Development of gold nanoparticles biosensor for ultrasensitive diagnosis of foot and mouth disease virus
© The Author(s) 2018
Received: 5 September 2017
Accepted: 28 April 2018
Published: 11 May 2018
Nano-PCR is a recent tool that is used in viral diseases diagnosis. The technique depends on the fundamental effects of gold nanoparticles (AuNPs) and is considered a very effective and sensitive tool in the diagnosis of different diseases including viral diseases. Although several techniques are currently available to diagnose foot and mouth disease virus (FMDV), a highly sensitive, highly specific technique is needed for specific diagnosis of the disease. In the present work, a novel AuNPs biosensor has been designed using thiol-linked oligonucleotides that recognize the conserved 3D gene of FMDV.
The AuNPs-FMDV biosensor specifically recognizes RNA standards of FMDV, but not that of swine vesicular disease virus (SVDV) isolates. The analytical sensitivity of the AuNPs-FMDV biosensor was 10 copy number RNA standards in RT-PCR and 1 copy number RNA standard in real-time rRT-PCR with a 94.5% efficiency, 0.989 R2, a − 3.544 slope and 100% specificity (no cross-reactivity with SVDV). These findings were confirmed by the specific and sensitive recognition of 31 Egyptian FMDV clinical isolates that represents the three FMDV serotypes (O, A, and SAT2).
The AuNPs-FMDV biosensor presents in this study demonstrates a superior analytical and clinical performance for FMDV diagnosis. In addition, this biosensor has a simple workflow and accelerates epidemiological surveillance, hence, it is qualified as an efficient FMDV diagnosis tool for quarantine stations and farms particularly in FMDV endemic areas.
PCR enhancers include small organic molecules (dimethyl sulphoxide, glycerol, betaine monohydrate, and formamide) , non-ionic detergents (0.1–1% Triton X-100, and Tween-20), proteins, such as bovine serum albumin (BSA), and single-stranded DNA binding proteins (SSBPs)  had been extensively used to improve PCR application for routine diagnostic purposes. However, there is still a need to develop more appropriate additives to enhance the specificity and efficiency of PCR, which is considered a great challenge . In recent years, huge research efforts have been directed to using various nanoparticle (NP)-based enhancers, that lead to the development of what so called nanomaterials-assisted PCR (nano-PCR) [4, 5]. NP-based enhancers such as gold nanoparticles (AuNPs) , semiconductor quantum dots , carbon nanotubes  and carbon nano powders , have shown to improve PCR specificity and efficiency by different working mechanisms that range from relieving the secondary DNA structure in GC-rich regions or in long amplification products, the reduction of the template melting temperature, the stabilization of DNA polymerases and the enhancement of its activity, to the prevention of adsorption of polymerases to plastic ware .
Out of these enhancers, AuNPs stands out as the most well-known and effective enhancer that is capable to improve two PCR rounds with respect to both yield and specificity . In the presence of the appropriate amount of AuNPs, the target product may be achieved after only six PCR cycles . In line with this, AuNPs have been reported to increase the sensitivity of PCR detection five- to tenfold in a conventional PCR system and 104-fold in a real-time PCR system . Moreover, AuNPs modulate the activity of DNA polymerases and achieve hot-start activity in the presence of conventional Pyrococcus furiosus (Pfu) polymerases . Besides, AuNPs have unique chemical and physical properties such as design flexibility, large surface-to-volume ratio, simple surface modification with multivalent ligands, catalytic effect for electrochemical reactions, improvement of electron transfer, and labelling of biomolecules, which make such enhancer particularly appropriate for designing new and improved biosensors [15, 16]. Indeed, AuNPs had been used as immunosensor , DNA sensors , streptavidin-AuNPs , and the layer-by-layer amino-thiophenol-AuNPs network .
AuNPs have been also used in RNA detection where a rapid label-free visual assay has been developed using peptide nucleic acid (PNA) probes and AuNPs. The specific agglomerative behaviour of PNA with AuNPs can detect as low as 5–10 ng of viral RNA from various biological samples, indicating the sensitivity of this assay . Hence, it was extensively used in viral diagnosis. A rapid and specific diagnosis of Japanese encephalitis virus (JEV) was achieved using an AuNPs-based RT-PCR and rRT-PCR assay . In addition, a specific label-free AuNPs immunosensor was optimized and applied in the diagnosis of dengue virus using , layer-by-layer AuNPs hybridization on a quartz crystal microbalance (QCM) DNA sensing system  and 4G2 antibody-AuNPs surface enhanced Raman spectroscopy (SERS) fingerprinting . Moreover, an AuNPs-immunochromatographic assay (AuNPs-ICA) was used for detecting severe fever with thrombocytopenia syndrome virus (SFTSV) infection with a sensitivity of 98.4% for IgM and 96.7% for IgG . This significant role of AuNPs in viral diagnosis was also highlighted in the colorimetric detection of influenza virus using AuNPs-RT loop-mediated isothermal amplification (AuNPs-RT-LAMP) that targets the virus M protein gene and showed 100% specificity and 98.6% sensitivity in comparison to conventional RT-LAMP . In addition, the influenza virus was detected using a portable AuNPs biosensor and based on surface-enhanced Raman scattering (SERS) with a 1 pg/µL detection limit [27, 28]. Influenza virus was detected also by biosensors based on dynamic light scattering (DLS) .
Foot and Mouth Disease (FMD) is one of the most infectious viral diseases with the potential for devastating economic, social and environmental impacts. The aetiological agent (FMDV) belongs to the Aphthovirus genus and family Picornaviridae and is present as seven serotypes (A, O, C, Asia1, and SAT 1, 2, and 3) with multiple antigenic and genetic variants. Currently, the reference method for the detection of all FMDV serotypes is real-time RT-PCR, which is based on protocols generated from Callahan et al.  and Reid et al.  that detect the virus RNA-dependent RNA polymerase (3D gene) and the 5′ untranslated region (5′UTR), respectively. Although, the two methods had been used extensively used for the virus detection and in-turn the disease diagnosis, they need further optimization. While there are several available methods to diagnose FMDV such as virus neutralization test, ELISA, and RT-PCR, there is still an essential need to improve these methods to make it more sensitive and specific.
In the present study, we evaluated AuNPs-based rRT-PCR for the detection of foot and mouth disease virus (FMDV). We found that AuNPs-FMDV biosensor was designed using thiol-linked primers of the 3D rRT-PCR . This biosensor has been validated with the FMDV RNA standard from the synthetic 3D gene of FMDV. Application of the AuNPs-FMDV biosensor in RT-PCR and rRT-PCR was conducted to test the enhancement effect of the AuNPs-FMDV biosensor in the specificity, the analytical sensitivity, dynamic range, efficiency and the limit of detection (LOD) of RT-PCR and rRT-PCR.
Synthesis and characterization of colloidal AuNPs (13 nm)
The synthesis of the AuNPs was conducted as previously described  and characterized using high resolution Transmission Electron Microscopy (TEM) (to examine the shape and size of AuNPs), UV–Vis spectrophotometer (to show absorption peaks of AuNPs), Dynamic light scattering (DLS) (to determine the size of the AuNPs using Zetasizer), and zeta potential distribution (to determine the net charge of the AuNPs using the Zetasizer).
Design and characterization of AuNPs-FMDV biosensor
Sequences of the 3D poly A thiol-linked oligonucleotides
Modification and sequence
(5′ Thiol group-AAAAAAAAAA-ACTGGG TTT TAC AAA CCT GTG A 3′)
(5′ Thiol group-AAAAAAAAAA-GCG AGT CCT GCC ACG GA 3′)
FAM (5′ TCCTT TGCAC GCCGT GGGAC 3′)
Real time RT-PCR
FMDV rRT-PCR was done using QuantiTect Kit (Qiagen) in a real-time PCR machine (StepOne, Applied Biosystems) with a thermal profile according to the manufacturer’s instructions. To study the effect of different thiol-linked oligonucleotides concentrations (400, 600, and 800 nM) during AuNPs-FMDV biosensor design a standard curve of rRT-PCR for each concentration was generated using an RNA standard (GeneArt, Thermo Fisher). For studying the effect of rRT-PCR conditions (salt concentration in PCR buffer and denaturation temperature) on the AuNPs-FMDV biosensor, PCR products were characterized with the UV–Vis–NIR spectrophotometer to compare the optical density of the AuNPs-FMDV biosensor before and after the rRT-PCR.
Validation and harmonization of the analytical and diagnostic sensitivity for the AuNPs-FMDV biosensor with rRT-PCR and RT-PCR
The AuNPs-FMDV biosensor was evaluated for the analytical sensitivity using the FMDV 3D gene synthesized by (GeneArt, Thermo Fisher). Additionally, the AuNPs-FMDV biosensor was evaluated for the diagnostic sensitivity using the previously sequenced FMDV isolates (A Iran 05, O1 Manisa, and SAT2 Gharbia). And swine vesicular disease virus (SVDV), which were used as a negative control to evaluate the optimal AuNPs oligonucleotides and probe concentrations. RNA extraction was conducted using a QIAamp viral RNA mini kit (Qiagen) according to the manufacturer’s instructions and one-step rRT-PCR was done as mentioned above.
Application of the AuNPs-FMDV biosensor with clinical samples
Thirty-one clinical field samples (unruptured and recently ruptured vesicles in the buccal cavity, vesicular fluid, epithelium and hearts) were collected from cattle, buffalos, and calves from Egypt from March 2012 to September 2015 and were tested with the FMDV-AuNPs biosensor using rRT-PCR technique.
Design and characterization of AuNPs-FMDV biosensor
To generate AuNPs-FMDV biosensor, thiol-linked poly(A) oligonucleotides that recognize FMDV 3D gene was conjugated to the colloidal AuNPs as described in “Methods” and the conjugation of the oligonucleotides to AuNPs was monitored by different characterization methods. TEM images of the conjugated AuNPs showed particles that are slightly bigger in size (17–20 nm diameter) than the unconjugated (naked) 13 nm in diameter AuNPs (compare Fig. 1A–C. Moreover, a light grey zone appears to surround each AuNPs conjugated particle (Fig. 1B, C), whereas this zone was not observed in naked AuNPs (Fig. 1A), suggesting that this may be the area representing the conjugation zone. To further confirm the conjugation process, the UV–Vis–NIR spectrophotometer absorbance of the conjugated AuNPs as well as thiol-linked poly(A) oligonucleotides was compared to that of the naked AuNPs. As shown in Fig. 2A, naked AuNPs showed a peak at absorbance–wavelength of 520 nm, whereas an absorbance peak of 260 nm was observed with thiol oligonucleotides alone (Fig. 2B). In contrast, the combination of AuNPs and the thiol oligonucleotides (conjugated AuNPs) showed two absorption peaks, one for the AuNPs at 520 nm and the other for conjugated thiol-linked poly(A) oligonucleotides at 260 nm (Fig. 2C), indicating the conjugation of AuNPs-FMDV oligonucleotides via thiol linkage. Moreover, DLS analysis confirmed such conjugation, where the addition of FMDV oligonucleotides shifted the naked AuNPs peak from 13 to 17–20 nm (compare Fig. 3a, b). In a final attempt to fully characterize the produced AuNPs-FMDV oligonucleotides conjugation, zeta potential of conjugated AuNPs particles was measured and compared to that of the naked AuNPs. As shown in Fig. 4 the conjugation of FMDV oligonucleotides to naked AuNPs induces a pronounced change in the zeta potential measurements from − 38.9 (naked AuNPs, Fig. 4a) to − 18.3 m V (AuNPs-FMDV, Fig. 4b). Collectively these findings ensure that we could produce AuNPs conjugated to FMDV oligonucleotides that can be used as a possible biosensor for FMDV detection.
Validation and harmonization of the analytical and diagnostic sensitivity for the AuNPs-FMDV biosensor with rRT-PCR and RT-PCR
Results of analytical sensitivity, dynamic range and limit of detection (LOD) with different conditions
AuNPs 0.7 nM-FMDV sensor primer Concentration
400 nM primer
600 nM primer
800 nM primer
Validation of the AuNPs 3D FMDV biosensor specificity
To exclude the possibility of cross reactivity diagnosis, the AuNPs-FMDV biosensor has been validated for specificity using RNA standard of 10 the FMDV closely related family member [swine vesicular disease virus isolates (SVDV)]. In parallel, serial dilution of the standard RNA for the FMDV 3D gene from Log 5–1 copy number was used as a positive control. Using conventional PCR or rRT-PCR reactions, we were not able to detect any specific bands or signals with SVDV isolates, respectively. In contrast, the AuNPs-FMDV biosensor specifically detected FMDV RNA as clearly shown by the very specific PCR bands representing 3D gene. (Additional file 1: Fig. S1). Moreover, PCR reaction could detect greater than Log 3 copy numbers with observed non-specific bands. Interestingly, rRT-PCR data showed an increase in the sensitivity up to tenfold indicating that the AuNPs-FMDV biosensor could detect greater than 100 copy numbers in the RT-PCR. This data, confirm that AuNPs-FMDV biosensor is a highly sensitive method that specifically detect FMDV without any cross reactivity with other related family members.
Application of the AuNPs-FMDV biosensor to detect FMDV in clinical samples
Results of clinical samples with classical rRT-PCR reagents and with AuNPs-FMDV biosensor
Type of sample
Classical reagents (C)
AuNPs-FMDV biosensor (S)
The mass culling of animals has generated interest in the development of sensitive diagnostic techniques and safe effective vaccines to confine outbreaks [36, 37], especially for FMDV, as it is highly variable, and its serotyping needs nucleotide sequencing and continuous monitoring of the primers’ sensitivity and specificity . The integration among science branches can develop new sensitive diagnostic techniques and increase the sensitivity, specificity and the efficiency of current diagnostic techniques. In the present study, an AuNPs-FMDV biosensor was designed. To obtain this biosensor, naked AuNPs were synthesized using a citrate reduction method . Characterization of the prepared particles was conducted by 4 techniques, which demonstrate an average diameter of 13 nm under transmission electron microscopy (TEM) (Fig. 1a), and UV–Vis spectrum analysis demonstrated the specific peak of the AuNPs at the absorption wavelength of 520 nm (Fig. 2a) and dynamic light scattering of 13 nm AuNPs (Fig. 3a). Moreover, the net charges of the AuNPs were characterized by the Zeta sizer, which showed a peak of zeta potential distribution representing the surface charges (− 38.9 m V) of the naked AuNPs (Fig. 4a). The AuNPs particles become stable and protected by the citrate capping layer and can be stored under sterile conditions for several months.
Citrate capping of AuNPs could be replaced easily with various ligands such as peptides, proteins and oligonucleotides . Thiol-linked oligonucleotides and peptides were employed to functionalize the AuNPs . The AuNPs-FMDV biosensor for the conserved region of RNA dependent RNA polymerase of the FMDV genome (3D gene) was designed according to  with some modifications. Functionalization of the AuNPs with thiol-linked oligonucleotides and peptides are the most common approaches . The prepared spherical 13 nm AuNPs have a maximum thiol-linked oligonucleotide loading density according to  who explored the relationship between the AuNPs size and the DNA loading density. The AuNPs-FMDV biosensor for the conserved region within the FMDV genome (3D gene) was designed according to  in which the thiol-linked polyA oligonucleotides for the 3D gene will replace the citrate ions and bind to the AuNPs. The thiol-linked poly(A) oligonucleotide length was 22 nucleotides for the forward primer and 17 nucleotides for reverse primer for the 3D gene with 10 poly(A) nucleotides with sequences as shown in (Table 1). This length ensured optimum immobilization according to . Poly(A) nucleotides were used as a spacer for organized immobilization . De-protection of the thiol-linked oligonucleotides (reduction by disulphide cleavage buffer) was conducted as previously reported . After that, the purification of freshly reduced thiol-linked oligonucleotides was conducted by using the Nap-5 column, and the thiol-linked oligonucleotides were desalted and purified according to the manufacturer’s instructions. Design and characterization of the 3D AuNPs-FMDV biosensor and the conjugation process of the AuNPs and functionalization of AuNPs with poly A thiol-linked oligonucleotides was conducted following the published protocol  with some validation modification of the conjugation of the 400, 600 and 800 nM concentration of poly(A) thiol-linked oligonucleotides with 0.7 nM of 13 nm AuNPs.
To study the effect of the thiol-linked oligonucleotide concentration during the AuNPs-FMDV biosensor design, a standard curve was generated using standard RNA with the Real-time RT-PCR (rRT-PCR) to determine the limit of detection (LOD) and in regard to the successful real-time parameters as the cut-off CT value of FMDV lower than 32 as recommended by ; the acceptable CT difference value between 2 dilutions (slope) ranges between 3.1 and 3.58 and the acceptable percentage for the ability to conduct exponential amplification (efficiency %) is 90–110% . In the present study, validation of the optimum thiol-linked oligonucleotide concentration, which has acceptable characteristics for the rRT-PCR assay, was tested with 400, 600 and 800 nM. The LOD, slope and efficiency % are illustrated in (Table 2). The thiol-linked oligonucleotide concentration of 400 nM showed acceptable success parameters with an efficiency of 94.5%, a slope − 3.544 and an LOD of 1 copy number as illustrated in Fig. 7a, b. Conversely, the thiol-linked oligonucleotide concentration of 600 and 800 nM had poor efficiency of 120 and 346% with a slope of − 2.8 and − 1.53; respectively, and with an LOD of 100 copy numbers for both as shown in Additional files 1, 2, and 3. Therefore, we recommend the conjugation with the 400 nM thiol-linked oligonucleotide concentration for designing the AuNPs-FMDV biosensor.
Moreover, to study the effect of the PCR conditions (the salt concentration in the PCR buffer and denaturation temperature), a comparison of optical densities of the AuNPs-FMDV biosensor before and after PCR was conducted. The peaks of the AuNPs after PCR have little variety from the peak intensity before PCR, but both have the same peak position at the 520 nm absorbance–wavelength; there was a slight disappearance of the immobilized thiol-linked oligonucleotide peak (260 nm) due to consumption of the primers in the PCR reaction (Fig. 5). This effect is observed is because the salt concentration in the PCR buffer was only three-tenths of that in Ref. . Moreover, the denaturation temperature of 95 °C did not cause aggregation of the AuNPs because the sodium citrate cannot be reacted until the boiling temperature is achieved . To study the stability of the AuNPs-FMDV biosensor, it was divided into aliquots and stored in a refrigerator at 4 °C for 6 months and each aliquot was tested by rRT-PCR to test the stability of the AuNPs-FMDV biosensor. The results revealed that the AuNPs-FMDV biosensor could be stored at 4 °C rather than − 20 °C for 6 months without changing its integrity and its analytical sensitivity.
The AuNPs-FMDV biosensor demonstrates a superior analytical and clinical performance for the FMDV diagnosis. The analytical sensitivity and dynamic range LOD of the AuNPs-FMDV biosensor was 10 copy numbers of the RNA standard in RT-PCR and 1 copy number of the RNA standard in the rRT-PCR with a 94.5% efficiency, 0.989 R2, a − 3.544 slope and 100% specificity without cross reactivity with SVDV. It has a simple workflow and it accelerates epidemiological surveillance. Hence, it is suitable for quarantine stations and farms for diagnosis, particularly in FMDV endemic areas.
Study conception and design: MEH, HAH, TAS, and AHE-D. Acquisition of data: MEH, HAH, MDC, GP, and TAS. Analysis and interpretation of data: MEH, HAH, TAS, DC and GP. Drafting of the manuscript: MEH and HAH. Critical revision: MEH, HAH, MME, GP, and DC. Approved of the version of the manuscript to be published: MEH, MDC, HAH, TAS, AHE-D, GP and DC. All authors read and approved the final manuscript.
Profound appreciation and the sincerest thanks are offered to Science and Technology Development Fund-Research Scientific Grant (STDF-RSG) Project No. (12718) for funding the current work.
The authors declare that they have no competing interests.
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This work was supported by the Science and Technology Development Fund-Research Scientific Grant (STDF-RSG) Project No. (12718). The funding sources had no role in the study design, collection or analysis of the data, writing of the manuscript, or in the decision to submit the manuscript for publication.
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