Limits...
One nanoprobe, two pathogens: gold nanoprobes multiplexing for point-of-care.

Veigas B, Pedrosa P, Carlos FF, Mancio-Silva L, Grosso AR, Fortunato E, Mota MM, Baptista PV - J Nanobiotechnology (2015)

Bottom Line: Following characterisation, the developed gold-nanoprobe allowed detection of either target in individual samples or in samples containing both DNA species with the same efficacy.Using one single probe via the non-cross-linking colorimetric methodology it is possible to identify multiple targets in one sample in one reaction.This proof-of-concept approach may easily be integrated into sensing platforms allowing for fast and simple multiplexing of Au-nanoprobe based detection at point-of-need.

View Article: PubMed Central - PubMed

Affiliation: Nanomedicine@FCT, Departamento de Ciências da Vida, Faculdade de Ciências e Tecnologia, CIGMH, UCIBIO, Universidade Nova de Lisboa, Campus de Caparica, 2829-516, Caparica, Portugal. bmrveigas@gmail.com.

ABSTRACT

Background: Gold nanoparticles have been widely employed for biosensing purposes with remarkable efficacy for DNA detection. Amongst the proposed systems, colorimetric strategies based on the remarkable optical properties have provided for simple yet effective sequence discrimination with potential for molecular diagnostics at point of need. These systems may also been used for parallel detection of several targets to provide additional information on diagnostics of pathogens.

Results: For the first time, we demonstrate that a single Au-nanoprobe may provide for detection of two distinct targets (pathogens) allowing colorimetric multi-target detection. We demonstrate this concept by using one single gold-nanoprobe capable to detect members of the Mycobacterium tuberculosis complex and Plasmodium sp., the etiologic agents of tuberculosis and malaria, respectively. Following characterisation, the developed gold-nanoprobe allowed detection of either target in individual samples or in samples containing both DNA species with the same efficacy.

Conclusions: Using one single probe via the non-cross-linking colorimetric methodology it is possible to identify multiple targets in one sample in one reaction. This proof-of-concept approach may easily be integrated into sensing platforms allowing for fast and simple multiplexing of Au-nanoprobe based detection at point-of-need.

No MeSH data available.


Related in: MedlinePlus

Single pathogen Au-nanoprobe detection assays using a MTBC probe; bPlasmodium sp. probe. The colorimetric assay consists of visual comparison of test solutions after salt induced Au-nanoprobe aggregation. Au-nanoprobes aggregation was measured by ratio of aggregation (ratio of SPR intensity at 525 and 600 nm) for the assay mixtures—2.5 nM Au-nanoprobe, 10 mM phosphate buffer (pH 8) + 0.1 M NaCl, purified dsDNA targets (PCR products) at a final concentration of 60 ng/µL. All spectrophotometric data was collected 30 min after salt addition and error bars represent the standard deviation of three independent assays. The horizontal line represents the threshold of 1 considered for discrimination between positive (rAbs ≥ 1) and negative (rAbs < 1) result. A representative colorimetric results is showed upon each result bar-red positive result; blue/purple negative result.
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4527100&req=5

Fig3: Single pathogen Au-nanoprobe detection assays using a MTBC probe; bPlasmodium sp. probe. The colorimetric assay consists of visual comparison of test solutions after salt induced Au-nanoprobe aggregation. Au-nanoprobes aggregation was measured by ratio of aggregation (ratio of SPR intensity at 525 and 600 nm) for the assay mixtures—2.5 nM Au-nanoprobe, 10 mM phosphate buffer (pH 8) + 0.1 M NaCl, purified dsDNA targets (PCR products) at a final concentration of 60 ng/µL. All spectrophotometric data was collected 30 min after salt addition and error bars represent the standard deviation of three independent assays. The horizontal line represents the threshold of 1 considered for discrimination between positive (rAbs ≥ 1) and negative (rAbs < 1) result. A representative colorimetric results is showed upon each result bar-red positive result; blue/purple negative result.

Mentions: First, we optimized the multiplex PCR amplification so that both two pathogens’ gene fragments are present with similar yields while maintaining PCR specificity. Primers were designed so as to produce amplicons showing similar sizes, since comparable hybridization efficiencies and profiles are desired [18]. The two loci were successfully amplified alone and then in a single multiplex reaction from DNA isolates of the pathogens (Additional file 1: Figure S1). Then, each Au-nanoprobe sequence was evaluated in terms of selectivity and specificity towards the respective target in solution (Fig. 2). Calibration of the Au-nanoprobe functionalization in presence of the respective target, but aggregated in its absence (see Additional file 1: Figures S3 and S4). No noteworthy loss in signal occurs when both pathogens amplicons are present in the mixture compared to each amplicon alone (Fig. 3).Fig. 2


One nanoprobe, two pathogens: gold nanoprobes multiplexing for point-of-care.

Veigas B, Pedrosa P, Carlos FF, Mancio-Silva L, Grosso AR, Fortunato E, Mota MM, Baptista PV - J Nanobiotechnology (2015)

Single pathogen Au-nanoprobe detection assays using a MTBC probe; bPlasmodium sp. probe. The colorimetric assay consists of visual comparison of test solutions after salt induced Au-nanoprobe aggregation. Au-nanoprobes aggregation was measured by ratio of aggregation (ratio of SPR intensity at 525 and 600 nm) for the assay mixtures—2.5 nM Au-nanoprobe, 10 mM phosphate buffer (pH 8) + 0.1 M NaCl, purified dsDNA targets (PCR products) at a final concentration of 60 ng/µL. All spectrophotometric data was collected 30 min after salt addition and error bars represent the standard deviation of three independent assays. The horizontal line represents the threshold of 1 considered for discrimination between positive (rAbs ≥ 1) and negative (rAbs < 1) result. A representative colorimetric results is showed upon each result bar-red positive result; blue/purple negative result.
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4527100&req=5

Fig3: Single pathogen Au-nanoprobe detection assays using a MTBC probe; bPlasmodium sp. probe. The colorimetric assay consists of visual comparison of test solutions after salt induced Au-nanoprobe aggregation. Au-nanoprobes aggregation was measured by ratio of aggregation (ratio of SPR intensity at 525 and 600 nm) for the assay mixtures—2.5 nM Au-nanoprobe, 10 mM phosphate buffer (pH 8) + 0.1 M NaCl, purified dsDNA targets (PCR products) at a final concentration of 60 ng/µL. All spectrophotometric data was collected 30 min after salt addition and error bars represent the standard deviation of three independent assays. The horizontal line represents the threshold of 1 considered for discrimination between positive (rAbs ≥ 1) and negative (rAbs < 1) result. A representative colorimetric results is showed upon each result bar-red positive result; blue/purple negative result.
Mentions: First, we optimized the multiplex PCR amplification so that both two pathogens’ gene fragments are present with similar yields while maintaining PCR specificity. Primers were designed so as to produce amplicons showing similar sizes, since comparable hybridization efficiencies and profiles are desired [18]. The two loci were successfully amplified alone and then in a single multiplex reaction from DNA isolates of the pathogens (Additional file 1: Figure S1). Then, each Au-nanoprobe sequence was evaluated in terms of selectivity and specificity towards the respective target in solution (Fig. 2). Calibration of the Au-nanoprobe functionalization in presence of the respective target, but aggregated in its absence (see Additional file 1: Figures S3 and S4). No noteworthy loss in signal occurs when both pathogens amplicons are present in the mixture compared to each amplicon alone (Fig. 3).Fig. 2

Bottom Line: Following characterisation, the developed gold-nanoprobe allowed detection of either target in individual samples or in samples containing both DNA species with the same efficacy.Using one single probe via the non-cross-linking colorimetric methodology it is possible to identify multiple targets in one sample in one reaction.This proof-of-concept approach may easily be integrated into sensing platforms allowing for fast and simple multiplexing of Au-nanoprobe based detection at point-of-need.

View Article: PubMed Central - PubMed

Affiliation: Nanomedicine@FCT, Departamento de Ciências da Vida, Faculdade de Ciências e Tecnologia, CIGMH, UCIBIO, Universidade Nova de Lisboa, Campus de Caparica, 2829-516, Caparica, Portugal. bmrveigas@gmail.com.

ABSTRACT

Background: Gold nanoparticles have been widely employed for biosensing purposes with remarkable efficacy for DNA detection. Amongst the proposed systems, colorimetric strategies based on the remarkable optical properties have provided for simple yet effective sequence discrimination with potential for molecular diagnostics at point of need. These systems may also been used for parallel detection of several targets to provide additional information on diagnostics of pathogens.

Results: For the first time, we demonstrate that a single Au-nanoprobe may provide for detection of two distinct targets (pathogens) allowing colorimetric multi-target detection. We demonstrate this concept by using one single gold-nanoprobe capable to detect members of the Mycobacterium tuberculosis complex and Plasmodium sp., the etiologic agents of tuberculosis and malaria, respectively. Following characterisation, the developed gold-nanoprobe allowed detection of either target in individual samples or in samples containing both DNA species with the same efficacy.

Conclusions: Using one single probe via the non-cross-linking colorimetric methodology it is possible to identify multiple targets in one sample in one reaction. This proof-of-concept approach may easily be integrated into sensing platforms allowing for fast and simple multiplexing of Au-nanoprobe based detection at point-of-need.

No MeSH data available.


Related in: MedlinePlus