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

Vetrone SA, Huarng MC, Alocilja EC - Sensors (Basel) (2012)

Bottom Line: Bacterial pathogens pose an increasing food safety and bioterrorism concern.Non-PCR amplified DNA was hybridized into sandwich-like structures (magnetic nanoparticles/DNA/AuNPs) and analyzed through detection of gold voltammetric peaks using differential pulse voltammetry.Future efforts will focus on further optimization of the DNA extraction method and AuNP-biosensors, to increase sensitivity at lower DNA target concentrations from food matrices comparable to PCR amplified DNA detection strategies.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Whittier College, 13406 E. Philadelphia St., Whittier, CA 90608, USA. svetrone@whittier.edu

ABSTRACT
Bacterial pathogens pose an increasing food safety and bioterrorism concern. Current DNA detection methods utilizing sensitive nanotechnology and biosensors have shown excellent detection, but require expensive and time-consuming polymerase chain reaction (PCR) to amplify DNA targets; thus, a faster, more economical method is still essential. In this proof-of-concept study, we investigated the ability of a gold nanoparticle-DNA (AuNP-DNA) biosensor to detect non-PCR amplified genomic Salmonella enterica serovar Enteritidis (S. enteritidis) DNA, from pure or mixed bacterial culture and spiked liquid matrices. Non-PCR amplified DNA was hybridized into sandwich-like structures (magnetic nanoparticles/DNA/AuNPs) and analyzed through detection of gold voltammetric peaks using differential pulse voltammetry. Our preliminary data indicate that non-PCR amplified genomic DNA can be detected at a concentration as low as 100 ng/mL from bacterial cultures and spiked liquid matrices, similar to reported PCR amplified detection levels. These findings also suggest that AuNP-DNA biosensors are a first step towards a viable detection method of bacterial pathogens, in particular, for resource-limited settings, such as field-based or economically limited conditions. Future efforts will focus on further optimization of the DNA extraction method and AuNP-biosensors, to increase sensitivity at lower DNA target concentrations from food matrices comparable to PCR amplified DNA detection strategies.

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Schematic of AuNP-DNAt-MNP sandwich structure. (A) The presence of specific DNAt (red wavy bar) will covalently bind with DNAt specific probes (black wavy lines) on AuNP (red Au labeled bead) and the MNP (grey M labeled bead), allowing the collection of AuNP. (B) Absence of specific DNAt will lead to the failure of AuNP collection (image used with permission from Michael J. Anderson at Michigan State University, 2012).
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f1-sensors-12-10487: Schematic of AuNP-DNAt-MNP sandwich structure. (A) The presence of specific DNAt (red wavy bar) will covalently bind with DNAt specific probes (black wavy lines) on AuNP (red Au labeled bead) and the MNP (grey M labeled bead), allowing the collection of AuNP. (B) Absence of specific DNAt will lead to the failure of AuNP collection (image used with permission from Michael J. Anderson at Michigan State University, 2012).

Mentions: The 2001 distribution of Bacillus anthracis (B. anthracis) through the United States postal system, which resulted in 22 cases of anthrax exposure and five deaths, brought much needed awareness to the significant effect of bacterial pathogens on public health [1]. Bioterrorism became a reality and identified critical needs in prevention, protection, and mitigation for homeland security, especially within the areas of food, water, and agricultural safety. To date, there are multiple commercially available methods for the detection of pathogenic agents, although none adequately comply with governmental food safety standards [2]. In addition, many of these methods utilize expensive reagents and equipment, and require a lengthy turn around period, as they often necessitate long incubation or detection times over a period of days [3,4]. Toward this end, much attention has been directed to the development of rapid, sensitive, low cost, portable biosensors for the biological detection of pathogens, such as those incorporating the use of nanoparticles for DNA detection [5–16]. Specifically, these analytical devices integrate a biological sensing element with a transducer to quantify a biological event, such as the presence of pathogenic microorganisms within a liquid or solid matrix, into an electrical output. The detection relies on the immobilization of single stranded DNA (ssDNA) probes that are complementary and specific for a DNA sequence of the pathogenic target, on two separate surfaces: magnetic nanoparticles (MNPs) and gold nanoparticles (AuNPs) (Figure 1). MNPs are used to extract the DNA target from the sample while AuNPs are used to report the sandwich hybridization. AuNPs are used here because of their ease of production and functionalization [17,18]. The nanoparticles conjugated with ssDNA probes specific for the pathogenic target of interest are then hybridized with the DNA test sample, isolated using magnetic separation, and detected through electrochemical analysis [16,19–21]. While biosensors using this detection strategy have been shown to detect specific DNA fragments from various pathogens [22–24], many of these biosensors have only been tested for the detection of purified and polymerase chain reaction (PCR) amplified DNA targets (DNAt), and not from genomic DNAt extracted from pure bacterial samples or contaminated food matrices. Similar to some of the limitations of commercially available detection strategies, PCR is often criticized for its complex, expensive, time-consuming, and labor-intensive procedure requirements. Consequently, the need of PCR for biosensor detection of pathogenic DNAt is greatly restrictive for both field-based and resource limited settings, resulting in the increased need for a PCR-independent biosensor detection methods.


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

Vetrone SA, Huarng MC, Alocilja EC - Sensors (Basel) (2012)

Schematic of AuNP-DNAt-MNP sandwich structure. (A) The presence of specific DNAt (red wavy bar) will covalently bind with DNAt specific probes (black wavy lines) on AuNP (red Au labeled bead) and the MNP (grey M labeled bead), allowing the collection of AuNP. (B) Absence of specific DNAt will lead to the failure of AuNP collection (image used with permission from Michael J. Anderson at Michigan State University, 2012).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3472839&req=5

f1-sensors-12-10487: Schematic of AuNP-DNAt-MNP sandwich structure. (A) The presence of specific DNAt (red wavy bar) will covalently bind with DNAt specific probes (black wavy lines) on AuNP (red Au labeled bead) and the MNP (grey M labeled bead), allowing the collection of AuNP. (B) Absence of specific DNAt will lead to the failure of AuNP collection (image used with permission from Michael J. Anderson at Michigan State University, 2012).
Mentions: The 2001 distribution of Bacillus anthracis (B. anthracis) through the United States postal system, which resulted in 22 cases of anthrax exposure and five deaths, brought much needed awareness to the significant effect of bacterial pathogens on public health [1]. Bioterrorism became a reality and identified critical needs in prevention, protection, and mitigation for homeland security, especially within the areas of food, water, and agricultural safety. To date, there are multiple commercially available methods for the detection of pathogenic agents, although none adequately comply with governmental food safety standards [2]. In addition, many of these methods utilize expensive reagents and equipment, and require a lengthy turn around period, as they often necessitate long incubation or detection times over a period of days [3,4]. Toward this end, much attention has been directed to the development of rapid, sensitive, low cost, portable biosensors for the biological detection of pathogens, such as those incorporating the use of nanoparticles for DNA detection [5–16]. Specifically, these analytical devices integrate a biological sensing element with a transducer to quantify a biological event, such as the presence of pathogenic microorganisms within a liquid or solid matrix, into an electrical output. The detection relies on the immobilization of single stranded DNA (ssDNA) probes that are complementary and specific for a DNA sequence of the pathogenic target, on two separate surfaces: magnetic nanoparticles (MNPs) and gold nanoparticles (AuNPs) (Figure 1). MNPs are used to extract the DNA target from the sample while AuNPs are used to report the sandwich hybridization. AuNPs are used here because of their ease of production and functionalization [17,18]. The nanoparticles conjugated with ssDNA probes specific for the pathogenic target of interest are then hybridized with the DNA test sample, isolated using magnetic separation, and detected through electrochemical analysis [16,19–21]. While biosensors using this detection strategy have been shown to detect specific DNA fragments from various pathogens [22–24], many of these biosensors have only been tested for the detection of purified and polymerase chain reaction (PCR) amplified DNA targets (DNAt), and not from genomic DNAt extracted from pure bacterial samples or contaminated food matrices. Similar to some of the limitations of commercially available detection strategies, PCR is often criticized for its complex, expensive, time-consuming, and labor-intensive procedure requirements. Consequently, the need of PCR for biosensor detection of pathogenic DNAt is greatly restrictive for both field-based and resource limited settings, resulting in the increased need for a PCR-independent biosensor detection methods.

Bottom Line: Bacterial pathogens pose an increasing food safety and bioterrorism concern.Non-PCR amplified DNA was hybridized into sandwich-like structures (magnetic nanoparticles/DNA/AuNPs) and analyzed through detection of gold voltammetric peaks using differential pulse voltammetry.Future efforts will focus on further optimization of the DNA extraction method and AuNP-biosensors, to increase sensitivity at lower DNA target concentrations from food matrices comparable to PCR amplified DNA detection strategies.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Whittier College, 13406 E. Philadelphia St., Whittier, CA 90608, USA. svetrone@whittier.edu

ABSTRACT
Bacterial pathogens pose an increasing food safety and bioterrorism concern. Current DNA detection methods utilizing sensitive nanotechnology and biosensors have shown excellent detection, but require expensive and time-consuming polymerase chain reaction (PCR) to amplify DNA targets; thus, a faster, more economical method is still essential. In this proof-of-concept study, we investigated the ability of a gold nanoparticle-DNA (AuNP-DNA) biosensor to detect non-PCR amplified genomic Salmonella enterica serovar Enteritidis (S. enteritidis) DNA, from pure or mixed bacterial culture and spiked liquid matrices. Non-PCR amplified DNA was hybridized into sandwich-like structures (magnetic nanoparticles/DNA/AuNPs) and analyzed through detection of gold voltammetric peaks using differential pulse voltammetry. Our preliminary data indicate that non-PCR amplified genomic DNA can be detected at a concentration as low as 100 ng/mL from bacterial cultures and spiked liquid matrices, similar to reported PCR amplified detection levels. These findings also suggest that AuNP-DNA biosensors are a first step towards a viable detection method of bacterial pathogens, in particular, for resource-limited settings, such as field-based or economically limited conditions. Future efforts will focus on further optimization of the DNA extraction method and AuNP-biosensors, to increase sensitivity at lower DNA target concentrations from food matrices comparable to PCR amplified DNA detection strategies.

Show MeSH
Related in: MedlinePlus