by Gubala et al. [36] . Some detection kits using AuNPs and their colorimetric sensing capability for human genetic tests can already be found in the market. For example, Nanosphere TM offers FDA-approved assays aimed at identifying typical mutations in coagulation factors F5 (1691G > A), F2 (20210G > A), and MTHFR (677C > T) without the need for nucleic acid amplification or to genotype polymorphisms associated with the drug-metabolizing enzyme CYP450 [90, 91] . Samples are processed through a cartridge where the sample is analyzed via an automated processor and reader.

AuNPs have become one of the most effective transducers and tags used in molecular diagnostics. In fact, the minimum number of AuNPs that can be detected by the naked eye against a white background is around roughly 10 10 . Assuming one molecule of target molecule per AuNP, ten femtomoles may be detected, which compares negatively to the amount of target analyte in a sample that is usually one million less [82] . An alternative to diminish this difference can combine detection with amplification techniques. These can either focus on the target, such as PCR or antigen concentration, or on the assay development, such as by silver enhancement. The conjugation of silver enhancement and microfluidics allows the exploitation of multiple protein and nucleic acid targets in a bio-barcode format. Nevertheless, the cost of manufacture of these microfluidic devices remains a problem, and the technology for POC use in detection protocols is still under development; thus, translation to the clinical setting remains a challenge.

Calculated absorption and scattering properties of gold nanoparticles of different size, shape, and composition: applications in biological imaging and biomedicine

Noble metals on the nanoscale: optical and photothermal properties and some applications in imaging, sensing, biology, and medicine

Nanodiagnostics: a new frontier for clinical laboratory medicine

A bio-barcode assay for on-chip attomolar-sensitivity protein detection

Finishing the euchromatic sequence of the human genome

A DNA-based method for rationally assembling nanoparticles into macroscopic materials

Homogeneous detection of unamplified genomic DNA sequences based on colorimetric scatter of gold nanoparticle probes

One-step label-free optical genosensing system for sequence-specific DNA related to the human immunodeficiency virus based on the measurements of light scattering signals of gold nanorods

Oligonucleotide-gold nanoparticle networks for detection of Cryptosporidium parvum heat shock protein 70 mRNA

Amplification-free detection of cryptosporidium nucleic acids using DNA/RNA-directed gold nanoparticle assemblies

Nanodiagnostic method for colorimetric detection of Mycobacterium tuberculosis 16S rRNA

A simple gold nanoparticle probes assay for identification of Mycobacterium tuberculosis and Mycobacterium tuberculosis complex from clinical specimens

Optical detection of human papillomavirus type 16 and type 18 by sequence sandwich hybridization with oligonucleotide-functionalized Au nanoparticles

Real-time colorimetric detection of target DNA using isothermal target and signaling probe amplification and gold nanoparticle cross-linking assay

Direct visual detection of Salmonella genomic DNA using gold nanoparticles

Multiplexed colorimetric detection of Kaposi's sarcoma associated herpesvirus and Bartonella DNA using gold and silver nanoparticles

A gold nanorod-based optical DNA biosensor for the diagnosis of pathogens

A broad-range method to detect genomic DNA of multiple pathogenic bacteria based on the aggregation strategy of gold nanorods

A plasmonic DNAzyme strategy for point-of-care genetic detection of infectious pathogens

Gold-nanoparticleprobe-based assay for rapid and direct detection of Mycobacterium tuberculosis DNA in clinical samples

Nanodiagnostics for tuberculosis

Au-nanoprobes for detection of SNPs associated with antibiotic resistance in Mycobacterium tuberculosis

Gold nanoprobe assay for the identification of mycobacteria of the Mycobacterium tuberculosis complex

Bio-microfluidic platform for gold nanoprobe based DNA detection-application to Mycobacterium tuberculosis

Inkjet printed and "doctor blade" TiO2 photodetectors for DNA biosensors

Portable optoelectronic biosensing platform for identification of mycobacteria from the Mycobacterium tuberculosis complex

Non-crosslinking gold nanoprobes for detection of nucleic acid sequence-based amplification products

An improved non-crosslinking gold nanoprobe-NASBA based on 16S rRNA for rapid discriminative bio-sensing of major salmonellosis pathogens

Direct detection of unamplified DNA from pathogenic mycobacteria using DNA-derivatized gold nanoparticles

A direct detection of Escherichia coli genomic DNA using gold nanoprobes

RNA quantification using gold nanoprobesapplication to cancer diagnostics

RNA quantification using noble metal nanoprobes: simultaneous identification of several different mRNA targets using color multiplexing and application to cancer diagnostics

Nanodiagnostics: fast colorimetric method for single nucleotide polymorphism/mutation detection

Point of care diagnostics: status and future

Lateral flow (immuno)assay: its strengths, weaknesses, opportunities and threats. A literature survey

Visual DNA microarrays for simultaneous detection of Ureaplasma urealyticum and Chlamydia trachomatis coupled with multiplex asymmetrical PCR

Optical detection of nanoparticle-enhanced human papillomavirus genotyping microarrays

DNA detection on lateral flow test strips: enhanced signal sensitivity using LNA-conjugated gold nanoparticles

Multiplexed, rapid detection of H5N1 using a PCR-free nanoparticle-based genomic microarray assay

Improving the sensitivity of immunoassays by tuning gold nanoparticles to the tipping point

RNA biosensor for the rapid detection of viable Escherichia coli in drinking water

A generic sandwich-type biosensor with nanomolar detection limits

Development of an improved PCR-ICT hybrid assay for direct detection of Legionellae and Legionella pneumophila from cooling tower water specimens

Development of a novel, rapid integrated Cryptosporidium parvum detection assay

New rapid detection test with a combination of polymerase chain reaction and immunochromatographic assay for Mycobacterium tuberculosis complex

Gold nanoparticle-based detection of genomic DNA targets on microarrays using a novel optical detection system

Colorimetric detection of DNA sequences based on electrostatic interactions with unmodified gold nanoparticles

Size-and distance-dependent nanoparticle surface-energy transfer (NSET) method for selective sensing of hepatitis C virus RNA

Direct detection of unamplified hepatitis C virus RNA using unmodified gold nanoparticles

Label-free optical detection of single-base mismatches by the combination of nuclease and gold nanoparticles

Colorimetric detection of HIV-1 ribonuclease H activity by gold nanoparticles

Long genomic DNA amplicons adsorption onto unmodified gold nanoparticles for colorimetric detection of Bacillus anthracis

Enhancement and quenching of single-molecule fluorescence

Why gold nanoparticles are more precious than pretty gold: noble metal surface plasmon resonance and its enhancement of the radiative and nonradiative properties of nanocrystals of different shapes

Theory of fluorophore-metallic surface interactions

Enhancement of single-molecule fluorescence using a gold nanoparticle as an optical nanoantenna

Noble metal nanoparticles for biosensing applications

Miniaturized sensor for microbial pathogens DNA and chemical toxins

Fluorescent bio-barcode DNA assay for the detection of Salmonella enterica serovar Enteritidis

Aggregation effects of gold nanoparticles for single-base mismatch detection in influenza A (H1N1) DNA sequences using fluorescence and Raman measurements

Single-mismatch detection using gold-quenched fluorescent oligonucleotides

Development of a gold nano-particle-based fluorescent molecular beacon for detection of cystic fibrosis associated mutation

Gold nanoparticle fluorescent molecular beacon for low-resolution DQ2 gene HLA typing

Optical detection of DNA hybridization based on fluorescence quenching of tagged oligonucleotide probes by gold nanoparticles

Gold nanoparticle enhanced fluorescence anisotropy for the assay of single nucleotide polymorphisms (SNPs) based on toehold-mediated strand-displacement reaction

Biosensing with plasmonic nanosensors

Nanoparticles with Raman spectroscopic fingerprints for DNA and RNA detection

Sub-attomolar HIV-1 DNA detection using surface-enhanced Raman spectroscopy

Surface-enhanced Raman gene probe for HIV detection

Using oligonucleotide-functionalized Au nanoparticles to rapidly detect foodborne pathogens on a piezoelectric biosensor

The Escherichia coli O157:H7 DNA detection on a gold nanoparticle-enhanced piezoelectric biosensor

DNA probe functionalized QCM biosensor based on gold nanoparticle amplification for Bacillus anthracis detection

Electron Transfer and Radical Processes in Transition Metal Chemistry

Integrated nanoparticle-biomolecule hybrid systems: synthesis, properties, and applications

Electrochemical sensing of DNA using gold nanoparticles

A multiplex nanoparticle-based bio-barcoded DNA sensor for the simultaneous detection of multiple pathogens

Specific detection of Mycobacterium sp. genomic DNA using dual labeled gold nanoparticle based electrochemical biosensor

Detection of non-PCR amplified S. enteritidis genomic DNA from food matrices using a gold-nanoparticle DNA biosensor: a proof-ofconcept study

Home tests to monitor fertility

The use of gold nanoparticles in diagnostics and detection

A biosensor assay for the detection of Mycobacterium avium subsp. paratuberculosis in fecal samples

Colloidal gold and other labels for lateral flow immunoassays

One step visual detection of PCR products with gold nanoparticles and a nucleic acid lateral flow (NALF) device

Microfluidic lab-on-a-chip platforms: requirements, characteristics and applications

A rapid DNA biosensor for the molecular diagnosis of infectious disease

A lateral flow assay for quantitative detection of amplified HIV-1 RNA

MicroRNA detection using lateral flow nucleic acid strips with gold nanoparticles

Warfarin genotyping using three different platforms

Comparison of assay systems for warfarin-related CYP2C9 and VKORC1 genotyping

Gold and silver nanoparticles for clinical diagnostics -from genomics to proteomics

Non-cross-linking gold nanoparticle aggregation as a detection method for single-base substitutions

Nanoparticle-based detection and quantification of DNA with single nucleotide polymorphism (SNP) discrimination selectivity

Gold nanoparticles with asymmetric polymerase chain reaction for colorimetric detection of DNA sequence

Direct colorimetric diagnosis of pathogen infections by utilizing thiol-labeled PCR primers and unmodified gold nanoparticles

One-pot colorimetric differentiation of polynucleotides with single base imperfections using gold nanoparticle probes

Dimeric gold nanoparticle assembly for detection and discrimination of single nucleotide mutation in Duchenne muscular dystrophy

Rolling circle amplification combined with gold nanoparticle aggregates for highly sensitive identification of singlenucleotide polymorphisms

Detection of HBV and HCV coinfection by TEM with Au nanoparticle gene probes

SNP identification in unamplified human genomic DNA with gold nanoparticle probes

Disposable nucleic acid biosensors based on gold nanoparticle probes and lateral flow strip

Multianalyte, dipstick-type, nanoparticle-based DNA biosensor for visual genotyping of single-nucleotide polymorphisms

Study of single-stranded DNA binding protein-nucleic acids interactions using unmodified gold nanoparticles and its application for detection of single nucleotide polymorphisms

Aptamer-based colorimetric detection of platelet-derived growth factor using unmodified gold nanoparticles

Unmodified gold nanoparticles for direct and rapid detection of Mycobacterium tuberculosis complex

Gold-nanobeacons for realtime monitoring of RNA synthesis

Detection of human viral RNA via a combined fluorescence and SERS molecular beacon assay

Single base extension reaction-based surface enhanced Raman spectroscopy for DNA methylation assay

PCR-free quantification of multiple splice variants in a cancer gene by surface-enhanced Raman spectroscopy

Catalytic signal amplification of gold nanoparticles combining with conformation-switched hairpin DNA probe for hepatitis C virus quantification

Electrochemical genosensor based on colloidal gold nanoparticles for the detection of Factor V Leiden mutation using disposable pencil graphite electrodes

A thermostabilized magnetogenosensing assay for DNA sequence-specific detection and quantification of Vibrio cholerae

Real-time detection of influenza a, influenza B, and respiratory syncytial virus a and B in respiratory specimens by use of nanoparticle probes

Simultaneous detection of attomolar pathogen DNAs by Bio-MassCode mass spectrometry