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Gold Nanoparticles for DNA/RNA-Based Diagnostics

  • Ricardo Franco
  • Pedro Pedrosa
  • Fábio Ferreira Carlos
  • Bruno Veigas
  • Pedro V. BaptistaEmail author
Reference work entry

Abstract

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.

Keywords

Au-nanoprobes Au-nanobeacons LSPR Nucleic acid Point of care SNP Pathogen 

List of Abbreviations

AuNP

Gold nanoparticle

ARMS

Amplification-refractory mutation system

AsPCR

Asymmetric PCR

HAV

Hepatitis A virus

HBV

Hepatitis B virus

HCV

Hepatitis C virus

HIV

Human immunodeficiency virus

iTPa

Isothermal target and probe amplification

LFA

Lateral flow assay

LOD

Limit of detection

LSPR

Localized surface plasmon resonance

MRSA

Methicillin-resistant staphylococcus aureus

MTBC

Mycobacterium tuberculosis complex

NASBA

Nucleic acid sequence based amplification

NP

Nanoparticle

PEXT

Primer extension

POC

Point of care

QCM

Quartz crystal microbalances

RCA

Rolling circle amplification

RT-PCR

Reverse transcription PCR

SERS

Surface–enhancement Raman spectroscopy

SNP

Single nucleotide polymorphism

SPCE

Screen-printed carbon electrode

SARS

Severe acute respiratory syndrome

SBE

Single base extension

TB

Tuberculosis

General Aspects of Gold Nanoparticles Application for DNA/RNA-Based Diagnostics

Gold nanoparticles (AuNPs) show unique properties for biodetection, namely, optical, electrochemical, and spectral properties. AuNPs can be readily synthesized and promptly bioconjugated with similarly sized oligonucleotides, antibodies, aptamers, and other biomolecules appropriate for biodetection. Due to their nanosized scale, AuNPs have a high surface-to-volume ratio, making them ideal platforms for multivalent interactions based on a high concentration of exposed bioactive molecules.

DNA and RNA characterization for diagnostics purposes relies on the hybridization of a probe to a given target exploring the strand complementarity resulting from specific and stable Watson–Crick pairing. Therefore, a key step toward the establishment of an AuNP-based diagnostics methodology is to create an AuNP nanoprobe which is generated by functionalizing AuNPs with bifunctional ligands in which a moiety binds to the particles’ surface, while the other allows for specific interaction with biomolecules. For example, AuNPs are easy functionalized with thiolated oligonucleotides [1] (Fig. 1).
Fig. 1

Au-nanoprobe synthesis. 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 single-stranded 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)

The interesting optical proprieties of AuNPs, i.e., their intense vibrant colors in solution, derive from the interaction with light. Upon influence of the oscillating 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).
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)

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.
Table 1

Gold nanoparticle based molecular diagnostics

Detection technique

Nucleic acid target

Biological target

Sample amplification vs. direct detection

Detection limit

References

Colorimetric (non-cross-linking aggregation)

Naked eye, UV–vis spectroscopy or optical monitoring system

DNA

Human

PCR

600 pM

Sato et al. [93]

DNA

Human

PCR

0.1 pmol

Qin and Yung [94]

DNA

Human

PCR

18 ng/μl

Doria et al. [35]

DNA

Human

AsPCR

10 pg

Deng et al. [95]

RNA

Human

Direct detection

10 ng/μl

Conde et al. [33]

DNA

M. tuberculosis spp.

Direct detection

2 ng/μl

Liandris et al. [31]

DNA

Chlamydia sp.

PCR

Jung et al. [96]

DNA

MTBC

PCR

0.75 ug

Baptista et al. [21] Costa et al. [24]; Veigas et al. [22, 23]; Silva et al. [27]; Bernacka-Wojcik et al. [25]

DNA

E. coli

Direct detection

54 ng

Padmavathy et al. [32]

RNA

S. typhimurium

NASBA

Mollasalehi and Yazdanparast [29]

RNA

S. Enteritidis; S. Typhimurium

NASBA

5 CFUs

Mollasalehi and Yazdanparast [30]

Colorimetric (cross-linking aggregation)

Light scattering imaging

DNA

Human

SBE

Storhoff et al. [97]

DNA

MRSA

Direct detection

33 fM

Storhoff et al. [8]

DNA

HIV; M. Tuberculosis; B. glucanase

Direct detection

80 pM

Wei He et al. [9]

DNA; RNA

C. parvum

PCR; RT-PCR

10 amol

Javier et al. [10]

Naked eye, UV–vis spectroscopy or optical monitoring system

DNA

Human

PCR

Qin et al. [98]

DNA

Human

RCA

70 fM

Li et al. [99]

DNA

MTBC

PCR

0.5 pmol

Soo et al. [13]

DNA

C. trachomatis

PCR

250 pM

Parab et al. [18]

DNA

C. trachomatis

iTPA

102 copies of Plasmid DNA

Jung et al. [15]

DNA

P. aeruginosa; S. aureus; S. epidermidis; K. pneumonia; S. marcescens; B. cereus

PCR

 

Wang et al. [19]

DNA

N. gonorrhoeae; T. pallidum; P. falciparum; HBV vírus

Non-enzimatic (MNAzyme)

1 nM

Zagorovsky and Chan [20]

DNA

Salmonella sp

Direct detection

37 fM

Kalidasan et al. [16]

RNA

M. Tuberculosis

NASBA

10 CPU/mL

Gill et al. [12]

 

RNA

C. parvum

Direct detection

1.2 × 107 copies/μL

Weigum et al. [11]

DNA

HPV

AsPCR

14 pM

Chen et al. [14]

DNA

Kaposi’s sarcoma-associated herpes virus

Direct detection

1 nM

Mancuso et al. [17]

Colorimetric (sandwich assay)

Colorimetric detection using gold label silver stain

DNA

U. urealyticum; C. trachomatis

Multiplex asPCR

5 pM

Cao et al. [38]

Naked eye or CCD camera (sandwich hybridization)

DNA

HBV

PCR

10 pM

Xi et al. [100]

Colorimetric detection using gold label silver stain – micro array

DNA

Human

Direct detection

0.5 ug

Bao et al. [101]

DNA

Human

Direct detection

1 ug

Lefferts et al. [90]

RNA

Avian Influenza Virus (H5N1)

Direct detection

0.1 pM

Zhao et al. [41]

Lateral flow test strips

DNA

Human

PCR

50 pM

Mao et al. [102]

DNA

Human

PCR or PEXT

Litos et al. [103]

miRNA

Human

Direct detection

1 fmol, 5 amol

Hou et al. [89]

DNA

V. cholerae

PCR

5 ng

Chua et al. [87]

DNA

E. coli

Direct detection

~0.4 nM

Rastogi et al. [40]

RNA

HIV-1

NASBA

9.5 log10 RNA copies in 20 mL

Rohrman et al. [88]

Colorimetric (unmodified AuNPs)

Hyper-Rayleigh scattering (HRS)

DNA

Human

PCR

10 nM

Li et al. [49]

Naked eye or optical monitoring system

DNA

Human

Direct detection

Tan et al. [104]

DNA

Human

Direct detection

6 nM

Chang et al. [105]

DNA

MTBC

PCR

1 ng

Hussain et al. [106]

DNA

HBV

Direct detection

5 nM

Liu et al. [52]

DNA

B. anthracis

AsPCR

0.1 pmol

Deng et al. [54]

RNA-DNA

HIV-1

Enzymatic activity (HIV-1 RT)

Xie et al. [53]

RNA

HCV

Direct detection

<100 fM

Shawky et al. [51]

Fluorescence spectroscopy

Fluorescence spectroscopy Surface-energy transfer (NSET)

DNA

Human

Direct detection

1 nM

Beni et al. [64]

DNA

Human

ARMS

0.5 nM

Beni et al. [65]

DNA

Human

Direct detection

5–10 nM

Wang et al. [67]

RNA

Human

RT-PCR

10.3 fmol

Rosa et al. [107]

 

cDNA

HAV; HCV; West Nile; HIV; SARS

RT-PCR

Sha et al. [108]

DNA

S. enterica serovar Enteritidis

PCR

1 ng/mL

Zhang et al. [61]

RNA

HCV

Direct detection

0.3 fM

Griffin et al. [50]

Raman spectroscopy

Surface-enhanced Raman scattering (SERS)

DNA

Human

SBE

3 pM

Hu and Zhang [109]

RNA

Human

RT-PCR

2.3 pM

Sun and Irudayaraj [110]

DNA

HIV-1

Direct detection

0.1 aM

Hu et al. [70]

DNA/RNA

HAV; HBV; HIV; Ebola virus; Variola virus; B. anthracis

Direct detection

20 fM

Cao et al. [69]

Piezoelectric

DNA probe functionalized quartz crystal microbalance

DNA

E. coli

PCR

1.2×102 CFU/mL

Chen et al. [72]

DNA

E. coli

AsPCR

2.0×103 CFU/mL

Wang et al. [73]

DNA

B. anthracis

AsPCR

3.5 × 102 CFU/mL

Hao et al. [74]

Electrochemical

Square wave anodic stripping voltammetry on screen-printed carbon electrode (SPCE) chips)

DNA

B. anthraci; S. enteritidis

PCR

0.5 ng/mL for S. enteritidis; 50 pg/mL for B. anthracis

Zhang et al. [78]

Automated System (Light scattering imaging)

DNA

Mycobacterium spp.

Direct detection

1.25 ng/mL

Thiruppathiraja et al. [79]

Differential pulse voltammetry

DNA

S. enterica serovar Enteritidis

Direct detection

7 ng/mL

Vetrone et al. [80]

Electrocatalytic signal amplification of AuNPs

cDNA

HCV

RT-PCR

Li et al. [111]

Differential pulse anodic stripping voltammetry

DNA

Human

PCR

Ozsoz et al. [112]

DNA

V. cholerae

AsPCR

3.9 nM

Low et al. [113]

Other

Automated System (Light scattering imaging)

DNA

Influenza A virus; Influenza B virus; respiratory syncytial virus A and B

PCR

Jannetto et al. [114]

Bio-MassCode mass spectrometry

DNA

HIV; HBV; HCV; T. pallidum

Direct detection

10−18 M

Yang et al. [115]

AuNPs for Colorimetric Sensing

Among detection methods based on AuNPs, colorimetric approaches are the most frequent, allowing for simplicity and portability, making them ideal for diagnostics 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′ and 5′ 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).
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)

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 × 105 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−1 with a sensitivity and specificity of 94.7 % and 96 %, respectively. Following the same detection approach, Soo et al. designed a set of 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: 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 sarcoma-associated herpes virus [17] and Salmonella sp. [16]. Kalidasan et al. reported the detection of unamplified DNA from Salmonella sp. with an LOD of 37 f. for visual detection.

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).
Fig. 4

Au-nanoprobe strategy for the detection of MTBC members. Schematic representation of detection of M. tuberculosis using Au-nanoprobes and a paper platform. The colorimetric assay consists of visual comparisons of test solutions after salt-induced Au-nanoprobe aggregation on a [MgCl2]-impregnated paper plate: MTBC Au-nanoprobe alone, blank; MTBC Au-nanoprobe in the presence of MTBC sample, M. tuberculosis; MTBC Au-nanoprobe in the presence of a non-MTBC sample; and MTBC Au-nanoprobe in the presence of a noncomplementary sample, nonrelated. After color development, a photo of the paper plate is captured and RGB image analysis is performed (Reproduced by permission of The Royal Society of Chemistry [28])

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-cross-linking 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 single-stranded 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′-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].
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)

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 multiplex asymmetrical PCR. This approach allowed for the simultaneous detection of Ureaplasma urealyticum and Chlamydia trachomatis. The surface immobilized 5′-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-target-intermediate hybridization, and AuNP triggered silver staining, without target amplification or any enzymatic reactions (LOD of 100 f. for purified PCR amplicons and TCID50 units for H5N1 RNA).

Non-functionalized AuNPs

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).

Gold Nanoparticles Modulation of Fluorescence

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).
Fig. 6

Fluorescent-based noble metal NPs biosensing, (a) molecular nanobeacons, (b) direct hybridization of a fluorophore-labeled target or sandwich assay using fluorophore-labeled probe. Distances are not represented to the scale (Reproduced from Doria et al. [59])

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 [1].

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 bio-barcode-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′ 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 100× 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].

Gold Nanoparticles for Plasmonic-Based Sensing

Plasmonic sensing here refers in the broader sense to detection based on light-scattering 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

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.

Piezoelectric Sensors Using AuNPs for DNA/RNA Recognition

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).
Fig. 7

Time-dependent frequency changes of the circulating-flow QCM sensor, (a) addition of Probe 1 (1 μM; P1-30/12 T) for immobilization on the surface of the QCM sensor via self-assembly, (b) complementary target oligonucleotides [0.5 μM; T-104(AS)] are subsequently introduced for DNA hybridization, (c) additional treatment of the DNA hybridized QCM with Probe 2 (P2-30/12 T)-capped Au nanoparticles. The sequences of Probe 1 and Probe 2 are complementary to the two ends of the analyte DNA (i.e., target sequences) (Reproduced from Chen et al. [72] with permission from Elsevier)

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 × 102 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-functionalized 70-nm AuNPs to further amplify the signal. This scheme was capable of detecting bacteria without sample enrichment showing an LOD of 2.0 × 103 CFU/mL. Recently, Hao et al. developed this method to detect Bacillus anthracis based on the recognition of a 168-bp 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 × 102 CFU/mL for B. anthracis vegetative cells without culture enrichment.

Electrochemical Detection of DNA/RNA Targets Using AuNPs

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 Cd2+ and 50 pg/mL for Pb2+.

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 oxide-coated 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 Au3+ 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 screen-printed carbon electrode chip together with bio-barcoded AuNPs and magnetic NPs, to detect B. anthracis and S. enteritidis [78].

AuNPs for Diagnostics at Point of Care: Lateral Flow Devices

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 resource-poor 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.
Fig. 8

Schematic representation of a LFA strip, (a) sample pad (sample inlet and filtering), conjugate pad (AuNP-antibody conjugates), incubation, and detection zone with test and control lines (antigen detection and functionality test) and final absorbent pad (liquid actuation); (b) sample is applied to the sample pad and the analyte binds to the AuNP-antibody conjugates and elutes with the buffer flow, and this analyte-AuNP-antibody conjugates bind to the test line (positive result). If the analyte is absent, the AuNP-antibody conjugates bind only to the control line (negative and control result) (Reprinted with permission from Gubala et al. [36]. Copyright 2011 American Chemical Society)

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, NanosphereTM 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.

Conclusion

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 1010. 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.

Notes

Acknowledgments

The authors acknowledge Fundação para a Ciência e Tecnologia (FCT/MEC) for funding: CIGMH (PEst-OE/SAU/UI0009/2011); REQUIMTE (PEst-C/EQB/LA0006/2011); UCIBIO (UID/Multi/04378/2013); SFRH/BD/78970/2011 for BV; SFRH/BD/51103/2010 for FC.

References

  1. 1.
    P.V. Baptista, G. Doria, P. Quaresma, M. Cavadas, C.S. Neves, I. Gomes, P. Eaton, E. Pereira, R. Franco, Nanoparticles in molecular diagnostics. Prog. Mol. Biol. Transl. Sci. 104, 427–488 (2011). doi:10.1016/B978-0-12-416020-0.00011-5CrossRefGoogle Scholar
  2. 2.
    P.K. Jain, K.S. Lee, I.H. El-Sayed, M.A. El-Sayed, Calculated absorption and scattering properties of gold nanoparticles of different size, shape, and composition: applications in biological imaging and biomedicine. J. Phys. Chem. B 110(14), 7238–7248 (2006). doi:10.1021/jp057170oCrossRefGoogle Scholar
  3. 3.
    P.K. Jain, X. Huang, I.H. El-Sayed, M.A. El-Sayed, Noble metals on the nanoscale: optical and photothermal properties and some applications in imaging, sensing, biology, and medicine. Acc. Chem. Res. 41(12), 1578–1586 (2008). doi:10.1021/ar7002804CrossRefGoogle Scholar
  4. 4.
    H.M. Azzazy, M.M. Mansour, S.C. Kazmierczak, Nanodiagnostics: a new frontier for clinical laboratory medicine. Clin. Chem. 52(7), 1238–1246 (2006). doi:10.1373/clinchem.2006.066654CrossRefGoogle Scholar
  5. 5.
    E.D. Goluch, J.M. Nam, D.G. Georganopoulou, T.N. Chiesl, K.A. Shaikh, K.S. Ryu, A.E. Barron, C.A. Mirkin, C. Liu, A bio-barcode assay for on-chip attomolar-sensitivity protein detection. Lab Chip 6(10), 1293–1299 (2006). doi:10.1039/b606294fCrossRefGoogle Scholar
  6. 6.
    Consortium IHGS, Finishing the euchromatic sequence of the human genome. Nature 431(7011), 931–945 (2004). doi:10.1038/nature03001CrossRefGoogle Scholar
  7. 7.
    C.A. Mirkin, R.L. Letsinger, R.C. Mucic, J.J. Storhoff, A DNA-based method for rationally assembling nanoparticles into macroscopic materials. Nature 382(6592), 607–609 (1996). doi:10.1038/382607a0CrossRefGoogle Scholar
  8. 8.
    J.J. Storhoff, A. Lucas, V. Garimella, Y.P. Bao, U.R. Müller, Homogeneous detection of unamplified genomic DNA sequences based on colorimetric scatter of gold nanoparticle probes. Nat. Biotechnol. 22(7), 883–887 (2004). doi:10.1038/nbt977CrossRefGoogle Scholar
  9. 9.
    W. He, C.Z. Huang, Y.F. Li, J.P. Xie, R.G. Yang, P.F. Zhou, J. Wang, 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. Anal. Chem. 80(22), 8424–8430 (2008). doi:10.1021/ac801005dCrossRefGoogle Scholar
  10. 10.
    D.J. Javier, A. Castellanos-Gonzalez, S.E. Weigum, A.C. White Jr., R. Richards-Kortum, Oligonucleotide-gold nanoparticle networks for detection of Cryptosporidium parvum heat shock protein 70 mRNA. J. Clin. Microbiol. 47(12), 4060–4066 (2009). doi:10.1128/JCM.00807-09CrossRefGoogle Scholar
  11. 11.
    S.E. Weigum, A. Castellanos-Gonzalez, C. White, R. Richards-Kortum, Amplification-free detection of cryptosporidium nucleic acids using DNA/RNA-directed gold nanoparticle assemblies. J. Parasitol. (2013). doi:10.1645/12-132.1Google Scholar
  12. 12.
    P. Gill, M. Ghalami, A. Ghaemi, N. Mosavari, H. Abdul-Tehrani, M. Sadeghizadeh, Nanodiagnostic method for colorimetric detection of Mycobacterium tuberculosis 16S rRNA. NanoBiotechnology 4(1–4), 28–35 (2009). doi:10.1007/s12030-009-9021-9Google Scholar
  13. 13.
    P.C. Soo, Y.T. Horng, K.C. Chang, J.Y. Wang, P.R. Hsueh, C.Y. Chuang, C.C. Lu, H.C. Lai, A simple gold nanoparticle probes assay for identification of Mycobacterium tuberculosis and Mycobacterium tuberculosis complex from clinical specimens. Mol. Cell. Probes 23(5), 240–246 (2009). doi:10.1016/j.mcp.2009.04.006CrossRefGoogle Scholar
  14. 14.
    S.H. Chen, K.I. Lin, C.Y. Tang, S.L. Peng, Y.C. Chuang, Y.R. Lin, J.P. Wang, C.S. Lin, Optical detection of human papillomavirus type 16 and type 18 by sequence sandwich hybridization with oligonucleotide-functionalized Au nanoparticles. IEEE Trans. Nanobioscience 8(2), 120–131 (2009). doi:10.1109/TNB.2008.2011733CrossRefGoogle Scholar
  15. 15.
    C. Jung, J.W. Chung, U.O. Kim, M.H. Kim, H.G. Park, Real-time colorimetric detection of target DNA using isothermal target and signaling probe amplification and gold nanoparticle cross-linking assay. Biosens. Bioelectron. 26(5), 1953–1958 (2011). doi:10.1016/j.bios.2010.07.088CrossRefGoogle Scholar
  16. 16.
    K. Kalidasan, J.L. Neo, M. Uttamchandani, Direct visual detection of Salmonella genomic DNA using gold nanoparticles. Mol. Biosyst. 9(4), 618–621 (2013). doi:10.1039/c3mb25527aCrossRefGoogle Scholar
  17. 17.
    M. Mancuso, L. Jiang, E. Cesarman, D. Erickson, Multiplexed colorimetric detection of Kaposi’s sarcoma associated herpesvirus and Bartonella DNA using gold and silver nanoparticles. Nanoscale 5(4), 1678–1686 (2013). doi:10.1039/c3nr33492aCrossRefGoogle Scholar
  18. 18.
    H.J. Parab, C. Jung, J.H. Lee, H.G. Park, A gold nanorod-based optical DNA biosensor for the diagnosis of pathogens. Biosens. Bioelectron. 26(2), 667–673 (2010). doi:10.1016/j.bios.2010.06.067CrossRefGoogle Scholar
  19. 19.
    X. Wang, Y. Li, J. Wang, Q. Wang, L. Xu, J. Du, S. Yan, Y. Zhou, Q. Fu, Y. Wang, L. Zhan, A broad-range method to detect genomic DNA of multiple pathogenic bacteria based on the aggregation strategy of gold nanorods. Analyst 137(18), 4267–4273 (2012). doi:10.1039/c2an35680eCrossRefGoogle Scholar
  20. 20.
    K. Zagorovsky, W.C. Chan, A plasmonic DNAzyme strategy for point-of-care genetic detection of infectious pathogens. Angew. Chem. Int. Ed. Engl. 52(11), 3168–3171 (2013). doi:10.1002/anie.201208715CrossRefGoogle Scholar
  21. 21.
    P.V. Baptista, M. Koziol-Montewka, J. Paluch-Oles, G. Doria, R. Franco, Gold-nanoparticle-probe-based assay for rapid and direct detection of Mycobacterium tuberculosis DNA in clinical samples. Clin. Chem. 52(7), 1433–1434 (2006). doi:10.1373/clinchem.2005.065391CrossRefGoogle Scholar
  22. 22.
    B. Veigas, G. Doria, V.P. Baptista, Nanodiagnostics for tuberculosis, in Understanding Tuberculosis – Global Experiences and Innovative Approaches to the Diagnosis, ed. by P.-J. Cardona (InTech, Croatia, 2012). doi:10.5772/30463Google Scholar
  23. 23.
    B. Veigas, D. Machado, J. Perdigao, I. Portugal, I. Couto, M. Viveiros, P.V. Baptista, Au-nanoprobes for detection of SNPs associated with antibiotic resistance in Mycobacterium tuberculosis. Nanotechnology 21(41), 415101 (2010). doi:10.1088/0957-4484/21/41/415101CrossRefGoogle Scholar
  24. 24.
    P. Costa, A. Amaro, A. Botelho, J. Inacio, P.V. Baptista, Gold nanoprobe assay for the identification of mycobacteria of the Mycobacterium tuberculosis complex. Clin. Microbiol. Infect. 16(9), 1464–1469 (2010). doi:10.1111/j.1469-0691.2009.03120.xCrossRefGoogle Scholar
  25. 25.
    I. Bernacka-Wojcik, P. Lopes, A. Catarina Vaz, B. Veigas, P. Jerzy Wojcik, P. Simoes, D. Barata, E. Fortunato, P. Viana Baptista, H. Aguas, R. Martins, Bio-microfluidic platform for gold nanoprobe based DNA detection-application to Mycobacterium tuberculosis. Biosens. Bioelectron. 48C, 87–93 (2013). doi:10.1016/j.bios.2013.03.079CrossRefGoogle Scholar
  26. 26.
    I. Bernacka-Wojcik, R. Senadeera, P.J. Wojcik, L.B. Silva, G. Doria, P. Baptista, H. Aguas, E. Fortunato, R. Martins, Inkjet printed and “doctor blade” TiO2 photodetectors for DNA biosensors. Biosens. Bioelectron. 25(5), 1229–1234 (2010). doi:10.1016/j.bios.2009.09.027CrossRefGoogle Scholar
  27. 27.
    L.B. Silva, B. Veigas, G. Doria, P. Costa, J. Inacio, R. Martins, E. Fortunato, P.V. Baptista, Portable optoelectronic biosensing platform for identification of mycobacteria from the Mycobacterium tuberculosis complex. Biosens. Bioelectron. 26(5), 2012–2017 (2011). doi:10.1016/j.bios.2010.08.078CrossRefGoogle Scholar
  28. 28.
    B. Veigas, J.M. Jacob, M.N. Costa, D.S. Santos, M. Viveiros, J. Inacio, R. Martins, P. Barquinha, E. Fortunato, P.V. Baptista, Gold on paper-paper platform for Au-nanoprobe TB detection. Lab Chip 12(22), 4802–4808 (2012). doi:10.1039/c2lc40739fCrossRefGoogle Scholar
  29. 29.
    H. Mollasalehi, R. Yazdanparast, Non-crosslinking gold nanoprobes for detection of nucleic acid sequence-based amplification products. Anal. Biochem. 425(2), 91–95 (2012). doi:10.1016/j.ab.2012.03.008CrossRefGoogle Scholar
  30. 30.
    H. Mollasalehi, R. Yazdanparast, An improved non-crosslinking gold nanoprobe-NASBA based on 16S rRNA for rapid discriminative bio-sensing of major salmonellosis pathogens. Biosens. Bioelectron. 47C, 231–236 (2013). doi:10.1016/j.bios.2013.03.012CrossRefGoogle Scholar
  31. 31.
    E. Liandris, M. Gazouli, M. Andreadou, M. Comor, N. Abazovic, L.A. Sechi, J. Ikonomopoulos, Direct detection of unamplified DNA from pathogenic mycobacteria using DNA-derivatized gold nanoparticles. J. Microbiol. Methods 78(3), 260–264 (2009). doi:10.1016/j.mimet.2009.06.009CrossRefGoogle Scholar
  32. 32.
    B. Padmavathy, R. Vinoth Kumar, B.M. Jaffar Ali, A direct detection of Escherichia coli genomic DNA using gold nanoprobes. J. Nanobiotechnol. 10(1), 8 (2012). doi:10.1186/1477-3155-10-8CrossRefGoogle Scholar
  33. 33.
    J. Conde, J.M. de la Fuente, P.V. Baptista, RNA quantification using gold nanoprobes – application to cancer diagnostics. J. Nanobiotechnol. 8, 5 (2010). doi:10.1186/1477-3155-8-5CrossRefGoogle Scholar
  34. 34.
    J. Conde, G. Doria, J.M. de la Fuente, P.V. Baptista, RNA quantification using noble metal nanoprobes: simultaneous identification of several different mRNA targets using color multiplexing and application to cancer diagnostics. Methods Mol. Biol. 906, 71–87 (2012). doi:10.1007/978-1-61779-953-2_6CrossRefGoogle Scholar
  35. 35.
    G. Doria, R. Franco, P. Baptista, Nanodiagnostics: fast colorimetric method for single nucleotide polymorphism/mutation detection. IET Nanobiotechnol. 1(4), 53–57 (2007). doi:10.1049/iet-nbt:20070001CrossRefGoogle Scholar
  36. 36.
    V. Gubala, L.F. Harris, A.J. Ricco, M.X. Tan, D.E. Williams, Point of care diagnostics: status and future. Anal. Chem. 84(2), 487–515 (2012). doi:10.1021/ac2030199CrossRefGoogle Scholar
  37. 37.
    G. Posthuma-Trumpie, J. Korf, A. Amerongen, Lateral flow (immuno)assay: its strengths, weaknesses, opportunities and threats. A literature survey. Anal. Bioanal. Chem. 393(2), 569–582 (2009). doi:10.1007/s00216-008-2287-2CrossRefGoogle Scholar
  38. 38.
    X. Cao, Y.F. Wang, C.F. Zhang, W.J. Gao, Visual DNA microarrays for simultaneous detection of Ureaplasma urealyticum and Chlamydia trachomatis coupled with multiplex asymmetrical PCR. Biosens. Bioelectron. 22(3), 393–398 (2006). doi:10.1016/j.bios.2006.05.011CrossRefGoogle Scholar
  39. 39.
    X.Z. Li, S. Kim, W. Cho, S.Y. Lee, Optical detection of nanoparticle-enhanced human papillomavirus genotyping microarrays. Biomed. Opt. Express. 4(2), 187–192 (2013). doi:10.1364/BOE.4.000187CrossRefGoogle Scholar
  40. 40.
    S.K. Rastogi, C.M. Gibson, J.R. Branen, D.E. Aston, A.L. Branen, P.J. Hrdlicka, DNA detection on lateral flow test strips: enhanced signal sensitivity using LNA-conjugated gold nanoparticles. Chem. Commun. (Camb.) 48(62), 7714–7716 (2012). doi:10.1039/c2cc33430eCrossRefGoogle Scholar
  41. 41.
    J. Zhao, S. Tang, J. Storhoff, S. Marla, Y.P. Bao, X. Wang, E.Y. Wong, V. Ragupathy, Z. Ye, I.K. Hewlett, Multiplexed, rapid detection of H5N1 using a PCR-free nanoparticle-based genomic microarray assay. BMC Biotechnol. 10, 74 (2010). doi:10.1186/1472-6750-10-74CrossRefGoogle Scholar
  42. 42.
    J. Aveyard, P. Nolan, R. Wilson, Improving the sensitivity of immunoassays by tuning gold nanoparticles to the tipping point. Anal. Chem. 80(15), 6001–6005 (2008). doi:10.1021/ac800699kCrossRefGoogle Scholar
  43. 43.
    A.J. Baeumner, R.N. Cohen, V. Miksic, J. Min, RNA biosensor for the rapid detection of viable Escherichia coli in drinking water. Biosens. Bioelectron. 18(4), 405–413 (2003). doi:10.1016/s0956-5663(02)00162-8CrossRefGoogle Scholar
  44. 44.
    A.J. Baeumner, C. Jones, C.Y. Wong, A. Price, A generic sandwich-type biosensor with nanomolar detection limits. Anal. Bioanal. Chem. 378(6), 1587–1593 (2004). doi:10.1007/s00216-003-2466-0CrossRefGoogle Scholar
  45. 45.
    Y.T. Horng, P.C. Soo, B.J. Shen, Y.L. Hung, K.Y. Lo, H.P. Su, J.R. Wei, S.C. Hsieh, P.R. Hsueh, H.C. Lai, Development of an improved PCR-ICT hybrid assay for direct detection of Legionellae and Legionella pneumophila from cooling tower water specimens. Water Res. 40(11), 2221–2229 (2006). doi:10.1016/j.watres.2006.03.033CrossRefGoogle Scholar
  46. 46.
    D. Kozwich, K.A. Johansen, K. Landau, C.A. Roehl, S. Woronoff, P.A. Roehl, Development of a novel, rapid integrated Cryptosporidium parvum detection assay. Appl. Environ. Microbiol. 66(7), 2711–2717 (2000). doi:10.1128/AEM.66.7.2711-2717.2000CrossRefGoogle Scholar
  47. 47.
    T. Suzuki, M. Tanaka, S. Otani, S. Matsuura, Y. Sakaguchi, T. Nishimura, A. Ishizaka, N. Hasegawa, New rapid detection test with a combination of polymerase chain reaction and immunochromatographic assay for Mycobacterium tuberculosis complex. Diagn. Microbiol. Infect. Dis. 56(3), 275–280 (2006). doi:10.1016/j.diagmicrobio.2006.04.009CrossRefGoogle Scholar
  48. 48.
    J.J. Storhoff, S.S. Marla, P. Bao, S. Hagenow, H. Mehta, A. Lucas, V. Garimella, T. Patno, W. Buckingham, W. Cork, U.R. Müller, Gold nanoparticle-based detection of genomic DNA targets on microarrays using a novel optical detection system. Biosens. Bioelectron. 19(8), 875–883 (2004). doi:10.1016/j.bios.2003.08.014CrossRefGoogle Scholar
  49. 49.
    H. Li, L. Rothberg, Colorimetric detection of DNA sequences based on electrostatic interactions with unmodified gold nanoparticles. Proc. Natl. Acad. Sci. U. S. A. 101(39), 14036–14039 (2004). doi:10.1073/pnas.0406115101CrossRefGoogle Scholar
  50. 50.
    J. Griffin, A.K. Singh, D. Senapati, P. Rhodes, K. Mitchell, B. Robinson, E. Yu, P.C. Ray, Size- and distance-dependent nanoparticle surface-energy transfer (NSET) method for selective sensing of hepatitis C virus RNA. Chemistry 15(2), 342–351 (2009). doi:10.1002/chem.200801812CrossRefGoogle Scholar
  51. 51.
    S.M. Shawky, D. Bald, H.M. Azzazy, Direct detection of unamplified hepatitis C virus RNA using unmodified gold nanoparticles. Clin. Biochem. 43(13–14), 1163–1168 (2010). doi:10.1016/j.clinbiochem.2010.07.001CrossRefGoogle Scholar
  52. 52.
    M. Liu, M. Yuan, X. Lou, H. Mao, D. Zheng, R. Zou, N. Zou, X. Tang, J. Zhao, Label-free optical detection of single-base mismatches by the combination of nuclease and gold nanoparticles. Biosens. Bioelectron. 26(11), 4294–4300 (2011). doi:10.1016/j.bios.2011.04.014CrossRefGoogle Scholar
  53. 53.
    X. Xie, W. Xu, T. Li, X. Liu, Colorimetric detection of HIV-1 ribonuclease H activity by gold nanoparticles. Small 7(10), 1393–1396 (2011). doi:10.1002/smll.201002150CrossRefGoogle Scholar
  54. 54.
    H. Deng, X. Zhang, A. Kumar, G. Zou, X. Zhang, X.J. Liang, Long genomic DNA amplicons adsorption onto unmodified gold nanoparticles for colorimetric detection of Bacillus anthracis. Chem. Commun. (Camb.) 49(1), 51–53 (2013). doi:10.1039/c2cc37037aCrossRefGoogle Scholar
  55. 55.
    P. Anger, P. Bharadwaj, L. Novotny, Enhancement and quenching of single-molecule fluorescence. Phys. Rev. Lett. 96(11), 113002 (2006). doi:10.1103/PhysRevLett.96.113002CrossRefGoogle Scholar
  56. 56.
    S. Eustis, M.A. el-Sayed, 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. Chem. Soc. Rev. 35(3), 209–217 (2006). doi:10.1039/b514191eCrossRefGoogle Scholar
  57. 57.
    J. Gersten, Theory of fluorophore-metallic surface interactions, in Radiative Decay Engineering, ed. by C. Geddes, J. Lakowicz. Topics in Fluorescence Spectroscopy, vol. 8 (Springer, New York, 2005), pp. 197–221. doi:10.1007/0-387-27617-3_6CrossRefGoogle Scholar
  58. 58.
    S. Kühn, U. Håkanson, L. Rogobete, V. Sandoghdar, Enhancement of single-molecule fluorescence using a gold nanoparticle as an optical nanoantenna. Phys. Rev. Lett. 97(1), 017402 (2006). doi:10.1103/PhysRevLett.97.017402CrossRefGoogle Scholar
  59. 59.
    G. Doria, J. Conde, B. Veigas, L. Giestas, C. Almeida, M. Assuncao, J. Rosa, P.V. Baptista, Noble metal nanoparticles for biosensing applications. Sensors (Basel) 12(2), 1657–1687 (2012). doi:10.3390/s120201657CrossRefGoogle Scholar
  60. 60.
    G.K. Darbha, E. Lee, Y.R. Anderson, P. Fowler, K. Mitchell, P.C. Ray, Miniaturized sensor for microbial pathogens DNA and chemical toxins. IEEE Sens. J. 8(6), 693–700 (2008). doi:10.1109/jsen.2008.922727CrossRefGoogle Scholar
  61. 61.
    D. Zhang, D.J. Carr, E.C. Alocilja, Fluorescent bio-barcode DNA assay for the detection of Salmonella enterica serovar Enteritidis. Biosens. Bioelectron. 24(5), 1377–1381 (2009). doi:10.1016/j.bios.2008.07.081CrossRefGoogle Scholar
  62. 62.
    E.O. Ganbold, T. Kang, K. Lee, S.Y. Lee, S.W. Joo, Aggregation effects of gold nanoparticles for single-base mismatch detection in influenza A (H1N1) DNA sequences using fluorescence and Raman measurements. Colloids Surf. B Biointerfaces 93, 148–153 (2012). doi:10.1016/j.colsurfb.2011.12.026CrossRefGoogle Scholar
  63. 63.
    B. Dubertret, M. Calame, A.J. Libchaber, Single-mismatch detection using gold-quenched fluorescent oligonucleotides. Nat. Biotechnol. 19(4), 365–370 (2001). doi:10.1038/86762CrossRefGoogle Scholar
  64. 64.
    V. Beni, K. Hayes, T.M. Lerga, C.K. O’Sullivan, Development of a gold nano-particle-based fluorescent molecular beacon for detection of cystic fibrosis associated mutation. Biosens. Bioelectron. 26(2), 307–313 (2010). doi:10.1016/j.bios.2010.08.043CrossRefGoogle Scholar
  65. 65.
    V. Beni, T. Zewdu, H. Joda, I. Katakis, C.K. O’Sullivan, Gold nanoparticle fluorescent molecular beacon for low-resolution DQ2 gene HLA typing. Anal. Bioanal. Chem. 402(3), 1001–1009 (2012). doi:10.1007/s00216-011-5493-2CrossRefGoogle Scholar
  66. 66.
    Z.S. Wu, J.H. Jiang, L. Fu, G.L. Shen, R.Q. Yu, Optical detection of DNA hybridization based on fluorescence quenching of tagged oligonucleotide probes by gold nanoparticles. Anal. Biochem. 353(1), 22–29 (2006). doi:10.1016/j.ab.2006.01.018CrossRefGoogle Scholar
  67. 67.
    X. Wang, M. Zou, H. Huang, Y. Ren, L. Li, X. Yang, N. Li, Gold nanoparticle enhanced fluorescence anisotropy for the assay of single nucleotide polymorphisms (SNPs) based on toehold-mediated strand-displacement reaction. Biosens. Bioelectron. 41, 569–575 (2013). doi:10.1016/j.bios.2012.09.023CrossRefGoogle Scholar
  68. 68.
    J.N. Anker, W.P. Hall, O. Lyandres, N.C. Shah, J. Zhao, R.P. Van Duyne, Biosensing with plasmonic nanosensors. Nat. Mater. 7(6), 442–453 (2008). doi:10.1038/nmat2162CrossRefGoogle Scholar
  69. 69.
    Y.C. Cao, R. Jin, C.A. Mirkin, Nanoparticles with Raman spectroscopic fingerprints for DNA and RNA detection. Science 297(5586), 1536–1540 (2002). doi:10.1126/science.297.5586.1536CrossRefGoogle Scholar
  70. 70.
    J. Hu, P.C. Zheng, J.H. Jiang, G.L. Shen, R.Q. Yu, G.K. Liu, Sub-attomolar HIV-1 DNA detection using surface-enhanced Raman spectroscopy. Analyst 135(5), 1084–1089 (2010). doi:10.1039/b920358cCrossRefGoogle Scholar
  71. 71.
    N.R. Isola, D.L. Stokes, T. Vo-Dinh, Surface-enhanced Raman gene probe for HIV detection. Anal. Chem. 70(7), 1352–1356 (1998). doi:10.1021/ac970901zCrossRefGoogle Scholar
  72. 72.
    S.H. Chen, V.C. Wu, Y.C. Chuang, C.S. Lin, Using oligonucleotide-functionalized Au nanoparticles to rapidly detect foodborne pathogens on a piezoelectric biosensor. J. Microbiol. Methods 73(1), 7–17 (2008). doi:10.1016/j.mimet.2008.01.004CrossRefGoogle Scholar
  73. 73.
    L. Wang, Q. Wei, C. Wu, Z. Hu, J. Ji, P. Wang, The Escherichia coli O157:H7 DNA detection on a gold nanoparticle-enhanced piezoelectric biosensor. Chin. Sci. Bull. 53(8), 1175–1184 (2008). doi:10.1007/s11434-007-0529-xGoogle Scholar
  74. 74.
    R.Z. Hao, H.B. Song, G.M. Zuo, R.F. Yang, H.P. Wei, D.B. Wang, Z.Q. Cui, Z. Zhang, Z.X. Cheng, X.E. Zhang, DNA probe functionalized QCM biosensor based on gold nanoparticle amplification for Bacillus anthracis detection. Biosens. Bioelectron. 26(8), 3398–3404 (2011). doi:10.1016/j.bios.2011.01.010CrossRefGoogle Scholar
  75. 75.
    D. Astruc, Electron Transfer and Radical Processes in Transition Metal Chemistry (VCH, New York, 1995)Google Scholar
  76. 76.
    E. Katz, I. Willner, Integrated nanoparticle–biomolecule hybrid systems: synthesis, properties, and applications. Angew. Chem. Int. Ed. 43(45), 6042–6108 (2004). doi:10.1002/anie.200400651CrossRefGoogle Scholar
  77. 77.
    M.T. Castañeda, S. Alegret, A. Merkoçi, Electrochemical sensing of DNA using gold nanoparticles. Electroanalysis 19(7–8), 743–753 (2007). doi:10.1002/elan.200603784CrossRefGoogle Scholar
  78. 78.
    D. Zhang, M.C. Huarng, E.C. Alocilja, A multiplex nanoparticle-based bio-barcoded DNA sensor for the simultaneous detection of multiple pathogens. Biosens. Bioelectron. 26(4), 1736–1742 (2010). doi:10.1016/j.bios.2010.08.012CrossRefGoogle Scholar
  79. 79.
    C. Thiruppathiraja, S. Kamatchiammal, P. Adaikkappan, D.J. Santhosh, M. Alagar, Specific detection of Mycobacterium sp. genomic DNA using dual labeled gold nanoparticle based electrochemical biosensor. Anal. Biochem. 417(1), 73–79 (2011). doi:10.1016/j.ab.2011.05.034CrossRefGoogle Scholar
  80. 80.
    S.A. Vetrone, M.C. Huarng, E.C. Alocilja, Detection of non-PCR amplified S. enteritidis genomic DNA from food matrices using a gold-nanoparticle DNA biosensor: a proof-of-concept study. Sensors (Basel) 12(8), 10487–10499 (2012). doi:10.3390/s120810487CrossRefGoogle Scholar
  81. 81.
    K. May, Home tests to monitor fertility. Am. J. Obstet. Gynecol. 165(6), 2000–2002 (1991). doi:10.1016/s0002-9378(11)90566-3CrossRefGoogle Scholar
  82. 82.
    R. Wilson, The use of gold nanoparticles in diagnostics and detection. Chem. Soc. Rev. 37(9), 2028–2045 (2008). doi:10.1039/b712179mCrossRefGoogle Scholar
  83. 83.
    V. Kumanan, S.R. Nugen, A.J. Baeumner, Y.F. Chang, A biosensor assay for the detection of Mycobacterium avium subsp. paratuberculosis in fecal samples. J. Vet. Sci. 10(1), 35–42 (2009). doi:10.4142/jvs.2009.10.1.35CrossRefGoogle Scholar
  84. 84.
    P. Chun, Colloidal gold and other labels for lateral flow immunoassays, in Lateral Flow Immunoassay, ed. by R. Wong, H. Tse (Humana Press, Totowa, 2009), pp. 1–19. doi:10.1007/978-1-59745-240-3CrossRefGoogle Scholar
  85. 85.
    J. Aveyard, M. Mehrabi, A. Cossins, H. Braven, R. Wilson, One step visual detection of PCR products with gold nanoparticles and a nucleic acid lateral flow (NALF) device. Chem. Commun. 41, 4251–4253 (2007). doi:10.1039/B708859KCrossRefGoogle Scholar
  86. 86.
    D. Mark, S. Haeberle, G. Roth, F. von Stetten, R. Zengerle, Microfluidic lab-on-a-chip platforms: requirements, characteristics and applications. Chem. Soc. Rev. 39(3), 1153–1182 (2010). doi:10.1039/b820557bCrossRefGoogle Scholar
  87. 87.
    A. Chua, C.Y. Yean, M. Ravichandran, B. Lim, P. Lalitha, A rapid DNA biosensor for the molecular diagnosis of infectious disease. Biosens. Bioelectron. 26(9), 3825–3831 (2011). doi:10.1016/j.bios.2011.02.040CrossRefGoogle Scholar
  88. 88.
    B.A. Rohrman, V. Leautaud, E. Molyneux, R.R. Richards-Kortum, A lateral flow assay for quantitative detection of amplified HIV-1 RNA. PLoS One 7(9), e45611 (2012). doi:10.1371/journal.pone.0045611CrossRefGoogle Scholar
  89. 89.
    S.Y. Hou, Y.L. Hsiao, M.S. Lin, C.C. Yen, C.S. Chang, MicroRNA detection using lateral flow nucleic acid strips with gold nanoparticles. Talanta 99, 375–379 (2012). doi:10.1016/j.talanta.2012.05.067CrossRefGoogle Scholar
  90. 90.
    J.A. Lefferts, M.C. Schwab, U.B. Dandamudi, H.K. Lee, L.D. Lewis, G.J. Tsongalis, Warfarin genotyping using three different platforms. Am. J. Transl. Res. 2(4), 441–446 (2010)Google Scholar
  91. 91.
    C.B. Maurice, P.K. Barua, D. Simses, P. Smith, J.G. Howe, G. Stack, Comparison of assay systems for warfarin-related CYP2C9 and VKORC1 genotyping. Clin. Chim. Acta 411(13–14), 947–954 (2010). doi:10.1016/j.cca.2010.03.005CrossRefGoogle Scholar
  92. 92.
    M. Larguinho, P.V. Baptista, Gold and silver nanoparticles for clinical diagnostics – from genomics to proteomics. J. Proteomics 75(10), 2811–2823 (2012). doi:10.1016/j.jprot.2011.11.007CrossRefGoogle Scholar
  93. 93.
    K. Sato, K. Hosokawa, M. Maeda, Non-cross-linking gold nanoparticle aggregation as a detection method for single-base substitutions. Nucleic Acids Res. 33(1), e4 (2005). doi: 10.1093/nar/gni007CrossRefGoogle Scholar
  94. 94.
    W.J. Qin, L.Y. Yung, Nanoparticle-based detection and quantification of DNA with single nucleotide polymorphism (SNP) discrimination selectivity. Nucleic Acids Res. 35(17), e111 (2007). doi: 10.1093/nar/gkm602CrossRefGoogle Scholar
  95. 95.
    H. Deng, Y. Xu, Y. Liu, Z. Che, H. Guo, S. Shan, Y. Sun, X. Liu, Gold nanoparticles with asymmetric polymerase chain reaction for colorimetric detection of DNA sequence. Anal. Chem. 84(3), 1253–1258 (2012). doi: 10.1021/ac201713tCrossRefGoogle Scholar
  96. 96.
    Y.L. Jung, C. Jung, H. Parab, T. Li, H.G. Park, Direct colorimetric diagnosis of pathogen infections by utilizing thiol-labeled PCR primers and unmodified gold nanoparticles. Biosens. Bioelectron. 25(8), 1941–1946 (2010). doi: 10.1016/j.bios.2010.01.010CrossRefGoogle Scholar
  97. 97.
    J.J. Storhoff, R. Elghanian, R.C. Mucic, C.A. Mirkin, R.L. Letsinger, One-pot colorimetric differentiation of polynucleotides with single base imperfections using gold nanoparticle probes. J. Am. Chem. Soc. 120(9), 1959–1964 (1998). doi: 10.1021/ja972332iCrossRefGoogle Scholar
  98. 98.
    W.J. Qin, O.S. Yim, P.S. Lai, L.Y. Yung, Dimeric gold nanoparticle assembly for detection and discrimination of single nucleotide mutation in Duchenne muscular dystrophy. Biosens. Bioelectron. 25(9), 2021–2025 (2010). doi: 10.1016/j.bios.2010.01.028CrossRefGoogle Scholar
  99. 99.
    J. Li, T. Deng, X. Chu, R. Yang, J. Jiang, G. Shen, R Yu, Rolling circle amplification combined with gold nanoparticle aggregates for highly sensitive identification of single-nucleotide polymorphisms. Anal. Chem. 82(7), 2811–2816 (2010). doi: 10.1021/ac100336nCrossRefGoogle Scholar
  100. 100.
    D. Xi, X. Luo, Q. Ning, Detection of HBV and HCV coinfection by TEM with Au nanoparticle gene probes. J. Huazhong Univ. Sci. Technolog. Med. Sci. 27(5), 532–534 (2007). doi: 10.1007/s11596-007-0514-2CrossRefGoogle Scholar
  101. 101.
    Y.P. Bao, M. Huber, T.F. Wei, S.S. Marla, J.J. Storhoff, U.R. Müller, SNP identification in unamplified human genomic DNA with gold nanoparticle probes. Nucleic Acids Res. 33(2), e15 (2005). doi: 10.1093/nar/gni017CrossRefGoogle Scholar
  102. 102.
    X. Mao, Y. Ma, A. Zhang, L. Zhang, L. Zeng, G. Liu, Disposable nucleic acid biosensors based on gold nanoparticle probes and lateral flow strip. Anal. Chem. 81(4), 1660–1668 (2009). doi: 10.1021/ac8024653CrossRefGoogle Scholar
  103. 103.
    I.K. Litos, P.C. Ioannou, T.K. Christopoulos, J. Traeger-Synodinos, E. Kanavakis, Multianalyte, dipstick-type, nanoparticle-based DNA biosensor for visual genotyping of single-nucleotide polymorphisms. Biosens. Bioelectron. 24(10), 3135–3139 (2009). doi: 10.1016/j.bios.2009.03.010CrossRefGoogle Scholar
  104. 104.
    Y.N. Tan, K.H. Lee, X. Su, Study of single-stranded DNA binding protein-nucleic acids interactions using unmodified gold nanoparticles and its application for detection of single nucleotide polymorphisms. Anal. Chem. 83(11), 4251–4257 (2011). doi: 10.1021/ac200525aCrossRefGoogle Scholar
  105. 105.
    C.C. Chang, S.C. Wei, T.H. Wu, C.H. Lee, C.W. Lin, Aptamer-based colorimetric detection of platelet-derived growth factor using unmodified gold nanoparticles. Biosens. Bioelectron. 42, 119–123 (2013). doi: 10.1016/j.bios.2012.10.072CrossRefGoogle Scholar
  106. 106.
    M.M. Hussain, T.M. Samir, H.M. Azzazy, Unmodified gold nanoparticles for direct and rapid detection of Mycobacterium tuberculosis complex. Clin. Biochem. 46(7–8):633–637 (2013). doi: 10.1016/j.clinbiochem.2012.12.020CrossRefGoogle Scholar
  107. 107.
    J. Rosa, J. Conde, J.M. de la Fuente, J.C. Lima, P.V. Baptista, Gold-nanobeacons for real-time monitoring of RNA synthesis. Biosens. Bioelectron. 36(1), 161–167 (2012). doi: 10.1016/j.bios.2012.04.006CrossRefGoogle Scholar
  108. 108.
    M.Y. Sha, S. Penn, G. Freeman, W.E. Doering, Detection of human viral RNA via a combined fluorescence and SERS molecular beacon assay. NanoBiotechnol. 3(1), 23–30 (2007). doi: 10.1007/s12030-007-0003-5CrossRefGoogle Scholar
  109. 109.
    J. Hu, C.Y. Zhang, Single base extension reaction-based surface enhanced Raman spectroscopy for DNA methylation assay. Biosens. Bioelectron. 31(1), 451–457 (2012). doi: 10.1016/j.bios.2011.11.014CrossRefGoogle Scholar
  110. 110.
    L. Sun, J. Irudayaraj, PCR-free quantification of multiple splice variants in a cancer gene by surface-enhanced Raman spectroscopy. J. Phys. Chem. B 113(42), 14021–14025 (2009). doi: 10.1021/jp908225fCrossRefGoogle Scholar
  111. 111.
    W. Li, P. Wu, H. Zhang, C. Cai, Catalytic signal amplification of gold nanoparticles combining with conformation-switched hairpin DNA probe for hepatitis C virus quantification. Chem. Commun. (Camb) 48(63), 7877–7879 (2012). doi: 10.1039/c2cc33635aCrossRefGoogle Scholar
  112. 112.
    M. Ozsoz, A. Erdem, K. Kerman, D. Ozkan, B. Tugrul, N. Topcuoglu, H. Ekren, M. Taylan, Electrochemical genosensor based on colloidal gold nanoparticles for the detection of Factor V Leiden mutation using disposable pencil graphite electrodes. Anal. Chem. 75(9), 2181–2187 (2003). doi: 10.1021/ac026212rCrossRefGoogle Scholar
  113. 113.
    K.F. Low, A. Karimah, C.Y. Yean, A thermostabilized magnetogenosensing assay for DNA sequence-specific detection and quantification of Vibrio cholerae. Biosens. Bioelectron. 47, 38–44 (2013). doi:10.1016/j.bios.2013.03.004CrossRefGoogle Scholar
  114. 114.
    P.J. Jannetto, B.W. Buchan, K.A. Vaughan, J.S. Ledford, D.K. Anderson, D.C. Henley, N.B. Quigley, N.A. Ledeboer, Real-time detection of influenza a, influenza B, and respiratory syncytial virus a and B in respiratory specimens by use of nanoparticle probes. J. Clin. Microbiol. 48(11), 3997–4002 (2010). doi: 10.1128/JCM.01118-10CrossRefGoogle Scholar
  115. 115.
    B. Yang, K. Gu, X. Sun, H. Huang, Y. Ding, F. Wang, G. Zhou, L.L. Huang, Simultaneous detection of attomolar pathogen DNAs by Bio-MassCode mass spectrometry. Chem. Commun. (Camb). 46(43), 8288–8290 (2010). doi: 10.1039/c0cc03156aCrossRefGoogle Scholar

Copyright information

© Springer International Publishing Switzerland 2016

Authors and Affiliations

  • Ricardo Franco
    • 1
  • Pedro Pedrosa
    • 2
  • Fábio Ferreira Carlos
    • 2
    • 3
  • Bruno Veigas
    • 2
    • 4
  • Pedro V. Baptista
    • 5
    Email author
  1. 1.REQUIMTE, Department of Chemistry, Faculdade de Ciências e TecnologiaUniversidade Nova de LisboaCaparicaPortugal
  2. 2.CIGMH, Department of Life Sciences, Faculdade de Ciências e TecnologiaUniversidade Nova de LisboaCaparicaPortugal
  3. 3.STABVIDACaparicaPortugal
  4. 4.CENIMAT-I3N, Department of Material Sciences, Faculdade de Ciências e TecnologiaUniversidade Nova de LisboaCaparicaPortugal
  5. 5.UCIBIO, CIGMH, Departamento de Ciências da Vida Faculdade de Ciências e TecnologiaUniversidade Nova de LisboaCaparicaPortugal

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