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Label-Free Nanoplasmonic-Based Short Noncoding RNA Sensing at Attomolar Concentrations Allows for Quantitative and Highly Specific Assay of MicroRNA-10b in Biological Fluids and Circulating Exosomes.

Joshi GK, Deitz-McElyea S, Liyanage T, Lawrence K, Mali S, Sardar R, Korc M - ACS Nano (2015)

Bottom Line: Here, an ultrasensitive localized surface plasmon resonance (LSPR)-based microRNA sensor with single nucleotide specificity was developed using chemically synthesized gold nanoprisms attached onto a solid substrate with unprecedented long-term stability and reversibility.We show that microRNA-10b levels were significantly higher in plasma-derived exosomes from pancreatic ductal adenocarcinoma patients when compared with patients with chronic pancreatitis or normal controls.Our findings suggest that this unique technique can be used to design novel diagnostic strategies for pancreatic and other cancers based on the direct quantitative measurement of plasma and exosome microRNAs, and can be readily extended to other diseases with identifiable microRNA signatures.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Chemical Biology, Indiana University-Purdue University Indianapolis , 402 North Blackford Street, LD 326, Indianapolis, Indiana 46202, United States.

ABSTRACT
MicroRNAs are short noncoding RNAs consisting of 18-25 nucleotides that target specific mRNA moieties for translational repression or degradation, thereby modulating numerous biological processes. Although microRNAs have the ability to behave like oncogenes or tumor suppressors in a cell-autonomous manner, their exact roles following release into the circulation are only now being unraveled and it is important to establish sensitive assays to measure their levels in different compartments in the circulation. Here, an ultrasensitive localized surface plasmon resonance (LSPR)-based microRNA sensor with single nucleotide specificity was developed using chemically synthesized gold nanoprisms attached onto a solid substrate with unprecedented long-term stability and reversibility. The sensor was used to specifically detect microRNA-10b at the attomolar (10(-18) M) concentration in pancreatic cancer cell lines, derived tissue culture media, human plasma, and media and plasma exosomes. In addition, for the first time, our label-free and nondestructive sensing technique was used to quantify microRNA-10b in highly purified exosomes isolated from patients with pancreatic cancer or chronic pancreatitis, and from normal controls. We show that microRNA-10b levels were significantly higher in plasma-derived exosomes from pancreatic ductal adenocarcinoma patients when compared with patients with chronic pancreatitis or normal controls. Our findings suggest that this unique technique can be used to design novel diagnostic strategies for pancreatic and other cancers based on the direct quantitative measurement of plasma and exosome microRNAs, and can be readily extended to other diseases with identifiable microRNA signatures.

No MeSH data available.


Related in: MedlinePlus

Schematic representation of the fabrication of the mixed -S-PEG6:-SC6-ssDNA-functionalized gold nanoprisms to prepare LSPR-based sensing platform for miR-10b detection in various biological compartments. Three different edge lengths gold nanoprisms (34, 42, and 47 nm) were synthesized via our previously published chemical reduction method and then attached onto silanized glass surface.45 All the surface modifications were performed in PBS buffer (pH = 7.4) under normal laboratory conditions. Detailed procedure for sensor fabrication and RNA isolation are provided in the Materials and Methods. The figure is not to scale.
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fig1: Schematic representation of the fabrication of the mixed -S-PEG6:-SC6-ssDNA-functionalized gold nanoprisms to prepare LSPR-based sensing platform for miR-10b detection in various biological compartments. Three different edge lengths gold nanoprisms (34, 42, and 47 nm) were synthesized via our previously published chemical reduction method and then attached onto silanized glass surface.45 All the surface modifications were performed in PBS buffer (pH = 7.4) under normal laboratory conditions. Detailed procedure for sensor fabrication and RNA isolation are provided in the Materials and Methods. The figure is not to scale.

Mentions: Here, we report a highly specific, ultrasensitive, and regenerative localized surface plasmon resonance (LSPR)-based miR-10b sensing approach that overcomes the above limitations. The solid-state LSPR-based sensors were developed using glass substrate-bound gold nanoprisms functionalized with complementary oligonucleotides (Figure 1). Importantly, our sensor was able to distinguish between miR-10b and miR-10a, indicating that it provides for single nucleotide specificity. We also demonstrate the usefulness of miR quantification by LSPR-based technique in cultured PCCs (AsPC-1, BxPC-3, and PANC-1), derived conditioned media, and exosomes. The attomolar (aM) limit of detection (LOD), at least 1000-fold lower than current label-free methods,22−24,26,28,29,31,32 of our LSPR-based sensor allowed us to readily differentiate between miR-10b levels in PDAC and CP patients, and normal controls in plasma, exosomes, and post-ultracentrifugation supernatants. We propose that our label-free and ultrasensitive assay, which detects very small increases in miR-10b levels, could allow for early stage PDAC detection and permit monitoring for PDAC recurrence following therapy or resection.


Label-Free Nanoplasmonic-Based Short Noncoding RNA Sensing at Attomolar Concentrations Allows for Quantitative and Highly Specific Assay of MicroRNA-10b in Biological Fluids and Circulating Exosomes.

Joshi GK, Deitz-McElyea S, Liyanage T, Lawrence K, Mali S, Sardar R, Korc M - ACS Nano (2015)

Schematic representation of the fabrication of the mixed -S-PEG6:-SC6-ssDNA-functionalized gold nanoprisms to prepare LSPR-based sensing platform for miR-10b detection in various biological compartments. Three different edge lengths gold nanoprisms (34, 42, and 47 nm) were synthesized via our previously published chemical reduction method and then attached onto silanized glass surface.45 All the surface modifications were performed in PBS buffer (pH = 7.4) under normal laboratory conditions. Detailed procedure for sensor fabrication and RNA isolation are provided in the Materials and Methods. The figure is not to scale.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4660391&req=5

fig1: Schematic representation of the fabrication of the mixed -S-PEG6:-SC6-ssDNA-functionalized gold nanoprisms to prepare LSPR-based sensing platform for miR-10b detection in various biological compartments. Three different edge lengths gold nanoprisms (34, 42, and 47 nm) were synthesized via our previously published chemical reduction method and then attached onto silanized glass surface.45 All the surface modifications were performed in PBS buffer (pH = 7.4) under normal laboratory conditions. Detailed procedure for sensor fabrication and RNA isolation are provided in the Materials and Methods. The figure is not to scale.
Mentions: Here, we report a highly specific, ultrasensitive, and regenerative localized surface plasmon resonance (LSPR)-based miR-10b sensing approach that overcomes the above limitations. The solid-state LSPR-based sensors were developed using glass substrate-bound gold nanoprisms functionalized with complementary oligonucleotides (Figure 1). Importantly, our sensor was able to distinguish between miR-10b and miR-10a, indicating that it provides for single nucleotide specificity. We also demonstrate the usefulness of miR quantification by LSPR-based technique in cultured PCCs (AsPC-1, BxPC-3, and PANC-1), derived conditioned media, and exosomes. The attomolar (aM) limit of detection (LOD), at least 1000-fold lower than current label-free methods,22−24,26,28,29,31,32 of our LSPR-based sensor allowed us to readily differentiate between miR-10b levels in PDAC and CP patients, and normal controls in plasma, exosomes, and post-ultracentrifugation supernatants. We propose that our label-free and ultrasensitive assay, which detects very small increases in miR-10b levels, could allow for early stage PDAC detection and permit monitoring for PDAC recurrence following therapy or resection.

Bottom Line: Here, an ultrasensitive localized surface plasmon resonance (LSPR)-based microRNA sensor with single nucleotide specificity was developed using chemically synthesized gold nanoprisms attached onto a solid substrate with unprecedented long-term stability and reversibility.We show that microRNA-10b levels were significantly higher in plasma-derived exosomes from pancreatic ductal adenocarcinoma patients when compared with patients with chronic pancreatitis or normal controls.Our findings suggest that this unique technique can be used to design novel diagnostic strategies for pancreatic and other cancers based on the direct quantitative measurement of plasma and exosome microRNAs, and can be readily extended to other diseases with identifiable microRNA signatures.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Chemical Biology, Indiana University-Purdue University Indianapolis , 402 North Blackford Street, LD 326, Indianapolis, Indiana 46202, United States.

ABSTRACT
MicroRNAs are short noncoding RNAs consisting of 18-25 nucleotides that target specific mRNA moieties for translational repression or degradation, thereby modulating numerous biological processes. Although microRNAs have the ability to behave like oncogenes or tumor suppressors in a cell-autonomous manner, their exact roles following release into the circulation are only now being unraveled and it is important to establish sensitive assays to measure their levels in different compartments in the circulation. Here, an ultrasensitive localized surface plasmon resonance (LSPR)-based microRNA sensor with single nucleotide specificity was developed using chemically synthesized gold nanoprisms attached onto a solid substrate with unprecedented long-term stability and reversibility. The sensor was used to specifically detect microRNA-10b at the attomolar (10(-18) M) concentration in pancreatic cancer cell lines, derived tissue culture media, human plasma, and media and plasma exosomes. In addition, for the first time, our label-free and nondestructive sensing technique was used to quantify microRNA-10b in highly purified exosomes isolated from patients with pancreatic cancer or chronic pancreatitis, and from normal controls. We show that microRNA-10b levels were significantly higher in plasma-derived exosomes from pancreatic ductal adenocarcinoma patients when compared with patients with chronic pancreatitis or normal controls. Our findings suggest that this unique technique can be used to design novel diagnostic strategies for pancreatic and other cancers based on the direct quantitative measurement of plasma and exosome microRNAs, and can be readily extended to other diseases with identifiable microRNA signatures.

No MeSH data available.


Related in: MedlinePlus