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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

Scanning electron microscopy (SEM) images of three different edge-length gold nanoprisms were used for LSPR-based miR-10b sensors fabrication: (A) 34 nm, (B) 42 nm, and (C) 47 nm. Scale bars: 100 nm. (D) Atomic force microscopy image of (on the average) 42 nm edge length gold nanoprisms. (E) Changes in the LSPR dipole peak (λLSPR) position of averagely 42 nm edge length gold nanoprisms before (black) and after (red) functionalization with mixed HS-PEG6:HSC6-ssDNA-10b. The sensing platform was then rinsed with PBS buffer and incubated with 10 nM of miR-10b solution in human plasma; then, rinsed with PBS buffer and dipole peak position (blue) was determined. (F) Average λLSPR shift (ΔλLSPR) of the sensing platforms, which were prepared with three different edge lengths nanoprisms, 34 nm (purple triangles), 42 nm (red diamonds), and 47 nm (blue dots) as a function of miR-10b concentration. All extinction spectra were measured in PBS buffer to determine the ΔλLSPR. The green bar represents three times the standard deviation (σ) of the blank (mixed HS-PEG6:HSC6-ssDNA-10b functionalized gold nanoprisms attached onto silanized glass substrate). Inasmuch as log scale is commonly used in the LSPR biosensing literature,35,40,46 in order to investigate nonspecific absorption at a lower concentration range, concentrations were plotted on the axis in log scale.
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fig2: Scanning electron microscopy (SEM) images of three different edge-length gold nanoprisms were used for LSPR-based miR-10b sensors fabrication: (A) 34 nm, (B) 42 nm, and (C) 47 nm. Scale bars: 100 nm. (D) Atomic force microscopy image of (on the average) 42 nm edge length gold nanoprisms. (E) Changes in the LSPR dipole peak (λLSPR) position of averagely 42 nm edge length gold nanoprisms before (black) and after (red) functionalization with mixed HS-PEG6:HSC6-ssDNA-10b. The sensing platform was then rinsed with PBS buffer and incubated with 10 nM of miR-10b solution in human plasma; then, rinsed with PBS buffer and dipole peak position (blue) was determined. (F) Average λLSPR shift (ΔλLSPR) of the sensing platforms, which were prepared with three different edge lengths nanoprisms, 34 nm (purple triangles), 42 nm (red diamonds), and 47 nm (blue dots) as a function of miR-10b concentration. All extinction spectra were measured in PBS buffer to determine the ΔλLSPR. The green bar represents three times the standard deviation (σ) of the blank (mixed HS-PEG6:HSC6-ssDNA-10b functionalized gold nanoprisms attached onto silanized glass substrate). Inasmuch as log scale is commonly used in the LSPR biosensing literature,35,40,46 in order to investigate nonspecific absorption at a lower concentration range, concentrations were plotted on the axis in log scale.

Mentions: Chemically synthesized gold nanoprisms, which displayed λLSPR at 750, 800, and 820 nm in acetonitrile (Figure S1) with average edge-lengths of 34, 42, and 47 nm, respectively, as determined from scanning electron microscopy images (Figure 2A–C), were used in sensor fabrication. Figure 2E illustrates the change of λLSPR position during the functionalization of gold nanoprisms with 42 nm of average edge-length, which were attached onto silanized glass. The red-shift of the λLSPR position suggested an increase in local refractive index36−38,41,42,46−49 from the attachment of molecular species on the gold nanoprism’s surface. The LODs of miR-10b detection for 34, 42, and 47 nm edge-length nanoprisms were calculated in human plasma and were found to be 47.5, 0.091, and 0.083 fM, respectively (see Figure 2F and Table S2). The LODs were calculated by measuring the ΔλLSPR for the blank sample (mixed -S-PEG6:-SC6-ssDNA-10b functionalized gold nanoprisms attached onto silanized glass substrate) and then calculating the Z (mean + 3σ) value. The Z value was then converted into the relative concentration using the calibration curve. The data suggest that as the edge-lengths of the nanoprisms increase, their sensing volume40,43,50−52 also increases, thereby enhancing the LSPR sensitivity of the nanoprisms. This result is also in agreement with the literature where largest gold nanoparticles demonstrated highest LSPR-based sensing ability toward the detection of proteins.35 Thus, a minute change in a nanoprism’s local dielectric environment due to analyte absorption can dramatically influence the LSPR properties and λLSPR position. It is important to mention that the final λLSPR values after -ssDNA-miR-10b and miR-10b hybridization were determined in PBS buffer (wet LSPR-based sensors) instead of air in order to avoid the effects of bulk refractive index caused by the surrounding media (water). Moreover, our lowest LOD of 83 aM was more than 106-, 104-, and 103-fold lower than the label-free fluorescent-,23,53 microring resonator-,26,29 and nanopore-based31,32 miR sensors, respectively. To the best of our knowledge, this is the lowest LOD reported in the literature for LSPR-based sensors for detecting any-type of biomolecules in complex physiological media such as human plasma. This label-free technique has also proven to be more sensitive than metal nanoparticle-based surface-enhanced Raman scattering sensing (LOD = 1.5 fM) of mouse pancreatic tumor.54 At higher concentrations we observed large error bars (Figure 2F), which points to limitations of our sensing platform. However, the concentration range (10 nM to 10 pM) at which we observed this enhanced variability is well above the concentration range of the values seen in PDAC and CP patients as shown later in this article.


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)

Scanning electron microscopy (SEM) images of three different edge-length gold nanoprisms were used for LSPR-based miR-10b sensors fabrication: (A) 34 nm, (B) 42 nm, and (C) 47 nm. Scale bars: 100 nm. (D) Atomic force microscopy image of (on the average) 42 nm edge length gold nanoprisms. (E) Changes in the LSPR dipole peak (λLSPR) position of averagely 42 nm edge length gold nanoprisms before (black) and after (red) functionalization with mixed HS-PEG6:HSC6-ssDNA-10b. The sensing platform was then rinsed with PBS buffer and incubated with 10 nM of miR-10b solution in human plasma; then, rinsed with PBS buffer and dipole peak position (blue) was determined. (F) Average λLSPR shift (ΔλLSPR) of the sensing platforms, which were prepared with three different edge lengths nanoprisms, 34 nm (purple triangles), 42 nm (red diamonds), and 47 nm (blue dots) as a function of miR-10b concentration. All extinction spectra were measured in PBS buffer to determine the ΔλLSPR. The green bar represents three times the standard deviation (σ) of the blank (mixed HS-PEG6:HSC6-ssDNA-10b functionalized gold nanoprisms attached onto silanized glass substrate). Inasmuch as log scale is commonly used in the LSPR biosensing literature,35,40,46 in order to investigate nonspecific absorption at a lower concentration range, concentrations were plotted on the axis in log scale.
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Related In: Results  -  Collection

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Show All Figures
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fig2: Scanning electron microscopy (SEM) images of three different edge-length gold nanoprisms were used for LSPR-based miR-10b sensors fabrication: (A) 34 nm, (B) 42 nm, and (C) 47 nm. Scale bars: 100 nm. (D) Atomic force microscopy image of (on the average) 42 nm edge length gold nanoprisms. (E) Changes in the LSPR dipole peak (λLSPR) position of averagely 42 nm edge length gold nanoprisms before (black) and after (red) functionalization with mixed HS-PEG6:HSC6-ssDNA-10b. The sensing platform was then rinsed with PBS buffer and incubated with 10 nM of miR-10b solution in human plasma; then, rinsed with PBS buffer and dipole peak position (blue) was determined. (F) Average λLSPR shift (ΔλLSPR) of the sensing platforms, which were prepared with three different edge lengths nanoprisms, 34 nm (purple triangles), 42 nm (red diamonds), and 47 nm (blue dots) as a function of miR-10b concentration. All extinction spectra were measured in PBS buffer to determine the ΔλLSPR. The green bar represents three times the standard deviation (σ) of the blank (mixed HS-PEG6:HSC6-ssDNA-10b functionalized gold nanoprisms attached onto silanized glass substrate). Inasmuch as log scale is commonly used in the LSPR biosensing literature,35,40,46 in order to investigate nonspecific absorption at a lower concentration range, concentrations were plotted on the axis in log scale.
Mentions: Chemically synthesized gold nanoprisms, which displayed λLSPR at 750, 800, and 820 nm in acetonitrile (Figure S1) with average edge-lengths of 34, 42, and 47 nm, respectively, as determined from scanning electron microscopy images (Figure 2A–C), were used in sensor fabrication. Figure 2E illustrates the change of λLSPR position during the functionalization of gold nanoprisms with 42 nm of average edge-length, which were attached onto silanized glass. The red-shift of the λLSPR position suggested an increase in local refractive index36−38,41,42,46−49 from the attachment of molecular species on the gold nanoprism’s surface. The LODs of miR-10b detection for 34, 42, and 47 nm edge-length nanoprisms were calculated in human plasma and were found to be 47.5, 0.091, and 0.083 fM, respectively (see Figure 2F and Table S2). The LODs were calculated by measuring the ΔλLSPR for the blank sample (mixed -S-PEG6:-SC6-ssDNA-10b functionalized gold nanoprisms attached onto silanized glass substrate) and then calculating the Z (mean + 3σ) value. The Z value was then converted into the relative concentration using the calibration curve. The data suggest that as the edge-lengths of the nanoprisms increase, their sensing volume40,43,50−52 also increases, thereby enhancing the LSPR sensitivity of the nanoprisms. This result is also in agreement with the literature where largest gold nanoparticles demonstrated highest LSPR-based sensing ability toward the detection of proteins.35 Thus, a minute change in a nanoprism’s local dielectric environment due to analyte absorption can dramatically influence the LSPR properties and λLSPR position. It is important to mention that the final λLSPR values after -ssDNA-miR-10b and miR-10b hybridization were determined in PBS buffer (wet LSPR-based sensors) instead of air in order to avoid the effects of bulk refractive index caused by the surrounding media (water). Moreover, our lowest LOD of 83 aM was more than 106-, 104-, and 103-fold lower than the label-free fluorescent-,23,53 microring resonator-,26,29 and nanopore-based31,32 miR sensors, respectively. To the best of our knowledge, this is the lowest LOD reported in the literature for LSPR-based sensors for detecting any-type of biomolecules in complex physiological media such as human plasma. This label-free technique has also proven to be more sensitive than metal nanoparticle-based surface-enhanced Raman scattering sensing (LOD = 1.5 fM) of mouse pancreatic tumor.54 At higher concentrations we observed large error bars (Figure 2F), which points to limitations of our sensing platform. However, the concentration range (10 nM to 10 pM) at which we observed this enhanced variability is well above the concentration range of the values seen in PDAC and CP patients as shown later in this article.

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