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LetterNovember 7, 2014

Highly Specific Plasmonic Biosensors for Ultrasensitive MicroRNA Detection in Plasma from Pancreatic Cancer Patients
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Cite this: Nano Lett. 2014, 14, 12, 6955–6963

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https://doi.org/10.1021/nl503220s

Published November 7, 2014

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Abstract

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MicroRNAs (miRs) are small noncoding RNAs that regulate mRNA stability and/or translation. Because of their release into the circulation and their remarkable stability, miR levels in plasma and other biological fluids can serve as diagnostic and prognostic disease biomarkers. However, quantifying miRs in the circulation is challenging due to issues with sensitivity and specificity. This Letter describes for the first time the design and characterization of a regenerative, solid-state localized surface plasmon resonance (LSPR) sensor based on highly sensitive nanostructures (gold nanoprisms) that obviates the need for labels or amplification of the miRs. Our direct hybridization approach has enabled the detection of subfemtomolar concentration of miR-X (X = 21 and 10b) in human plasma in pancreatic cancer patients. Our LSPR-based measurements showed that the miR levels measured directly in patient plasma were at least 2-fold higher than following RNA extraction and quantification by reverse transcriptase-polymerase chain reaction. Through LSPR-based measurements we have shown nearly 4-fold higher concentrations of miR-10b than miR-21 in plasma of pancreatic cancer patients. We propose that our highly sensitive and selective detection approach for assaying miRs in plasma can be applied to many cancer types and disease states and should allow a rational approach for testing the utility of miRs as markers for early disease diagnosis and prognosis, which could allow for the design of effective individualized therapeutic approaches.

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Pancreatic ductal adenocarcinoma (PDAC)-related deaths are a major health concern in the United States since the five-year survival rate is only 6%. (1) A crucial contributor to this dismal statistic is the absence of a biomarker for early PDAC detection. Moreover, most patients with PDAC do not develop specific symptoms until the disease is quite advanced. Therefore, at clinical presentation, PDAC patients often have locally advanced and/or metastatic disease, which precludes effective therapy in the vast majority of patients. In this context microRNAs (miRs), (2) which are small single-stranded, noncoding RNAs often play a major role in cell proliferation, survival, migration, invasion, and metastasis in various cancers, (3-5) including PDAC. (6, 7) Moreover, miRs are released into the circulation, where they exhibit remarkable stability. Therefore, the development of sensitive and specific detection techniques, which precisely and quantitatively measure the concentration of miRs in their native environments such as blood or plasma, may provide a unique opportunity for developing diagnostic and prognostic markers in PDAC. (6-8)

Microarrays (9) and quantitative reverse transcription polymerase chain reaction (qRT-PCR) (10, 11) assays are routinely used to detect miRs. However, these methods are semiquantitative, require sequence-based amplification and radioactive labeling steps, and suffer from cross-hybridization and invalid internal controls. Other analytical techniques such as electrochemical (12, 13) and fluorescence-based assays (14) are also used to quantify the miRs. However, such techniques require either additional amplification or labeling, or complex electron/energy transfer processes, and cannot be performed in physiological media. A few label-free techniques such as photonic microring resonators, (15) nanopores, (16, 17) and nanoparticle-based biobarcode gel assay (18) can detect miRs associated with cancer patients. However, microring resonators suffer from low sensitivity and do not work in physiological media. Although nanopore-based sensors have shown the ability to detect miRs in the circulation of lung cancer patients, the technique requires a complicated fabrication procedure, a high probe concentration, and a specific probe signature. (16) The biobarcode gel technique relies on complex sandwich-type capturing methods, uses of the toxic chemical potassium cyanide, and may not be applicable to clinically relevant patient samples.

Plasmonic nanostructures have gained significant attention because of their geometrical feature-dependent localized surface plasmon resonance (LSPR) properties, (19-23) which can be further controlled by modulating their local dielectric environment. (21, 24) Utilizing these properties, several molecular (25, 26) and biological (27-34) sensors have been developed where analyte binding to nanostructure surface-bound receptors results in an increase in refractive index and consequently a LSPR peak shift. (35, 36) In this context, it has not been possible to detect and quantify sequence specific miRs by their direct hybridization to nanostructure probes followed by monitoring the LSPR properties of nanostructures without using labeling steps. We now report for the first time the fabrication of label-free, solid-state plasmonic biosensors for miR detection. The biosensing involves the direct hybridization of PDAC-relevant miRs in plasma to their complementary single-stranded DNAs (HS-C6-ssDNA) that were functionalized on the surface of gold nanoprisms attached onto a glass substrate. This construct serves as a plasmonic biosensor through monitoring the LSPR dipole peak (λ_LSPR). We also demonstrate that our sensors are extremely specific in miR detection and that addition of DNA-RNA duplex cleaving enzymes regenerates the sensor, allowing for multiple uses without compromising sensing efficiency.

Fabrication of the Plasmonic Biosensor for miRs Detection

Figure 1 represents the schematic diagram of our solid-state, label-free plasmonic biosensor fabrication for miR detection. Chemically synthesized gold nanoprisms (Figure 1a), which displayed their λ_LSPR at ∼797 nm upon attachment to solid substrate immersed in PBS buffer, were selected as nanoantennas for our biosensor fabrication because (1) their λ_LSPR peak position (in the 700–900 nm wavelength range) is particularly suitable for biomolecule detection because of negligible background scattering and adsorption of endogeneous chromophores from physiological media such as blood and plasma; (37, 38) (2) they have strong electromagnetic (EM) field enhancement at the sharp tips; (39, 40) (3) they exhibits a strong LSPR response to small changes in their surrounding environment; (36, 41-46) (4) their atomically smooth surface allows formation of a self-assembled monolayer (SAM) (47) of receptors with both a tightly packed lower layer of alkylthiols and a more loosely packed upper layer that provide the required space for duplex formation with complementary miR strands; (5) gold is nontoxic and stable under extreme physiological conditions; (48) and (6) the gold–sulfur bond is very stable with thiol-modified receptors making a strong covalent bond with the gold surface. Details describing the synthesis of gold nanoprisms and their attachment onto the silanized glass substrate are provided in Supporting Information. Recently, (49) we have shown that a molecular sensor fabricated using an ∼35 nm average edge-length gold nanoprisms displayed an unprecedentedly large 21 nm reversible shift upon a minor 0.6 nm increase in the thickness of the local dielectric environment. Therefore, gold nanoprisms of this size and geometry are unique and should provide extremely high sensitivity if plasmonic biosensors are fabricated using them, which is the scope of this Letter. This Letter provides the first example of LSPR-based miRs sensing in physiological media.

Figure 1. Design of plasmonic biosensors and detecting miR-X in various physiological media. (a) Chemically synthesized and freshly prepared gold nanoprisms were covalently attached onto a 3-mercaptopropyltriethoxysilane-functionalized glass coverslip (substrate). (b) Surface of gold nanoprisms was chemically modified with a 1.0 μM 1:1 mixture of SH-C6-ssDNA-X and PEG₆-SH in PBS buffer (pH 7.4) to prepare the plasmonic biosensor. (c) Incubation of sensor in miR-X solution and formation of DNA duplex. (d) Schematic of the extinction spectrum of the biosensor collected in PBS buffer after modification with a 1.0 μM 1:1 mixture of SH-C6-ssDNA-X and PEG₆-SH (blue curve). The extinction spectrum was again collected after incubation in miR-X solution and careful rinsing with PBS buffer to determine the new peak position (red curve). The extent of LSPR dipole peak shift (Δλ_LSPR) depends on the concentration of miR-X used during the incubation in (c), which ranged from 100 nM to 50 fM. (e) Plot of Δλ_LSPR versus log of miR-X concentrations used to determine the limit of detection. The image is not to scale.

For detection and quantification, our targets were miR-21 and miR-10b, because we have shown by locked nuclei acid–based in situ hybridization that they are overexpressed in pancreatic cells (PCCs) within the tumor mass (50, 51) and that circulating miR-10b may serve as biomarker for diagnosis of PDAC. (8) We designed the sensing strategy based on the hybridization between complementary probes (-C6-ssDNA-X, X = 21 and 10b) attached to gold nanoprisms and their target miRs. The introduction of spacers in-between the DNA probes was included to reduce steric hindrance between the probes and the miRs and therefore to enhance the hybridization and ultimately the sensitivity. As shown in Figure 1b, poly(ethylene glycol)₆-thiols (PEG₆-SH) were used as spacers because they avoid nonspecific adsorption of extraneous materials onto the nanoprism’s surface and are not reactive toward miRs or other biological constituents present in plasma. Previously, we demonstrated that functionalization of a nanoantenna’s surface with an equal mole ratio of receptor and spacer provided the best sensitivity and lowest limit of detection (LOD). (42) Therefore, a 1:1 ratio of HS–C6-ssDNA-X:PEG₆-SH was used to prepare the plasmonic biosensors (Figure 1b). All the miRs and oligonucleotides sequences used in these studies are provided in Supporting Information, Table S1.

As illustrated in Figure 2a, we used UV–vis spectroscopy to monitor the changes in λ_LSPR of the gold nanoprisms at different functionalization steps. The functionalization of substrate-bound nanoprisms with 1:1 ratio of HS–C6-ssDNA-21:PEG₆-SH resulted in an ∼20.5 ± 3.2 nm red-shift of λ_LSPR as a result of the increase in local refractive index, which suggested the attachment of both molecular species onto the nanoprism’s surface. These plasmonic biosensors were utilized for miR detection by incubating miR-21 (obtained from Sigma-Aldrich, U.S.A.) with concentration ranging from 100 nM to 50 fM in PBS buffer, 40% bovine plasma, or 40% human plasma. The λ_LSPR response of gold nanoprisms for each miR-21 concentration was measured where the highest 18.8 ± 1.9 nm λ_LSPR red shift was observed for 100 nM miR-21 (Figure 2a, blue) in PBS buffer. We hypothesize that the λ_LSPR red-shift is due to hybridization between ssDNA-21 and miR-21. It was found that the magnitude of the λ_LSPR shift was concentration dependent, where 50 fM miR-21 caused a 3.7 ± 0.3 nm λ_LSPR red shift in PBS buffer. Figure 2b illustrates the magnitude of the λ_LSPR shift (Δλ_LSPR) upon DNA/RNA duplex formation for various miR-21 concentrations in the three different media. Evidently higher concentrations of miR-21 induced a larger number of ssDNA-21 strands to convert to DNA/RNA duplexes and consequently a larger change in the local refractive index around the nanoprisms, which results in a larger value of Δλ_LSPR.

Figure 2. MicroRNA detection using label-free plasmonic biosensors. (a) Monitoring LSPR dipole peak (λ_LSPR) changes by UV–visible absorption spectra of gold nanoprisms during various functionalization steps: before (black, λ_LSPR = 800 nm) and after functionalized with a 1 μM/1 μM ratio of HS-C6-ssDNA-21/PEG₆-SH (red, λ_LSPR = 821 nm), and after incubation with 100 nM miR-21 solution in PBS buffer (blue, λ_LSPR = 839 nm). (b) average λ_LSPR peak shift (Δλ_LSPR) of gold nanoprisms functionalized with a 1 μM/1 μM ratio of HS-C6-ssDNA-21/PEG₆-SH after incubation in different concentrations of miR-21 in PBS buffer (red triangles), 40% human plasma (black squares), and 40% bovine plasma (blue diamonds). The Δλ_LSPR were calculated by taking the difference between the λ_LSPR peak position of the plasmonic biosensor after and before the hybridization with miR-21 in the various media. (c) Average Δλ_LSPR of the plasmonic biosensor functionalized with a 1 μM/1 μM ratio of HS-C6-ssDNA-10b/PEG₆-SH after hybridization with different miR-10b concentrations in PBS buffer (red triangles), in 40% human plasma (black squares), and in 40% bovine plasma (blue diamonds). All extinction spectra recorded after miR-X incubation were measured in PBS buffer after rinsing with PBS buffer.

The highest red shift of 18.8 ± 1.9 nm we observed for 100 nM miR-21 incubation with our plasmonic biosensor is significant considering only an ∼5% change in the refractive index upon ssDNA/RNA duplex formation. (52) We believe such a high sensitivity of our plasmonic biosensors is because of the unique LSPR properties of our gold nanoprisms and the possibility of electron delocalization as the ssDNA forms the duplex and becomes double-strand. The atomically flat surface, extremely small height (∼8 nm), and sharp tips of our nanoprisms display strong EM field enhancement near their surface and therefore are expected to be extremely sensitive to small changes of their local dielectric environment. (35, 40) Moreover, transformation of ssDNA into double-strand DNA significantly changes the refractive index because of the high charge density and polarizability of the DNAs. (52) The duplex DNA is capable of long-range charge transfer and alters the electron density around the nanoprisms thus influencing their LSPR properties. This interesting phenomenon requires further scientific study, which is currently under our investigation.

Our sensing mechanism is based on the hypothesis that the attachment of complementary target miRs to our plasmonic biosensor will shift the λ_LSPR to higher wavelength (Figure 1c). The total shift (Δλ_LSPR) depended on the miR concentration (Figure 1d) and could be used to determine the limit of detection (LOD) (Figure 1e). The LODs calculated for miR-21 in three different media were found to be in the range of 23–35 fM, which was more than 1000 and 3 fold lower than with the label-free microring resonator (150 fmol) (15) and the nanopore based (100 fM) (16) miR sensors, respectively. Importantly, these techniques detected miRs in PBS buffer whereas we have demonstrated here for the first time a sensing approach in physiological media. Utilizing our same direct hybridization-based detection approach, plasmonic biosensors were constructed with of −C6-ssDNA-10, while keeping other parameters constant. The LOD for miR-10b in the above media was determined over a concentration range from 100 nM to 50 fM. The average Δλ_LSPR and LODs for miR-10b in three diverse media are shown in Figure 2c.

The principle underlying the actions of plasmonic biosensors is based on the successful hybridization between miRs and ssDNA attached to nanoprisms, where a higher number of duplex formations will result in a larger change in the refractive index surrounding the nanoprisms resulting in larger Δλ_LSPR and higher sensitivity. Therefore, it would be expected that functionalization of gold nanoprisms with 100% HS–C6-ssDNA-X (without the PEG₆-thiol spacer) should reduce the LOD values because of steric hindrance and low attachment of miRs. To investigate this, gold nanoprisms were functionalized with 100% −C6-ssDNA-21 resulting in an ∼15.0 ± 1.8 nm λ_LSPR red shift (Figure 3a). The sensor was then incubated in different concentrations of miR-21 prepared in 40% human plasma. As illustrated in Figure 3a, an ∼9.6 ± 1.1 nm red shift was observed for a 100 nM miR-21 concentrations and the lowest concentration that can be repeatedly detected was 10 pM from a Δλ_LSPR of 3.4 ± 0.5 nm. Figure 3b shows the Δλ_LSPR versus concentration plot. Evidently, functionalization of the nanoprism’s surface with 100% −C6-ssDNA-21 resulted in a 200-fold increase in detection limit in comparison to the 1:1 ratio −C6-ssDNA-21/PEG₆-SH mixed functionalization (Supporting Information Table S2). These experimental data further highlight our rationale for using spacers that increase the likelihood of hybridization. We believe fully covered gold nanoprisms were obtained when 100% −C6-ssDNA-21 was used for functionalization, which creates steric hindrance and does not allow the maximum number of miR-21 strands to come into close proximity with −C6-ssDNA-21 for hybridization. Therefore, not all the −C6-ssDNA-21 attached on the gold nanoprisms’ surface was hybridized with miR-21 strands resulting in low sensing response. Thus, if we introduce a spacer between the −C6-ssDNA-21, it will allow the maximum -ssDNA-21 strands to be freely available for hybridization without any interference and ultimately enhance the sensitivity of the biosensor. Accordingly, for the remaining of our investigation, we used a 1:1 mixed −C6-ssDNA-X:PEG₆-SH to functionalize the gold nanoprisms.

Figure 3. Determining the optimum detection condition. (a) UV–visible extinction spectra monitoring the LSPR dipole peak (λ_LSPR) of gold nanoprisms attached to silanized glass substrate before (black, λ_LSPR = 796 nm) and after (red, λ_LSPR = 811 nm) functionalization with 1 μM of HS-C6-ssDNA-21 without PEG₆-SH spacers and after incubation in 100 nM miR-21 solution in 40% human plasma (blue, λ_LSPR = 822 nm). (b) Average Δλ_LSPR of these HS-C6-ssDNA-21 functionalized gold nanoprisms upon hybridization with different miR-21 concentrations in 40% human plasma. The Δλ_LSPR were calculated by taking the difference between the λ_LSPR peak position of the nanoprisms after and before the incubation with miR-21 in PBS buffer.

In order to confirm the hybridization of miR-X with −C6-ssDNA-X that resulted in the Δλ_LSPR, the enzyme RNase H was used to selectively cleave the DNA: RNA duplex and potentially reverse the Δλ_LSPR. Initially, the plasmonic biosensor for miR-21 was incubated in a 100 nM solution of miR-21, which resulted in red-shift of λ_LSPR potentially reflecting hybridization. The biosensor was then immersed in 15 units of RNase H solution for 2 h. Afterward the λ_LSPR showed a blue shift and reverted back to its original position before miR-21 incubation (Figure 4a). When the 1:1 ratio −C6-ssDNA-21:PEG₆-SH mixed functionalized biosensor was incubated with RNase H solution alone overnight, no noticeable change in λ_LSPR value was observed (Supporting Information Figure S7). These experimental results validate our previous observation that the λ_LSPR blue shift was due to the cleavage of heteroduplex done by RNase H. The biosensors were rinsed with RNase free water to PBS buffer and again incubated in 100 nM miR-21 solution for rehybridization where an ∼14 nm red shift of the λ_LSPR was observed. These experiments confirm our working hypothesis that hybridization between the nanoprism’s surface ligands (−C6-ssDNA-X) and the miR-X resulted in changes in the local dielectric environment around the nanoprisms causing wavelength shift. As shown in Figure 4b, the λ_LSPR responses were identical for several cycles due to hybridization and dehybridization of miR-21 over a period of 6 days. The same experiments were done for the miR-10b biosensor and similar results were observed, underscoring the long-term stability of the sensors and their potential for being developed into cost-effective point of care diagnostic tools.

Figure 4. Characterization of the plasmonic biosensors regeneration ability. (a) UV–visible extinction spectra of gold nanoprisms functionalized with a 1 μM/1 μM ratio of HS-C6-ssDNA-21/PEG₆-SH (red, λ_LSPR = 818 nm) attached to silanized glass, after incubation with 100 nM of miR-21 (blue, λ_LSPR = 832 nm), after treatment with 15 units of RNase H for 2 h (red dotted, λ_LSPR = 818 nm), and after rehybridized with 100 nM of miR-21 (blue dotted, λ_LSPR = 832 nm). (b) Changes in LSPR dipole peak position (λ_LSPR) of gold nanoprisms functionalized with a 1 μM/1 μM ratio of HS-C6-ssDNA-21/PEG₆-SH upon hybridization and dehybridization with miR-21 for several cycles. The λ_LSPR peak shifts back and forth upon sensor regeneration with RNase H by cleaving DNA/RNA duplex and rehybridization after incubation into 100 nM miR-21 in 40% human plasma. After each of the dehybridization steps, the plasmonic biosensors were thoroughly rinsed with PBS buffer to completely remove enzyme RNase H.

The hybridization takes place at the 5′ end of −C6-ssDNA-21 and the 3′ end of miR-21, which evidently increased the refractive index. Additionally such hybridization would also increase the thickness of the local dielectric environment of the nanoprisms. Together, a significantly large Δλ_LSPR was generated for both miR-21 and miR-10b. Atomic force microscopy (AFM) analysis was conducted to characterize our plasmonic biosensors and also to verify the change in surface area caused by miR-21 incubation with mixed −C6-ssDNA-21 and PEG₆-SH-functioanlized gold nanoprisms. After analyzing 40 different nanoprisms (Figure 5 and Supporting Information Figure S8) – the exact same area of four different sections of the sensor - an average 2.4 × 10^–15 m² increase in surface area was observed by AFM. Thus, attachment of miRs to plasmonic biosensors has increased the thickness of local dielectric environment around the gold nanoprisms and influenced their LSPR properties. Ultrasensitive refractive index-induced LSPR response of nanoprisms allows us to fabricate label-free plasmonic biosensor.

Figure 5. Surface characterization of the plasmonic biosensors. Atomic force microscopy images of gold nanoprisms bound to silanized glass substrate (a) after functionalization with 1:1 ratio of HS-C6-ssDNA-21/PEG₆-SH (b) and after hybridization with100 nM miR-21 in 40% human plasma (c). The measurements were conducted in air. (d) Changes in the surface area of gold nanoprisms after each functionalization steps. Forty nanoprisms were selected to determine the average change in the surface area. Detailed method of surface area calculation is provided in the Supporting Information file.

The successful implementation of plasmonic biosensors for use with real biological samples mandates documentation of their specificity toward target miRs in that patient samples containing multiple miR species. The mix functionalized (−C6-ssDNA-21 and PEG₆-SH) biosensors were incubated overnight in 40% human plasma solution containing 100 nM each of miR-16, miR-122, miR-126, and miR-141 because these miRs are commonly present in human plasma. The λ_LSPR response was measured before and after incubation (Supporting Information Figure S9) and resulted in an ∼2.5 ± 0.3 nm λ_LSPR red shift, which is within the instrument noise level and/or minor nonspecific adsorption of extraneous materials present in human plasma. In another control experiment, gold nanoprisms attached as before to glass substrate were functionalized with 100% PEG₆-SH by incubation in a 1 μM aqueous solution, and after rinsing with large amounts of water, incubated in a 40% human plasma solution of 100 nM miR-21 for 12 h. This procedure resulted in only an ∼0.9 ± 0.7 nm λ_LSPR red shift (Supporting Information Figure S10), confirming that the plasmonic biosensors we designed are highly specific toward the target miRs.

Detection of miR Levels in in Plasma from Pancreatic Cancer Patients

Pancreatic cancer is the fourth-leading cause of cancer death in the United States with an annual mortality of nearly 40 000 and a dismal five-year survival rate of 6%. (1) PDAC is characterized by chemotherapeutic resistance and by the absence of an effective screening procedure for early disease. It is generally accepted that early diagnosis could reduce mortality rates substantially and thus a noninvasive early PDAC test must be developed. Several miRs (such as miR-21, -10b, -103, -155, -196a, 210, and -221) were found to be overexpressed in PDAC. (6-8, 53) Given their resistance to degradation, plasma miRs have the potential to serve as biomarkers for the noninvasive diagnosis of PDAC. Previously, nanopore sensors were used to detect miRs in lung cancer patients, but to the best of our knowledge no sensors have been developed to date to detect PDAC-related miRs in human plasma.

Utilizing our plasmonic biosensors we detected miR-21 and miR-10b in plasma from PDAC patients. Plasma samples were collected from six patients and six normal control subjects. Total plasma RNAs including miRs were extracted from 100 μL of each plasma sample using a TRIZOL kit with a final elution volume of 28 μL. Next, 14 μL volumes were used for miR quantification by the plasmonic biosensor and the remaining 14 μL were used in the qRT-PCR assay. The plasmonic biosensors were fabricated as described before for both miR-21 and miR-10b detection. The extracted human miR-21 or miR-10b samples were diluted in PBS buffer and incubated with the biosensors were for 12 h, followed by rinsing with PBS buffer and measurement of the λ_LSPR response in PBS buffer. The observed λ_LSPR shift for each miR-21 and miR-10b sample was converted into the corresponding concentration using the calibration curve derived for miR-21 or 10b under optimized conditions and compared with the value from normal control subjects (Figure 6a,c). The concentrations of miR-21 and miR-10b determined from plasmonic biosensors were also compared with the values obtained from the qRT-PCR assay (Figure 6b and Supporting Information Figure S16). Importantly, for the first time through a label-free technique we have shown that miR-10b concentration is nearly 4-fold higher than the miR-21 level in patient samples. Inasmuch as both miR-21 and miR-10b are overexpressed in PDAC, it is possible that miR-10b is released more efficiently by pancreatic cancer cells than miR-21, allowing it to achieve higher levels in the circulation. It is therefore possible that miR-10b levels are also increased within the pancreatic tumor microenvironment, where it could be acting to enhance PDAC biological aggressiveness.

Figure 6. Determination of microRNA concentration in PDAC patients and normal control subjects. (a) The average λ_LSPR peak shifts of gold nanoprisms functionalized with a 1:1 ratio of HS-C6-ssDNA-21/PEG₆-SH upon hybridization with miR-21 from the total RNAs extracted from plasma samples of PDAC patients (blue diamonds) and normal control subjects (blue squares). The respective λ_LSPR peak shifts were converted to concentrations using the calibration curve established for miR-21 in PBS buffer as shown in Figure 2b [PDAC patients (red triangles), and for normal control subjects (red circles)]. (b) Comparison of miR-21 concentration for six PDAC patients determined using plasmonic biosensors (blue diamond) and qRT-PCR (red square). (c) Similar experiments were conducted to detect miR-10b where the λ_LSPR peak shifts and concentrations for PDAC patients are shown in blue diamonds and red triangles, respectively. The λ_LSPR peak shifts (blue squares) and concentrations (red circles) for normal controls are shown for comparison. (d) The average λ_LSPR peak shifts (blue diamonds) and concentration (red triangles) for the miR-21 in plasma samples from PDAC patients without any purification.

We also detected miR-21 levels directly in human plasma samples collected from PDAC patients without RNAs extraction. Thus, human plasma (50 μL/sample) from six pancreatic cancer patients were diluted in PBS buffer followed by incubation with our plasmonic biosensors for 12 h. The λ_LSPR response of each sample was measured through UV–vis spectroscopy and showed a steady increase in concentration from sample 6 to 1 (Figure 6d). Both plasmonic biosensor and qRT-PCR results indicated that miR-10b levels were higher in PDAC patients compared to normal control subjects and that the levels of miR-21 and miR-10b can be quantified with high accuracy using our gold nanoprism-based plasmonic biosensor without any modification, amplification, or labeling. Importantly, the miR-21 concentration in extracted samples was at least 2-fold lower than in the pure plasma samples. We believe this is due to loss of miRs during the RNA extraction process, which requires multiple steps for RNA purification. Therefore, the most widely used qRT-PCR method to determine the concentration of miRs in patients may not accurately represent the actual concentration. This limitation and imprecise quantification can be avoided by using our newly developed plasmonic biosensors, which can provide a unique opportunity as potential diagnostic and prognostic markers in PDAC, other cancers, and potentially other disease states.

Conclusion

We have designed, fabricated, and characterized a plasmonic biosensor that was able to detect PDAC relevant miRs in human plasma without using RNAs extraction, which opens a new avenue for the direct detection and quantification of miR levels in clinical samples without any form of sample preparation. To our knowledge, this is the first LSPR-based, label-free, direct hybridization method for miR detection, which eliminates all the current drawbacks such as labeling, tagging, amplification, use of highly toxic chemical, and further modification of the sensor. Furthermore, it vastly simplifies the detection approach without requiring detailed knowledge of the electron or energy transfer processes involved as in other more complicated techniques. Additionally, this ultrasensitive, plasmonic-based, direct hybridization-controlled detection approach is applicable to any type of miRs that are relevant to various diseases. It was found that our plasmonic biosensor can be regenerated through several cycles and is stable for several days without compromising its sensitivity and selectivity, which should enable the development of simple, cost-effective tools for the early detection of miRs and thus facilitate the early diagnosis of various cancers. Finally, the large EM-field enhancement at the nanoprism’s sharp tips (39) will enhance the Raman scattering intensity of the analytes. In theory, therefore, nanoprisms can be used to design effective substrates for surface-enhanced Raman spectroscopy-based (54-57) detection and quantification of multiple miRs simultaneously through integration of their spectral characteristic with the λ_LSPR shifts.

Materials and Methods

All synthetic DNA probes and microRNAs were purchased from Sigma-Aldrich (U.S.A.). PBS buffer prepared with RNase-free water was used to dilute oligonucleotides and miRs solutions. Patient plasma was obtained from the Indiana University Simon Cancer Center Solid Tissue Bank (Indianapolis, Indiana).

Fabrication of LSPR-Based miR Sensors and Detection

The gold nanoprism-based miR sensors were designed using our published procedure with modification. (49) The attachment of gold nanoprisms on silanized glass substrates is described in the Supporting Information file. The substrate-bound nanoprisms were incubated in PBS buffer solution containing 1 μM each of HS–C6-ssDNA-X and PEG₆-SH overnight and rinsed with PBS buffer. The initial LSPR peak position of each sensing platform was determined using UV–visible spectroscopy in PBS buffer and then was incubated in the different concentrations of miR solutions, for exmaple, either in PBS buffer, 40% bovine plasma, or 40% human plasma for 12 h at room temperature. The plasmonic biosensors were thoroughly washed with PBS buffer to remove any nonspecifically adsorbed species. The miR bound biosensor was then placed in PBS buffer for 10 min before the LSPR peak position was determined. For UV–vis extinction spectra measurement, one particular solvent was chosen to avoid the solvent dielectric constant effect, which is known to shift the LSPR peak. (21, 58, 59)

Total RNA Extraction and Quantification of MicroRNA by qRT-PCR

Total RNA was isolated from plasma samples that were obtained from the Indiana University Simon Cancer Center Solid Tissue Bank (Indianapolis, IN, U.S.A.) using Trizol-LS reagent (Life Technologies, Carlsbad, CA, U.S.A.). cDNA was generated using 10 ng of RNA and miR-10b, miR-21, or miR-425–5p RT primers and a miR reverse transcription kit (Life Technologies) as per the manufacturer’s recommendations. Quantitative PCR (qPCR) was performed using Taqman miR expression assay reagents. Expression levels as determined by qPCR were normalized to miR-425-5p, since this miR was expressed at similar levels in all samples and exhibited <1 cycle threshold (Ct) difference across all samples. After normalization to miR-425-5p (ΔCt), the ΔCt values for miRs in controls were averaged and subtracted from the ΔCt values of each individual sample (ΔΔCt). miR levels were then calculated using the 2^–ΔΔCt method. (60)

Supporting Information

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Experimental detail of nanoprisms synthesis, fabrication of plasmonic biosensors, and additional UV–visible spectra, AFM images, and histograms. This material is available free of charge via the Internet at http://pubs.acs.org.

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Corresponding Authors
- Murray Korc - Department of Chemistry and Chemical Biology, Indiana University-Purdue University Indianapolis, 402 N. Blackford Street, LD 326, Indianapolis, Indiana 46202, United States; ‡Department of Medicine, §Department of Biochemistry and Molecular Biology, and ∥the Center for Pancreatic Cancer Research at the IU Simon Cancer Center, Indiana University School of Medicine, 980 W. Walnut Street, C549, Indianapolis, Indiana 46202, United States; Department of Physics, Indiana University-Purdue University Indianapolis, 402 N. Blackford Street, LD 326, Indianapolis, Indiana 46202, United States; Integrated Nanosystems Development Institute, Indiana University-Purdue University Indianapolis, 402 N. Blackford Street, Indianapolis, Indiana 46202, United States; Email: [email protected]
- Rajesh Sardar - Department of Chemistry and Chemical Biology, Indiana University-Purdue University Indianapolis, 402 N. Blackford Street, LD 326, Indianapolis, Indiana 46202, United States; Integrated Nanosystems Development Institute, Indiana University-Purdue University Indianapolis, 402 N. Blackford Street, Indianapolis, Indiana 46202, United States; Email: [email protected]
Authors
- Gayatri K. Joshi - Department of Chemistry and Chemical Biology, Indiana University-Purdue University Indianapolis, 402 N. Blackford Street, LD 326, Indianapolis, Indiana 46202, United States
- Samantha Deitz-McElyea - Department of Chemistry and Chemical Biology, Indiana University-Purdue University Indianapolis, 402 N. Blackford Street, LD 326, Indianapolis, Indiana 46202, United States; ‡Department of Medicine, §Department of Biochemistry and Molecular Biology, and ∥the Center for Pancreatic Cancer Research at the IU Simon Cancer Center, Indiana University School of Medicine, 980 W. Walnut Street, C549, Indianapolis, Indiana 46202, United States; Department of Physics, Indiana University-Purdue University Indianapolis, 402 N. Blackford Street, LD 326, Indianapolis, Indiana 46202, United States; Integrated Nanosystems Development Institute, Indiana University-Purdue University Indianapolis, 402 N. Blackford Street, Indianapolis, Indiana 46202, United States
- Merrell Johnson - Department of Physics, Indiana University-Purdue University Indianapolis, 402 N. Blackford Street, LD 326, Indianapolis, Indiana 46202, United States
- Sonali Mali - Department of Chemistry and Chemical Biology, Indiana University-Purdue University Indianapolis, 402 N. Blackford Street, LD 326, Indianapolis, Indiana 46202, United States
Notes
The authors declare no competing financial interest.

Acknowledgment

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The authors would like to thank B. Muhoberac and S. Savant for invaluable advice regarding the manuscript and K. Lawrence for providing PEG₆-thiol. We also thank the IUSCC Cancer Center at Indiana University School of Medicine for the use of the Tissue Procurement and Distribution Core, which provided patient and control plasma samples for microRNA assays. The research was partially supported by Indiana University-Purdue University Indianapolis startup funds (R.S), Purdue Research Foundation (R.S), the Indiana Economic Development Fund (IEDF) and the National Institute of Health Grant R37-CA-075059 (M.K). The authors also thank Amy Trotch (from Affymetrix) for providing RNase H sample and the Wallace lab (IUPUI) for the use of the AFM instrument. S.M. thanks ACS Project SEED program for financial support.

References

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Localized surface plasmon resonance sensors
Mayer, Kathryn M.; Hafner, Jason H.
Chemical Reviews (Washington, DC, United States) (2011), 111 (6), 3828-3857CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)
A review. Topics include physics of localized surface plasmon resonance (LSPR); sensing with LSPR, effect of particle size, shape, and material; LSPR-based biol. and chem. sensors; technol. advances in LSPR sensing; maximizing the mol. detection sensitivity of LSPR; and future directions like mol. biol. or diagnostic tool application.
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Photoinduced conversion of silver nanospheres to nanoprisms
Jin, Rongchao; Cao, YunWei; Mirkin, Chad A.; Kelly, K. L.; Schatz, George C.; Zheng, J. G.
Science (Washington, DC, United States) (2001), 294 (5548), 1901-1903CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
A photoinduced method for converting large quantities of Ag nanospheres into triangular nanoprisms is reported. The photo-process was characterized by time-dependent UV-visible spectroscopy and TEM, allowing for the observation of several key intermediates in and characteristics of the conversion process. This light-driven process results in a colloid with distinctive optical properties that directly relate to the nanoprism shape of the particles. Theor. calcns. coupled with exptl. observations allow for the assignment of the nanoprism plasmon bands and for the 1st identification of 2 distinct quadrupole plasmon resonances for a nanoparticle. Unlike the spherical particles they are derived from that Rayleigh light-scatter in the blue, these nanoprisms exhibit scattering in the red, which could be useful in developing multicolor diagnostic labels on the basis not only of nanoparticle compn. and size but also of shape.
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Sun, Y.; Xia, Y. Science 2002, 298, 2176– 2179
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Shape-Controlled Synthesis of Gold and Silver Nanoparticles
Sun, Yugang; Xia, Younan
Science (Washington, DC, United States) (2002), 298 (5601), 2176-2179CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
Monodisperse samples of silver nanocubes were synthesized in large quantities by reducing silver nitrate with ethylene glycol in the presence of poly(vinyl pyrrolidone) (PVP). These cubes were single crystals and were characterized by a slightly truncated shape bounded by {100}, {110}, and {111} facets. The presence of PVP and its molar ratio (in terms of repeating unit) relative to silver nitrate both played important roles in detg. the geometric shape and size of the product. The silver cubes could serve as sacrificial templates to generate single-cryst. nanoboxes of gold: hollow polyhedra bounded by six {100} and eight {111} facets. Controlling the size, shape, and structure of metal nanoparticles is technol. important because of the strong correlation between these parameters and optical, elec., and catalytic properties.
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Biosensing with plasmonic nanosensors
Anker, Jeffrey N.; Hall, W. Paige; Lyandres, Olga; Shah, Nilam C.; Zhao, Jing; Van Duyne, Richard P.
Nature Materials (2008), 7 (6), 442-453CODEN: NMAACR; ISSN:1476-1122. (Nature Publishing Group)
A review. Light incident on metallic nanoparticles can induce a collective motion of electrons that can lead to a strong amplification of the local electromagnetic field. As reviewed here, these plasmonic resonances have important applications in biosensing where they push resoln. and sensitivity towards the single-mol. detection limit. Recent developments have greatly improved the sensitivity of optical sensors based on metal nanoparticle arrays and single nanoparticles. The authors introduce the localized surface plasmon resonance (LSPR) sensor and describe how its exquisite sensitivity to size, shape and environment can be harnessed to detect mol. binding events and changes in mol. conformation. The authors then describe recent progress in three areas representing the most significant challenges: pushing sensitivity towards the single-mol. detection limit, combining LSPR with complementary mol. identification techniques such as surface-enhanced Raman spectroscopy, and practical development of sensors and instrumentation for routine use and high-throughput detection. This review highlights several exceptionally promising research directions and discusses how diverse applications of plasmonic nanoparticles can be integrated in the near future.
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Photoswitchable Oligonucleotide-Modified Gold Nanoparticles: Controlling Hybridization Stringency with Photon Dose
Yan, Yunqi; Chen, Jennifer I. L.; Ginger, David S.
Nano Letters (2012), 12 (5), 2530-2536CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)
We describe a new class of stimulus-responsive DNA-functionalized gold nanoparticles that incorporate azobenzene-modified oligonucleotides. Beyond the classic directed assembly and sensing behaviors assocd. with oligonucleotide-modified nanoparticles, these particles also exhibit reversible photoswitching of their assembly behavior. Exposure to UV light induces a trans-cis isomerization of the azobenzene which destabilizes the DNA duplex, resulting in dissocn. of the nanoparticle assemblies. The isomerization is reversible upon exposure to blue light, resulting in rehybridization and reassembly of the DNA-linked nanoparticle clusters. We show that perfectly complementary and partially mismatched strands exhibit clearly distinguishable photoinduced melting properties, and we demonstrate that photon dose can thus be used in place of temp. or ionic strength to control hybridization stringency with the ability to discriminate single-base mismatches.
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Gold nanoparticles in chemical and biological sensing
Saha, Krishnendu; Agasti, Sarit S.; Kim, Chaekyu; Li, Xiaoning; Rotello, Vincent M.
Chemical Reviews (Washington, DC, United States) (2012), 112 (5), 2739-2779CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)
A review. Topics include synthesis ans surface functionalization; phys. properties; colorimetric and fluorimetric sensing; elec. al and electrochem. sensing; SERS; gold nanoparticles in quartz crystal microbalance-based sensing; application of gold nanoparticles in bio-barcode assays.
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Super-resolution Optical Imaging of Single-Molecule SERS Hot Spots
Stranahan, Sarah M.; Willets, Katherine A.
Nano Letters (2010), 10 (9), 3777-3784CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)
The authors present the 1st super-resoln. optical images of single-mol. surface-enhanced Raman scattering (SM-SERS) hot spots, using super-resoln. imaging as a powerful new tool for understanding the interaction between single mols. and nanoparticle hot spots. Using point spread function fitting, the authors map the centroid position of SM-SERS with ±10 nm resoln., revealing a spatial relation between the SM-SERS centroid position and the highest SERS intensity. The authors are also able to measure the unique position of the SM-SERS centroid relative to the centroid assocd. with nanoparticle photoluminescence, which allows the authors to speculate on the presence of multiple hot spots within a single diffraction-limited spot. These measurements allow the authors to follow dynamic movement of the SM-SERS centroid position over time as it samples different locations in space and explores regions larger than the expected size of a SM-SERS hot spot. Probably the movement of the SERS centroid is due to diffusion of a single mol. on the surface of the nanoparticle, which leads to changes in coupling between the scattering dipole and the optical near field of the nanoparticle.
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Elghanian, R.; Storhoff, J. J.; Mucic, R. C.; Letsinger, R. L.; Mirkin, C. A. Science 1997, 277, 1078– 1081
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Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles
Elghanian, Robert; Storhoff, James J.; Mucic, Robert C.; Letsinger, Robert L.; Mirkin, Chad A.
Science (Washington, D. C.) (1997), 277 (5329), 1078-1080CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
A highly selective, colorimetric polynucleotide detection method based on mercaptoalkyloligonucleotide-modified gold nanoparticle probes is reported. Introduction of a single-stranded target oligonucleotide (30 bases) into a soln. contg. the appropriate probes resulted in the formation of a polymeric network of nanoparticles with a concomitant red-to-pinkish/purple color change. Hybridization was facilitated by freezing and thawing of the solns., and the denaturation of these hybrid materials showed transition temps. over a narrow range that allowed differentiation of a variety of imperfect targets. Transfer of the hybridization mixt. to a reverse-phase silica plate resulted in a blue color upon drying that could be detected visually. The unoptimized system can detect about 10 fmol in oligonucleotide.
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Localized surface plasmon resonance spectroscopy and sensing
Willets, Katherine A.; Van Duyne, Richard P.
Annual Review of Physical Chemistry (2007), 58 (), 267-297CODEN: ARPLAP; ISSN:0066-426X. (Annual Reviews Inc.)
A review. Localized surface plasmon resonance (LSPR) spectroscopy of metallic nanoparticles is a powerful technique for chem. and biol. sensing expts. Also, the LSPR is responsible for the electromagnetic-field enhancement that leads to surface-enhanced Raman scattering (SERS) and other surface-enhanced spectroscopic processes. This review describes recent fundamental spectroscopic studies that reveal key relations governing the LSPR spectral location and its sensitivity to the local environment, including nanoparticle shape and size. The authors also describe studies on the distance dependence of the enhanced electromagnetic field and the relation between the plasmon resonance and the Raman excitation energy. Lastly, the authors introduce a new form of LSPR spectroscopy, involving the coupling between nanoparticle plasmon resonances and adsorbate mol. resonances. The results from these fundamental studies guide the design of new sensing expts., illustrated through applications in which researchers use both LSPR wavelength-shift sensing and SERS to detect mols. of chem. and biol. relevance.
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Malinsky, M. D.; Kelly, K. L.; Schatz, G. C.; Van Duyne, R. P. J. Am. Chem. Soc. 2001, 123, 1471– 1482
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Jackson, J. B.; Westcott, S. L.; Hirsch, L. R.; West, J. L.; Halas, N. J. Appl. Phys. Lett. 2003, 82, 257– 259
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Hao, E.; Schatz, G. C. J. Chem. Phys. 2004, 120, 357– 366
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Electromagnetic fields around silver nanoparticles and dimers
Hao, Encai; Schatz, George C.
Journal of Chemical Physics (2004), 120 (1), 357-366CODEN: JCPSA6; ISSN:0021-9606. (American Institute of Physics)
We use the discrete dipole approxn. to investigate the electromagnetic fields induced by optical excitation of localized surface plasmon resonances of silver nanoparticles, including monomers and dimers, with emphasis on what size, shape, and arrangement leads to the largest local elec. field (E-field) enhancement near the particle surfaces. The results are used to det. what conditions are most favorable for producing enhancements large enough to observe single mol. surface enhanced Raman spectroscopy. Most of the calcns. refer to triangular prisms, which exhibit distinct dipole and quadrupole resonances that can easily be controlled by varying particle size. In addn., for the dimer calcns. we study the influence of dimer sepn. and orientation, esp. for dimers that are sepd. by a few nanometers. We find that the largest |E|2 values for dimers are about a factor of 10 larger than those for all the monomers examd. For all particles and particle orientations, the plasmon resonances which lead to the largest E-fields are those with the longest wavelength dipolar excitation. The spacing of the particles in the dimer plays a crucial role, and we find that the spacing needed to achieve a given |E|2 is proportional to nanoparticle size for particles below 100 nm in size. Particle shape and curvature are of lesser importance, with a head to tail configuration of two triangles giving enhanced fields comparable to head to head, or rounded head to tail. The largest |E|2 values we have calcd. for spacings of 2 nm or more is ∼105.
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Sherry, L. J.; Jin, R.; Mirkin, C. A.; Schatz, G. C.; Van Duyne, R. P. Nano Lett. 2006, 6, 2060– 2065
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Chen, H.; Shao, L.; Woo, K. C.; Ming, T.; Lin, H.-Q.; Wang, J. J. Phys. Chem. C 2009, 113, 17691– 17697
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Joshi, G. K.; McClory, P. J.; Muhoberac, B. B.; Kumbhar, A.; Smith, K. A.; Sardar, R. J. Phys. Chem. C 2012, 116, 20990– 21000
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Haes, A. J.; Zou, S.; Zhao, J.; Schatz, G. C.; Van Duyne, R. P. J. Am. Chem. Soc. 2006, 128, 10905– 10914
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Localized Surface Plasmon Resonance Spectroscopy near Molecular Resonances
Haes, Amanda J.; Zou, Shengli; Zhao, Jing; Schatz, George C.; Van Duyne, Richard P.
Journal of the American Chemical Society (2006), 128 (33), 10905-10914CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
The peak location of the localized surface plasmon resonance (LSPR) of noble metal nanoparticles is highly dependent upon the refractive index of the nanoparticles' surrounding environment. New phenomena are revealed by exploring the influence of interacting mol. resonances and nanoparticle resonances. The LSPR peak shift and line shape induced by a resonant mol. vary with wavelength. In most instances, the oscillatory dependence of the peak shift on wavelength tracks with the wavelength dependence of the real part of the refractive index, as detd. by a Kramers-Kronig transformation of the mol. resonance absorption spectrum. A quant. assessment of this shift based on discrete dipole approxn. calcns. shows that the Kramers-Kronig index must be scaled to match expt.
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Hall, W. P.; Modica, J.; Anker, J.; Lin, Y.; Mrksich, M.; Van Duyne, R. P. Nano Lett. 2011, 11, 1098– 1105
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Single Silver Nanoparticles as Real-Time Optical Sensors with Zeptomole Sensitivity
McFarland, Adam D.; Van Duyne, Richard P.
Nano Letters (2003), 3 (8), 1057-1062CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)
This work utilizes dark-field optical microscopy to demonstrate the localized surface plasmon resonance λmax response of individual Ag nanoparticles to the formation of a monolayer of small-mol. adsorbates. The adsorption of fewer than 60 000 1-hexadecanethiol mols. on single Ag nanoparticles results in a localized surface plasmon resonance shift of 40.7 nm. Addnl., the kinetics of the single nanoparticle response was shown to be comparable to that of other real-time sensor technologies.
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Formation of monolayers by the coadsorption of thiols on gold: variation in the length of the alkyl chain
Bain, Colin D.; Whitesides, George M.
Journal of the American Chemical Society (1989), 111 (18), 7164-75CODEN: JACSAT; ISSN:0002-7863.
Mixts. of 2 long-chain alkanethiols, HS(CH2)nX and HS(CH2)mY (X, Y = CH3, OH; n > m), in which the alkyl chains have different lengths, adsorb from soln. on Au and form monolayers comprising a densely packed inner region adjacent to the Au surface and a disordered outer region in contact with the soln. When X = Y = Me (n ≠ m), this disordered phase makes the "mixed monolayer" more oleophilic than the ordered, pure (i.e., single-component) monolayers. When X = Y = OH, the pure monolayers are wetted by water, but the mixed monolayers are less hydrophilic because nonpolar polymethylene chains are exposed at the surface. When X = Me, Y = OH (n = 21, m = 11), a very sharp transition occurs from a monolayer composed largely of the longer, Me-terminated component to the shorter, OH-terminated component as the mol fraction of HS(CH2)11OH in the adsorption soln. is increased. From solns. contg. 2 thiols, adsorption of the thiol with the longer chain is preferred. This preference is greater when the monolayers are adsorbed from EtOH than from isooctane. The mixed monolayers do not act as ideal 2-dimensional solns. The adsorption isotherms suggest a pos. excess free energy of mixing of the 2 components in the monolayer. The compns. of the monolayers appear to be detd. largely by thermodn., although in some cases there is also a kinetic contribution. The 2 components in the mixed monolayers do not phase-segregate into macroscopic islands (greater than a few tens of angstroms across), but are probably not randomly dispersed within the monolayer. The wettability of mixed, Me-terminated monolayers can be rationalized partially by the geometric mean approxn., but a full description probably requires inclusion of the entropy of mixing at the monolayer-liq. interface. The hysteresis in the contact angle on these monolayers cannot be explained by theories of wetting based on macroscopic heterogeneity. Contact angles are more sensitive than optical ellipsometry or XPS to certain types of changes in the compn. and structure of these monolayers.
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Murphy, C. J.; Gole, A. M.; Stone, J. W.; Sisco, P. N.; Alkilany, A. M.; Goldsmith, E. C.; Baxter, S. C. Acc. Chem. Res. 2008, 41, 1721– 1730
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Gold Nanoparticles in Biology: Beyond Toxicity to Cellular Imaging
Murphy, Catherine J.; Gole, Anand M.; Stone, John W.; Sisco, Patrick N.; Alkilany, Alaaldin M.; Goldsmith, Edie C.; Baxter, Sarah C.
Accounts of Chemical Research (2008), 41 (12), 1721-1730CODEN: ACHRE4; ISSN:0001-4842. (American Chemical Society)
A review. Gold, enigmatically represented by the target-like design of its ancient alchem. symbol, has been considered a mystical material of great value for centuries. Nanoscale particles of gold now command a great deal of attention for biomedical applications. Depending on their size, shape, degree of aggregation, and local environment, gold nanoparticles can appear red, blue, or other colors. These visible colors reflect the underlying coherent oscillations of conduction-band electrons ("plasmons") upon irradn. with light of appropriate wavelengths. These plasmons underlie the intense absorption and elastic scattering of light, which in turn forms the basis for many biol. sensing and imaging applications of gold nanoparticles. The brilliant elastic light-scattering properties of gold nanoparticles are sufficient to detect individual nanoparticles in a visible light microscope with ∼102 nm spatial resoln. Despite the great excitement about the potential uses of gold nanoparticles for medical diagnostics, as tracers, and for other biol. applications, researchers are increasingly aware that potential nanoparticle toxicity must be investigated before any in vivo applications of gold nanoparticles can move forward. In this Account, we illustrate the importance of surface chem. and cell type for interpretation of nanoparticle cytotoxicity studies. We also describe a relatively unusual live cell application with gold nanorods. The light-scattering properties of gold nanoparticles, as imaged in dark-field optical microscopy, can be used to infer their positions in a living cell construct. Using this positional information, we can quant. measure the deformational mech. fields assocd. with living cells as they push and pull on their local environment. The local mech. environment experienced by cells is part of a complex feedback loop that influences cell metab., gene expression, and migration.
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Joshi, G. K.; Blodgett, K. N.; Muhoberac, B. B.; Johnson, M. A.; Smith, K. A.; Sardar, R. Nano Lett. 2014, 14, 532– 540
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50
Preis, M.; Gardner, T. B.; Gordon, S. R.; Pipas, J. M.; Mackenzie, T. A.; Klein, E. E.; Longnecker, D. S.; Gutmann, E. J.; Sempere, L. F.; Korc, M. Clin. Cancer Res. 2011, 17, 5812– 5821
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MicroRNA-10b Expression Correlates with Response to Neoadjuvant Therapy and Survival in Pancreatic Ductal Adenocarcinoma
Preis, Meir; Gardner, Timothy B.; Gordon, Stuart R.; Pipas, J. Marc; MacKenzie, Todd A.; Klein, Erin E.; Longnecker, Daniel S.; Gutmann, Edward J.; Sempere, Lorenzo F.; Korc, Murray
Clinical Cancer Research (2011), 17 (17), 5812-5821CODEN: CCREF4; ISSN:1078-0432. (American Association for Cancer Research)
INTRODUCTION: Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy. Diagnosis and management of PDAC are hampered by the absence of sensitive and specific disease biomarkers. MicroRNAs (miRNA) are noncoding regulatory RNAs involved in initiation and progression of human cancers. In this study, we sought to det. whether miR-10b could serve as a biomarker for PDAC. Exptl. Design: miRNA expression was characterized by fluorescence-based in situ hybridization using locked nucleic acid-modified DNA probes against miR-10b, miR-21, miR-155, miR-196a, and miR-210, followed by codetection of proteins by immunohistochem. on the same tissue sections. miRNA expression in surgically resected PDAC tissues and in endoscopic ultrasonog. (EUS)-guided fine-needle aspirate (EUS-FNA) samples was analyzed in cytokeratin 19 (CK19)-pos. epithelial cells using optical intensity anal. RESULTS: In 10 resected PDAC samples, miR-10b was the most frequently and consistently overexpressed miRNA among characterized miRNAs, exhibiting a four-fold increase in the cancer cells (P = 0.012). Given this preferential overexpression of miR-10b, we sought to det. whether miR-10b expression was clin. relevant. Accordingly, miR-10b expression was examd. in 106 EUS-FNA samples obtained from pancreatic lesions. miR-10b expression was increased in cancer cells compared with CK19-pos. epithelial cells in benign lesions (P = 0.0001). In patients with PDACs, lower levels of miR-10b were assocd. with improved response to multimodality neoadjuvant therapy, likelihood of surgical resection, delayed time to metastasis, and increased survival. CONCLUSION: miR-10b is a novel diagnostic biomarker for PDACs when assessing pancreatic lesions. Expression of miR-10b is predictive of response to neoadjuvant therapy and outcome in this disease. Clin Cancer Res; 17(17); 5812-21.
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Sempere, L. F.; Preis, M.; Yezefski, T.; Ouyang, H.; Suriawinata, A. A.; Silahtaroglu, A.; Conejo-Garcia, J. R.; Kauppinen, S.; Wells, W.; Korc, M. Clin. Cancer Res. 2010, 16, 4246– 4255
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Fluorescence-Based Codetection with Protein Markers Reveals Distinct Cellular Compartments for Altered MicroRNA Expression in Solid Tumors
Sempere, Lorenzo F.; Preis, Meir; Yezefski, Todd; Ouyang, Haoxu; Suriawinata, Arief A.; Silahtaroglu, Asli; Conejo-Garcia, Jose R.; Kauppinen, Sakari; Wells, Wendy; Korc, Murray
Clinical Cancer Research (2010), 16 (16), 4246-4255CODEN: CCREF4; ISSN:1078-0432. (American Association for Cancer Research)
PURPOSE: High-throughput profiling expts. have linked altered expression of microRNAs (miRNA) to different types of cancer. Tumor tissues are a heterogeneous mixt. of not only cancer cells, but also supportive and reactive tumor microenvironment elements. To clarify the clin. significance of altered miRNA expression in solid tumors, we developed a sensitive fluorescence-based in situ hybridization (ISH) method to visualize miRNA accumulation within individual cells in formalin-fixed, paraffin-embedded tissue specimens. This ISH method was implemented to be compatible with routine clin. immunohistochem. (IHC) assays to enable the detection of miRNAs and protein markers in the same tissue section for colocalization and functional studies. Exptl. Design: We used this combined ISH/IHC assay to study a subset of cancer-assocd. miRNAs, including miRNAs frequently detected at low (miR-34a and miR-126) and high (miR-21 and miR-155) levels, in a panel of breast, colorectal, lung, pancreas, and prostate carcinomas. RESULTS: Despite the distinct histopathol. alterations of each particular cancer type, general trends emerged that pinpointed distinct source cells of altered miRNA expression. Although altered expressions of miR-21 and miR-34a were manifested within cancer cells, those of miR-126 and miR-155 were predominantly confined to endothelial cells and immune cells, resp. These results suggest a heterogeneous participation of miRNAs in carcinogenesis by intrinsically affecting cancer cell biol. or by modulating stromal, vascular, and immune responses. CONCLUSIONS: We described a rapid and sensitive multicolor ISH/IHC assay and showed that it could be broadly applied as an investigational tool to better understand the etiol. relevance of altered miRNA expression in cancer.
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International Journal of Cancer (2010), 126 (1), 73-80CODEN: IJCNAW; ISSN:0020-7136. (Wiley-Liss, Inc.)
Pancreatic cancer is the eighth most common cancer and has an overall 5-yr survival rate lower than 10%. Because of their ability to regulate gene expression, microRNAs can act as oncogenes or tumor-suppressor genes and so have garnered interest as possible prognostic and therapeutic markers during the last decade. However, the prognostic value of microRNA expression in pancreatic cancer has not been thoroughly investigated. We measured the levels of miR-155, miR-203, miR-210, miR-216, miR-217 and miR-222 by quant. RT-PCR in a cohort of 56 microdissected pancreatic ductal adenocarcinomas (PDAC). These microRNAs were chosen as they had previously been shown to be differentially expressed in pancreatic tumors compared to normal tissues. The possible assocn. of microRNA expression and patients' survival was examd. using multivariate Cox's regression hazard analyses. Interestingly, significant correlations between elevated microRNA expression and overall survival were obsd. for miR-155 (RR = 2.50; p = 0.005), miR-203 (RR = 2.21; p = 0.017), miR-210 (RR = 2.48; p = 0.005) and miR-222 (RR = 2.05; p = 0.035). Furthermore, tumors from patients demonstrating elevated expression levels of all 4 microRNAs possessed a 6.2-fold increased risk of tumor-related death compared to patients whose tumors showed a lower expression of these microRNAs. This study provides the first evidence for an oncogenic activity of miR-155, miR-203, miR-210 and miR-222 in the development of pancreatic cancer as has been reported for other tumor types. Furthermore, the putative target genes for these microRNAs suggest a complex signaling network that can affect PDAC tumorigenesis and tumor progression.
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Annual Review of Analytical Chemistry (2008), 1 (), 601-626CODEN: ARACFU; ISSN:1936-1327. (Annual Reviews Inc.)
A review. The ability to control the size, shape, and material of a surface has reinvigorated the field of surface-enhanced Raman spectroscopy (SERS). Because excitation of the localized surface plasmon resonance of a nanostructured surface or nanoparticle lies at the heart of SERS, the ability to reliably control the surface characteristics has taken SERS from an interesting surface phenomenon to a rapidly developing anal. tool. This article first explains many fundamental features of SERS and then describes the use of nanosphere lithog. for the fabrication of highly reproducible and robust SERS substrates. In particular, we review metal film over nanosphere surfaces as excellent candidates for several expts. that were once impossible with more primitive SERS substrates (e.g., metal island films). The article also describes progress in applying SERS to the detection of chem. warfare agents and several biol. mols.
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A review. The electromagnetic theory of surface-enhanced Raman spectroscopy (SERS), despite its simplicity, can account for all major SERS observations, including: the need for a nanostructured material as the SERS-active system; the observation that some metals form good SERS-active systems while others do not; the observation that strongly interacting metal nanoparticles result in very much more effective SERS-active systems; the obsd. polarization sensitivity shown by nanoparticle aggregates; and the optical behavior of nanostructured metals in the absence of a mol. adsorbate. By extending the ideas inherent in the electromagnetic model one can also understand the seminal features reported for single-mol. SERS, including the puzzling observation that only a few Ag particles in an ensemble are hot (they are appropriately structured nanoparticle clusters) and that for a hot particle, once one is able to observe SERS, adding more adsorbate does not significantly alter the intensity (once the electromagnetic hot spot is occupied, adding adsorbate to other sites on the nanoparticle cluster will not add greatly to the obsd. intensity). However, the electromagnetic model does not account for all that is learned through SERS. Mol. resonances, charge-transfer transitions and other processes such as ballistic electrons transiently probing the region where the mol. resides and then modulating electronic processes of the metal as a result certainly contribute to the rich information SERS reports; and by virtue of the fact that these contributions will vary from mol. to mol., they will constitute the most interesting aspects reported by SERS. But, the overall reason why SERS produces such inordinate enhancements is largely an electromagnetic property of nanostructures.
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Singh, Anant Kumar; Khan, Sadia Afrin; Fan, Zhen; Demeritte, Teresa; Senapati, Dulal; Kanchanapally, Rajashekhar; Ray, Paresh Chandra
Journal of the American Chemical Society (2012), 134 (20), 8662-8669CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
Optical-ruler-based distance measurements are essential for tracking biomol. processes in a wide range of anal. biochem. applications. The normally used Forster resonance energy transfer (FRET) ruler is not useful for studying distance-dependent properties when distances are >10 nm. Driven by this limitation, the authors have developed a long-range surface-enhanced Raman spectroscopy (SERS) optical ruler using oval-shaped gold nanoparticles and Rh6G dye-modified rigid, variable-length double-strand DNAs. The bifunctional rigid dsDNA mol. serves as the SERS-active ruler. The authors' exptl. results show that one can tune the length of the SERS ruler between 8 and ∼18 nm by choosing the size of the oval-shaped gold nanoparticles. A possible mechanism for the authors' obsd. distance-dependent SERS phenomenon is discussed using the Gersten and Nitzan model. Ultimately, the authors' long-range SERS mol. rulers can be an important step toward understanding distance-dependent biol. processes.
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Surface-enhanced Raman scattering on tunable plasmonic nanoparticle substrates
Jackson, J. B.; Halas, N. J.
Proceedings of the National Academy of Sciences of the United States of America (2004), 101 (52), 17930-17935CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
Au and Ag nanoshells are studied as substrates for surface-enhanced Raman scattering (SERS). SERS enhancements on nanoshell films are dramatically different from those obsd. on colloidal aggregates, specifically that the Raman enhancement follows the plasmon resonance of the individual nanoparticles. Comparative finite difference time domain calcns. of fields at the surface of smooth and roughened nanoshells reveal that surface roughness contributes only slightly to the total enhancement. SERS enhancements as large as 2.5 × 1010 on Ag nanoshell films for the nonresonant mol. p-mercaptoaniline are measured.
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Heterogeneous catalysts were pivotal to the development of the modern chem. industry and are essential for catalyzing many industrial reactions. However, reaction rates are different for every individual catalyst particle and depend upon each particle's morphol. and size, crystal structure and compn. Measuring the rates of reaction on single nanocrystals will enable the role of catalyst structure to be quantified. Here, using surface plasmon spectroscopy, the authors have directly obsd. the kinetics of at. deposition onto a single gold nanocrystal and also monitored electron injection and extn. during a redox reaction involving the oxidn. of ascorbic acid on a gold nanocrystal surface. These results constitute the first direct measurement of the rates of redox catalysis on single nanocrystals.
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Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method
Livak, Kenneth J.; Schmittgen, Thomas D.
Methods (San Diego, CA, United States) (2001), 25 (4), 402-408CODEN: MTHDE9; ISSN:1046-2023. (Academic Press)
The two most commonly used methods to analyze data from real-time, quant. PCR expts. are abs. quantification and relative quantification. Abs. quantification dets. the input copy no., usually by relating the PCR signal to a std. curve. Relative quantification relates the PCR signal of the target transcript in a treatment group to that of another sample such as an untreated control. The 2-ΔΔCT method is a convenient way to analyze the relative changes in gene expression from real-time quant. PCR expts. The purpose of this report is to present the derivation, assumptions, and applications of the 2-ΔΔCT method. In addn., we present the derivation and applications of two variations of the 2-ΔΔCT method that may be useful in the anal. of real-time, quant. PCR data. (c) 2001 Academic Press.
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Abstract
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Figure 1
Figure 1. Design of plasmonic biosensors and detecting miR-X in various physiological media. (a) Chemically synthesized and freshly prepared gold nanoprisms were covalently attached onto a 3-mercaptopropyltriethoxysilane-functionalized glass coverslip (substrate). (b) Surface of gold nanoprisms was chemically modified with a 1.0 μM 1:1 mixture of SH-C6-ssDNA-X and PEG₆-SH in PBS buffer (pH 7.4) to prepare the plasmonic biosensor. (c) Incubation of sensor in miR-X solution and formation of DNA duplex. (d) Schematic of the extinction spectrum of the biosensor collected in PBS buffer after modification with a 1.0 μM 1:1 mixture of SH-C6-ssDNA-X and PEG₆-SH (blue curve). The extinction spectrum was again collected after incubation in miR-X solution and careful rinsing with PBS buffer to determine the new peak position (red curve). The extent of LSPR dipole peak shift (Δλ_LSPR) depends on the concentration of miR-X used during the incubation in (c), which ranged from 100 nM to 50 fM. (e) Plot of Δλ_LSPR versus log of miR-X concentrations used to determine the limit of detection. The image is not to scale.
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Figure 2
Figure 2. MicroRNA detection using label-free plasmonic biosensors. (a) Monitoring LSPR dipole peak (λ_LSPR) changes by UV–visible absorption spectra of gold nanoprisms during various functionalization steps: before (black, λ_LSPR = 800 nm) and after functionalized with a 1 μM/1 μM ratio of HS-C6-ssDNA-21/PEG₆-SH (red, λ_LSPR = 821 nm), and after incubation with 100 nM miR-21 solution in PBS buffer (blue, λ_LSPR = 839 nm). (b) average λ_LSPR peak shift (Δλ_LSPR) of gold nanoprisms functionalized with a 1 μM/1 μM ratio of HS-C6-ssDNA-21/PEG₆-SH after incubation in different concentrations of miR-21 in PBS buffer (red triangles), 40% human plasma (black squares), and 40% bovine plasma (blue diamonds). The Δλ_LSPR were calculated by taking the difference between the λ_LSPR peak position of the plasmonic biosensor after and before the hybridization with miR-21 in the various media. (c) Average Δλ_LSPR of the plasmonic biosensor functionalized with a 1 μM/1 μM ratio of HS-C6-ssDNA-10b/PEG₆-SH after hybridization with different miR-10b concentrations in PBS buffer (red triangles), in 40% human plasma (black squares), and in 40% bovine plasma (blue diamonds). All extinction spectra recorded after miR-X incubation were measured in PBS buffer after rinsing with PBS buffer.
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Figure 3
Figure 3. Determining the optimum detection condition. (a) UV–visible extinction spectra monitoring the LSPR dipole peak (λ_LSPR) of gold nanoprisms attached to silanized glass substrate before (black, λ_LSPR = 796 nm) and after (red, λ_LSPR = 811 nm) functionalization with 1 μM of HS-C6-ssDNA-21 without PEG₆-SH spacers and after incubation in 100 nM miR-21 solution in 40% human plasma (blue, λ_LSPR = 822 nm). (b) Average Δλ_LSPR of these HS-C6-ssDNA-21 functionalized gold nanoprisms upon hybridization with different miR-21 concentrations in 40% human plasma. The Δλ_LSPR were calculated by taking the difference between the λ_LSPR peak position of the nanoprisms after and before the incubation with miR-21 in PBS buffer.
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Figure 4
Figure 4. Characterization of the plasmonic biosensors regeneration ability. (a) UV–visible extinction spectra of gold nanoprisms functionalized with a 1 μM/1 μM ratio of HS-C6-ssDNA-21/PEG₆-SH (red, λ_LSPR = 818 nm) attached to silanized glass, after incubation with 100 nM of miR-21 (blue, λ_LSPR = 832 nm), after treatment with 15 units of RNase H for 2 h (red dotted, λ_LSPR = 818 nm), and after rehybridized with 100 nM of miR-21 (blue dotted, λ_LSPR = 832 nm). (b) Changes in LSPR dipole peak position (λ_LSPR) of gold nanoprisms functionalized with a 1 μM/1 μM ratio of HS-C6-ssDNA-21/PEG₆-SH upon hybridization and dehybridization with miR-21 for several cycles. The λ_LSPR peak shifts back and forth upon sensor regeneration with RNase H by cleaving DNA/RNA duplex and rehybridization after incubation into 100 nM miR-21 in 40% human plasma. After each of the dehybridization steps, the plasmonic biosensors were thoroughly rinsed with PBS buffer to completely remove enzyme RNase H.
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Figure 5
Figure 5. Surface characterization of the plasmonic biosensors. Atomic force microscopy images of gold nanoprisms bound to silanized glass substrate (a) after functionalization with 1:1 ratio of HS-C6-ssDNA-21/PEG₆-SH (b) and after hybridization with100 nM miR-21 in 40% human plasma (c). The measurements were conducted in air. (d) Changes in the surface area of gold nanoprisms after each functionalization steps. Forty nanoprisms were selected to determine the average change in the surface area. Detailed method of surface area calculation is provided in the Supporting Information file.
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Figure 6
Figure 6. Determination of microRNA concentration in PDAC patients and normal control subjects. (a) The average λ_LSPR peak shifts of gold nanoprisms functionalized with a 1:1 ratio of HS-C6-ssDNA-21/PEG₆-SH upon hybridization with miR-21 from the total RNAs extracted from plasma samples of PDAC patients (blue diamonds) and normal control subjects (blue squares). The respective λ_LSPR peak shifts were converted to concentrations using the calibration curve established for miR-21 in PBS buffer as shown in Figure 2b [PDAC patients (red triangles), and for normal control subjects (red circles)]. (b) Comparison of miR-21 concentration for six PDAC patients determined using plasmonic biosensors (blue diamond) and qRT-PCR (red square). (c) Similar experiments were conducted to detect miR-10b where the λ_LSPR peak shifts and concentrations for PDAC patients are shown in blue diamonds and red triangles, respectively. The λ_LSPR peak shifts (blue squares) and concentrations (red circles) for normal controls are shown for comparison. (d) The average λ_LSPR peak shifts (blue diamonds) and concentration (red triangles) for the miR-21 in plasma samples from PDAC patients without any purification.
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  Esquela-Kerscher, Aurora; Slack, Frank J.
  Nature Reviews Cancer (2006), 6 (4), 259-269CODEN: NRCAC4; ISSN:1474-175X. (Nature Publishing Group)
  A review. MicroRNAs (miRNAs) are an abundant class of small non-protein-coding RNAs that function as neg. gene regulators. They regulate diverse biol. processes, and bioinformatic data indicates that each miRNA can control hundreds of gene targets, underscoring the potential influence of miRNAs on almost every genetic pathway. Recent evidence has shown that miRNA mutations or mis-expression correlate with various human cancers and indicates that miRNAs can function as tumor suppressors and oncogenes. MiRNAs have been shown to repress the expression of important cancer-related genes and might prove useful in the diagnosis and treatment of cancer.
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5. 5
  He, L.; Thomson, J. M.; Hemann, M. T.; Hernando-Monge, E.; Mu, D.; Goodson, S.; Powers, S.; Cordon-Cardo, C.; Lowe, S. W.; Hannon, G. J.; Hammond, S. M. Nature 2005, 435, 828– 833
  5
  A microRNA polycistron as a potential human oncogene
  He, Lin; Thomson, J. Michael; Hemann, Michael T.; Hernando-Monge, Eva; Mu, David; Goodson, Summer; Powers, Scott; Cordon-Cardo, Carlos; Lowe, Scott W.; Hannon, Gregory J.; Hammond, Scott M.
  Nature (London, United Kingdom) (2005), 435 (7043), 828-833CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
  To date, more than 200 microRNAs have been described in humans; however, the precise functions of these regulatory, non-coding RNAs remains largely obscure. One cluster of microRNAs, the mir-17-92 polycistron, is located in a region of DNA that is amplified in human B-cell lymphomas. Here we compared B-cell lymphoma samples and cell lines to normal tissues, and found that the levels of the primary or mature microRNAs derived from the mir-17-92 locus are often substantially increased in these cancers. Enforced expression of the mir-17-92 cluster acted with c-myc expression to accelerate tumor development in a mouse B-cell lymphoma model. Tumors derived from haematopoietic stem cells expressing a subset of the mir-17-92 cluster and c-myc could be distinguished by an absence of apoptosis that was otherwise prevalent in c-myc-induced lymphomas. Together, these studies indicate that non-coding RNAs, specifically microRNAs, can modulate tumor formation, and implicate the mir-17-92 cluster as a potential human oncogene.
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6. 6
  Szafranska, A. E.; Doleshal, M.; Edmunds, H. S.; Gordon, S.; Luttges, J.; Munding, J. B.; Barth, R. J.; Gutmann, E. J.; Suriawinata, A. A.; Marc Pipas, J.; Tannapfel, A.; Korc, M.; Hahn, S. A.; Labourier, E.; Tsongalis, G. J. Clin. Chem. 2008, 54, 1716– 1724
  6
  Analysis of microRNAs in pancreatic fine-needle aspirates can classify benign and malignant tissues
  Szafranska, Anna E.; Doleshal, Martina; Edmunds, Hayward S.; Gordon, Stuart; Luttges, Jutta; Munding, Johanna B.; Barth, Richard J., Jr.; Gutmann, Edward J.; Suriawinata, Arief A.; Pipas, J. Marc; Tannapfel, Andrea; Korc, Murray; Hahn, Stephan A.; Labourier, Emmanuel; Tsongalis, Gregory J.
  Clinical Chemistry (Washington, DC, United States) (2008), 54 (10), 1716-1724CODEN: CLCHAU; ISSN:0009-9147. (American Association for Clinical Chemistry)
  MicroRNAs (miRNAs) are RNA mols. that are involved in the regulation of many cellular processes, including those related to human cancers. The aim of this study was to det., as a proof of principle, whether specific candidate miRNAs could be detected in fine-needle aspirate (FNA) biopsies of pancreatic ductal adenocarcinoma (PDAC) and could accurately differentiate malignant from benign pancreatic tissues. We used TaqMan assays to quantify miRNA levels in FNA samples collected in RNARetain (n = 16) and compared the results with a training set consisting of frozen macrodissected pancreatic samples (n = 20). Quant. reverse-transcription PCR anal. confirmed that miRNA levels are affected in PDAC FNAs and correlate well with the changes obsd. in the training set of frozen pancreatic samples. Anal. of the amts. produced for a few specific miRNAs enabled identification of PDAC samples. The combination of miR-196a and miR-217 biomarkers further improved the ability to distinguish between healthy tissue, PDAC, and chronic pancreatitis in the training set (P = 8.2 × 10-10), as well as segregate PDAC FNA samples from other FNA samples (P = 1.1 × 10-5). Furthermore, we showed that miR-196a prodn. is likely specific to PDAC cells and that its incidence paralleled the progression of PDAC. To the best of our knowledge, this study is the first to evaluate the diagnostic potential of miRNAs in a clin. setting and has shown that miRNA anal. of pancreatic FNA biopsy samples can aid in the pathol. evaluation of suspicious cases and may provide a new strategy for improving the diagnosis of pancreatic diseases.
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7. 7
  Setoyama, T.; Zhang, X.; Natsugoe, S.; Calin, G. A. Clin. Cancer Res. 2011, 17, 5527– 5529
  7
  microRNA-10b: A New Marker or the Marker of Pancreatic Ductal Adenocarcinoma?
  Setoyama, Tetsuro; Zhang, Xinna; Natsugoe, Shoji; Calin, George A.
  Clinical Cancer Research (2011), 17 (17), 5527-5529CODEN: CCREF4; ISSN:1078-0432. (American Association for Cancer Research)
  A review. MicroRNA-10b (miR-10b) expression in pancreatic ductal adenocarcinoma (PDAC), as identified by in situ hybridization, is highly correlated with cancer diagnosis, therapy response, and prognosis. If these findings are further confirmed in prospective studies, miR-10b could be used to improve the management of PDAC and decrease the mortality rate of this deadly cancer. Clin Cancer Res; 17(17); 5527-9.
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8. 8
  Ouyang, H.; Gore, J.; Deitz, S.; Korc, M. Oncogene 2014, 33, 4664– 4674
  There is no corresponding record for this reference.
9. 9
  Fang, S.; Lee, H. J.; Wark, A. W.; Corn, R. M. J. Am. Chem. Soc. 2006, 128, 14044– 14046
  9
  Attomole Microarray Detection of MicroRNAs by Nanoparticle-Amplified SPR Imaging Measurements of Surface Polyadenylation Reactions
  Fang, Shiping; Lee, Hye Jin; Wark, Alastair W.; Corn, Robert M.
  Journal of the American Chemical Society (2006), 128 (43), 14044-14046CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
  Multiple microRNAs (miRNAs) are detected in a microarray format using a novel approach that combines a surface enzyme reaction with nanoparticle-amplified SPR imaging (SPRI). The surface reaction of poly(A) polymerase creates poly(A) tails on miRNAs hybridized onto locked nucleic acid (LNA) microarrays. DNA-modified nanoparticles are then adsorbed onto the poly(A) tails and detected with SPRI. This ultrasensitive nanoparticle-amplified SPRI methodol. can be used for miRNA profiling at attomole levels.
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10. 10
  Li, J.; Yao, B.; Huang, H.; Wang, Z.; Sun, C.; Fan, Y.; Chang, Q.; Li, S.; Wang, X.; Xi, J. Anal. Chem. 2009, 81, 5446– 5451
  10
  Real-time polymerase chain reaction microRNA detection based on enzymatic stem-loop probes ligation
  Li, Juan; Yao, Bo; Huang, Huang; Wang, Zhao; Sun, Changhong; Fan, Yu; Chang, Qing; Li, Shaolu; Wang, Xiang; Xi, Jianzhong
  Analytical Chemistry (Washington, DC, United States) (2009), 81 (13), 5446-5451CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
  MiRNAs (microRNAs) are a group of endogenous, small noncoding RNA with the length of 18-25 nucleotides, which have recently been demonstrated to play important roles in a wide range of biol. processes. In this work, we developed a simple, sensitive, specific, and inexpensive assay through the combination of enzymic probe ligation and real-time PCR amplification for the measurement of mature miRNAs. A couple of novel DNA probes with a stem-loop structure were implemented to reduce nonspecific ligation by at least 100-fold. The assay has several remarkable features including wide dynamic range, low total RNA input (0.02-0.2 ng), distinct anti-interference from precursor miRNAs (signal-to-noise ratio > 500), and single-base mismatch discrimination among miRNA sequences. In addn., a one-tube assay could be accomplished by designing a couple of universal probes, which makes it feasible to examine the expression of a whole family of miRNA (such as let-7) at one time. Finally, we validated the method for quantifying the expression of four mature miRNAs including miR-122, miR-1, miR-34a, and let-7a across 10 mouse tissues, where U6 snRNA could be simultaneously examd. as an endogenous control. Thus, this method revealed a great potential for miRNA quantitation in ordinary lab. studies and clin. diagnoses.
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11. 11
  Chen, C.; Ridzon, D. A.; Broomer, A. J.; Zhou, Z.; Lee, D. H.; Nguyen, J. T.; Barbisin, M.; Xu, N. L.; Mahuvakar, V. R.; Andersen, M. R.; Lao, K. Q.; Livak, K. J.; Guegler, K. J. Nucleic Acids Res. 2005, 33, e179
  11
  Real-time quantification of microRNAs by stem-loop RT-PCR
  Chen, Caifu; Ridzon, Dana A.; Broomer, Adam J.; Zhou, Zhaohui; Lee, Danny H.; Nguyen, Julie T.; Barbisin, Maura; Xu, Nan Lan; Mahuvakar, Vikram R.; Andersen, Mark R.; Lao, Kai Qin; Livak, Kenneth J.; Guegler, Karl J.
  Nucleic Acids Research (2005), 33 (20), e179/1-e179/9CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)
  A novel microRNA (miRNA) quantification method has been developed using stem-loop RT followed by TaqMan PCR anal. Stem-loop RT primers are better than conventional ones in terms of RT efficiency and specificity. TaqMan miRNA assays are specific for mature miRNAs and discriminate among related miRNAs that differ by as little as one nucleotide. Furthermore, they are not affected by genomic DNA contamination. Precise quantification is achieved routinely with as little as 25 pg of total RNA for most miRNAs. In fact, the high sensitivity, specificity and precision of this method allows for direct anal. of a single cell without nucleic acid purifn. Like std. TaqMan gene expression assays, TaqMan miRNA assays exhibit a dynamic range of seven orders of magnitude. Quantification of five miRNAs in seven mouse tissues showed variation from less than 10 to more than 30,000 copies per cell. This method enables fast, accurate and sensitive miRNA expression profiling and can identify and monitor potential biomarkers specific to tissues or diseases. Stem-loop RT-PCR can be used for the quantification of other small RNA mols. such as short interfering RNAs (siRNAs). Furthermore, the concept of stem-loop RT primer design could be applied in small RNA cloning and multiplex assays for better specificity and efficiency.
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12. 12
  Johnson, B. N.; Mutharasan, R. Anal. Chem. 2012, 84, 10426– 10436
  There is no corresponding record for this reference.
13. 13
  Ramnani, P.; Gao, Y.; Ozsoz, M.; Mulchandani, A. Anal. Chem. 2013, 85, 8061– 8064
  There is no corresponding record for this reference.
14. 14
  Cissell, K. A.; Rahimi, Y.; Shrestha, S.; Hunt, E. A.; Deo, S. K. Anal. Chem. 2008, 80, 2319– 2325
  14
  Bioluminescence-Based Detection of MicroRNA, miR21 in Breast Cancer Cells
  Cissell, Kyle A.; Rahimi, Yasmeen; Shrestha, Suresh; Hunt, Eric A.; Deo, Sapna K.
  Analytical Chemistry (Washington, DC, United States) (2008), 80 (7), 2319-2325CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
  A hybridization assay for the detection of microRNA, miR21 in cancer cells using the bioluminescent enzyme Renilla luciferase (Rluc) as a label, has been developed. MicroRNAs are small RNAs found in plants, animals, and humans that perform key functions in gene silencing and affect early-stage cell development, cell differentiation, and cell death. MiRNAs are considered useful early diagnostic and prognostic markers of cancer, candidates for therapeutic intervention, and targets for basic biomedical research. However, methods for highly sensitive and rapid detection of miRNA directly from samples such as cells that can serve as a suitable diagnostics platform are lacking. In that regard, the utilization of the bioluminescent label, Rluc, that offers the advantage of high signal-to-noise ratio, allows for the development of highly sensitive assays for the detn. of miRNA in a variety of matrixes. In this paper, we have described the development of a competitive oligonucleotide hybridization assay for the detection of miR21 using the free miR21 and Rluc-labeled miR21 that competes to bind to an immobilized miR21 complementary probe. The miR21 microRNA chosen for this study is of biomedical significance because its levels are elevated in a variety of cancers. Using the optimized assay, a detection limit of 1 fmol was obtained. The assay was employed for the detection of miR21 in human breast adenocarcinoma MCF-7 cells and nontumorigenic epithelial MCF-10A cells. The comparison of miR21 expression level in two cell lines demonstrated higher expression of miR21 in breast cancer cell line MCF-7 compared to the nontumorigenic MCF-10A cells. Further, using the assay developed, the miR21 quantification could be performed directly in cell exts. The hybridization assay was developed in a microplate format with a total assay time of 1.5 h and without the need for sample PCR amplification. The need for early mol. markers and their detection methods in cancer diagnosis is tremendous. The characteristics of the assay developed in this work show its suitability for early cancer diagnosis based on miRNA as a biomarker.
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15. 15
  Qavi, A. J.; Bailey, R. C. Angew. Chem., Int. Ed. 2010, 49, 4608– 4611
  15
  Multiplexed detection and label-free quantitation of microRNAs using arrays of silicon photonic microring resonators
  Qavi, Abraham J.; Bailey, Ryan C.
  Angewandte Chemie, International Edition (2010), 49 (27), 4608-4611, S4608/1-S4608/21CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
  Herein is reported a label-free direct hybridization assay enabling the simultaneous detection of multiple different miRNAs from a single sample using com. fabricated and modularly multiplexable arrays of silicon photonic microring resonators. Using ssDNA capture probes, the authors were able to rapidly quantitate down to approx. 150 fmol of miRNA and were able to discriminate between single nucleotide polymorphisms within the biol. important let-7 family of miRNAs. The authors also demonstrated the applicability of this platform for quant. multiplexed expression profiling by detg. the concn. of four miRNAs from within a clin. relevant sample size of a cell line model of glioblastoma with minimal sample prepn.
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16. 16
  Wang, Y.; Zheng, D.; Tan, Q.; Wang, M. X.; Gu, L.-Q. Nat. Nanotechnol. 2011, 6, 668– 674
  16
  Nanopore-based detection of circulating microRNAs in lung cancer patients
  Wang, Yong; Zheng, Da-Li; Tan, Qiu-Lin; Wang, Michael X.; Gu, Li-Qun
  Nature Nanotechnology (2011), 6 (10), 668-674CODEN: NNAABX; ISSN:1748-3387. (Nature Publishing Group)
  MicroRNAs are short RNA mols. that regulate gene expression, and have been investigated as potential biomarkers because their expression levels are correlated with various diseases. However, detecting microRNAs in the bloodstream remains difficult because current methods are not sufficiently selective or sensitive. Here, we show that a nanopore sensor based on the α-haemolysin protein can selectively detect microRNAs at the single mol. level in plasma samples from lung cancer patients without the need for labels or amplification of the microRNA. The sensor, which uses a programmable oligonucleotide probe to generate a target-specific signature signal, can quantify subpicomolar levels of cancer-assocd. microRNAs and can distinguish single-nucleotide differences between microRNA family members. This approach is potentially useful for quant. microRNA detection, the discovery of disease markers and non-invasive early diagnosis of cancer.
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17. 17
  Wanunu, M.; Dadosh, T.; Ray, V.; Jin, J.; McReynolds, L.; Drndic, M. Nat. Nanotechnol. 2010, 5, 807– 814
  17
  Rapid electronic detection of probe-specific microRNAs using thin nanopore sensors
  Wanunu, Meni; Dadosh, Tali; Ray, Vishva; Jin, Jingmin; McReynolds, Larry; Drndic, Marija
  Nature Nanotechnology (2010), 5 (11), 807-814CODEN: NNAABX; ISSN:1748-3387. (Nature Publishing Group)
  Small RNA mols. have an important role in gene regulation and RNA silencing therapy, but it is challenging to detect these mols. without the use of time-consuming radioactive labeling assays or error-prone amplification methods. Here, we present a platform for the rapid electronic detection of probe-hybridized microRNAs from cellular RNA. In this platform, a target microRNA is first hybridized to a probe. This probe:microRNA duplex is then enriched through binding to the viral protein p19. Finally, the abundance of the duplex is quantified using a nanopore. Reducing the thickness of the membrane contg. the nanopore to 6 nm leads to increased signal amplitudes from biomols., and reducing the diam. of the nanopore to 3 nm allows the detection and discrimination of small nucleic acids based on differences in their phys. dimensions. We demonstrate the potential of this approach by detecting picogram levels of a liver-specific miRNA from rat liver RNA.
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18. 18
  Lee, H.; Park, J.-E.; Nam, J.-M. Nat. Commun. 2014, 5, 1– 7
  There is no corresponding record for this reference.
19. 19
  Jain, P. K.; Huang, X.; El-Sayed, I. H.; El-Sayed, M. A. Acc. Chem. Res. 2008, 41, 1578– 1586
  19
  Noble Metals on the Nanoscale: Optical and Photothermal Properties and Some Applications in Imaging, Sensing, Biology, and Medicine
  Jain, Prashant K.; Huang, Xiaohua; El-Sayed, Ivan H.; El-Sayed, Mostafa A.
  Accounts of Chemical Research (2008), 41 (12), 1578-1586CODEN: ACHRE4; ISSN:0001-4842. (American Chemical Society)
  A review. Noble metal nanostructures attract much interest because of their unique properties, including large optical field enhancements resulting in the strong scattering and absorption of light. The enhancement in the optical and photothermal properties of noble metal nanoparticles arises from resonant oscillation of their free electrons in the presence of light, also known as localized surface plasmon resonance (LSPR). The plasmon resonance can either radiate light (Mie scattering), a process that finds great utility in optical and imaging fields, or be rapidly converted to heat (absorption); the latter mechanism of dissipation has opened up applications in several new areas. The ability to integrate metal nanoparticles into biol. systems has had greatest impact in biol. and biomedicine. In this Account, the authors discuss the plasmonic properties of gold and silver nanostructures and present examples of how they are being utilized for biodiagnostics, biophys. studies, and medical therapy. For instance, taking advantage of the strong LSPR scattering of gold nanoparticles conjugated with specific targeting mols. allows the mol.-specific imaging and diagnosis of diseases such as cancer. The authors emphasize in particular how the unique tunability of the plasmon resonance properties of metal nanoparticles through variation of their size, shape, compn., and medium allows chemists to design nanostructures geared for specific bio-applications. The authors discuss some interesting nanostructure geometries, including nanorods, nanoshells, and nanoparticle pairs, that exhibit dramatically enhanced and tunable plasmon resonances, making them highly suitable for bio-applications. Tuning the nanostructure shape (e.g., nanoprisms, nanorods, or nanoshells) is another means of enhancing the sensitivity of the LSPR to the nanoparticle environment and, thereby, designing effective biosensing agents. Metal nanoparticle pairs or assemblies display distance-dependent plasmon resonances as a result of field coupling. A universal scaling model, relating the plasmon resonance frequency to the interparticle distance in terms of the particle size, becomes potentially useful for measuring nanoscale distances (and their changes) in biol. systems. The strong plasmon absorption and photothermal conversion of gold nanoparticles has been exploited in cancer therapy through the selective localized photothermal heating of cancer cells. For nanorods or nanoshells, the LSPR can be tuned to the near-IR region, making it possible to perform in vivo imaging and therapy. The examples of the applications of noble metal nanostructures provided herein can be readily generalized to other areas of biol. and medicine because plasmonic nanomaterials exhibit great range, versatility, and systematic tunability of their optical attributes.
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20. 20
  Halas, N. J.; Lal, S.; Chang, W.-S.; Link, S.; Nordlander, P. Chem. Rev. 2011, 111, 3913– 3961
  20
  Plasmons in Strongly Coupled Metallic Nanostructures
  Halas, Naomi J.; Lal, Surbhi; Chang, Wei-Shun; Link, Stephan; Nordlander, Peter
  Chemical Reviews (Washington, DC, United States) (2011), 111 (6), 3913-3961CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)
  A review examines the unique light-focusing properties of strongly coupled plasmonic systems, properties that resulted in an extraordinary increase in interest in these systems within the chem. community. It describes the concept of plasmon hybridization that takes advantage of the analogy between plasmons and the wave functions of simple quantum systems to provide a simple, intuitive explanation of the properties of complex plasmonic systems. It discusses coupled plasmonic systems where the classical electromagnetic description of coupled plasmons is no longer adequate and a quantum mech. description is necessary to understand their behavior.
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21. 21
  Mayer, K. M.; Hafner, J. H. Chem. Rev. 2011, 111, 3828– 3857
  21
  Localized surface plasmon resonance sensors
  Mayer, Kathryn M.; Hafner, Jason H.
  Chemical Reviews (Washington, DC, United States) (2011), 111 (6), 3828-3857CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)
  A review. Topics include physics of localized surface plasmon resonance (LSPR); sensing with LSPR, effect of particle size, shape, and material; LSPR-based biol. and chem. sensors; technol. advances in LSPR sensing; maximizing the mol. detection sensitivity of LSPR; and future directions like mol. biol. or diagnostic tool application.
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22. 22
  Jin, R.; Cao, Y.; Mirkin, C. A.; Kelly, K. L.; Schatz, G. C.; Zheng, J. G. Science 2001, 294, 1901– 1903
  22
  Photoinduced conversion of silver nanospheres to nanoprisms
  Jin, Rongchao; Cao, YunWei; Mirkin, Chad A.; Kelly, K. L.; Schatz, George C.; Zheng, J. G.
  Science (Washington, DC, United States) (2001), 294 (5548), 1901-1903CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
  A photoinduced method for converting large quantities of Ag nanospheres into triangular nanoprisms is reported. The photo-process was characterized by time-dependent UV-visible spectroscopy and TEM, allowing for the observation of several key intermediates in and characteristics of the conversion process. This light-driven process results in a colloid with distinctive optical properties that directly relate to the nanoprism shape of the particles. Theor. calcns. coupled with exptl. observations allow for the assignment of the nanoprism plasmon bands and for the 1st identification of 2 distinct quadrupole plasmon resonances for a nanoparticle. Unlike the spherical particles they are derived from that Rayleigh light-scatter in the blue, these nanoprisms exhibit scattering in the red, which could be useful in developing multicolor diagnostic labels on the basis not only of nanoparticle compn. and size but also of shape.
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23. 23
  Sun, Y.; Xia, Y. Science 2002, 298, 2176– 2179
  23
  Shape-Controlled Synthesis of Gold and Silver Nanoparticles
  Sun, Yugang; Xia, Younan
  Science (Washington, DC, United States) (2002), 298 (5601), 2176-2179CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
  Monodisperse samples of silver nanocubes were synthesized in large quantities by reducing silver nitrate with ethylene glycol in the presence of poly(vinyl pyrrolidone) (PVP). These cubes were single crystals and were characterized by a slightly truncated shape bounded by {100}, {110}, and {111} facets. The presence of PVP and its molar ratio (in terms of repeating unit) relative to silver nitrate both played important roles in detg. the geometric shape and size of the product. The silver cubes could serve as sacrificial templates to generate single-cryst. nanoboxes of gold: hollow polyhedra bounded by six {100} and eight {111} facets. Controlling the size, shape, and structure of metal nanoparticles is technol. important because of the strong correlation between these parameters and optical, elec., and catalytic properties.
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24. 24
  Anker, J. N.; Hall, W. P.; Lyandres, O.; Shah, N. C.; Zhao, J.; Van Duyne, R. P. Nat. Mater. 2008, 7, 442– 453
  24
  Biosensing with plasmonic nanosensors
  Anker, Jeffrey N.; Hall, W. Paige; Lyandres, Olga; Shah, Nilam C.; Zhao, Jing; Van Duyne, Richard P.
  Nature Materials (2008), 7 (6), 442-453CODEN: NMAACR; ISSN:1476-1122. (Nature Publishing Group)
  A review. Light incident on metallic nanoparticles can induce a collective motion of electrons that can lead to a strong amplification of the local electromagnetic field. As reviewed here, these plasmonic resonances have important applications in biosensing where they push resoln. and sensitivity towards the single-mol. detection limit. Recent developments have greatly improved the sensitivity of optical sensors based on metal nanoparticle arrays and single nanoparticles. The authors introduce the localized surface plasmon resonance (LSPR) sensor and describe how its exquisite sensitivity to size, shape and environment can be harnessed to detect mol. binding events and changes in mol. conformation. The authors then describe recent progress in three areas representing the most significant challenges: pushing sensitivity towards the single-mol. detection limit, combining LSPR with complementary mol. identification techniques such as surface-enhanced Raman spectroscopy, and practical development of sensors and instrumentation for routine use and high-throughput detection. This review highlights several exceptionally promising research directions and discusses how diverse applications of plasmonic nanoparticles can be integrated in the near future.
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25. 25
  Weiss, P. S. Acc. Chem. Res. 2008, 41, 1772– 1781
  There is no corresponding record for this reference.
26. 26
  Yan, Y.; Chen, J. I. L.; Ginger, D. S. Nano Lett. 2012, 12, 2530– 2536
  26
  Photoswitchable Oligonucleotide-Modified Gold Nanoparticles: Controlling Hybridization Stringency with Photon Dose
  Yan, Yunqi; Chen, Jennifer I. L.; Ginger, David S.
  Nano Letters (2012), 12 (5), 2530-2536CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)
  We describe a new class of stimulus-responsive DNA-functionalized gold nanoparticles that incorporate azobenzene-modified oligonucleotides. Beyond the classic directed assembly and sensing behaviors assocd. with oligonucleotide-modified nanoparticles, these particles also exhibit reversible photoswitching of their assembly behavior. Exposure to UV light induces a trans-cis isomerization of the azobenzene which destabilizes the DNA duplex, resulting in dissocn. of the nanoparticle assemblies. The isomerization is reversible upon exposure to blue light, resulting in rehybridization and reassembly of the DNA-linked nanoparticle clusters. We show that perfectly complementary and partially mismatched strands exhibit clearly distinguishable photoinduced melting properties, and we demonstrate that photon dose can thus be used in place of temp. or ionic strength to control hybridization stringency with the ability to discriminate single-base mismatches.
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27. 27
  Saha, K.; Agasti, S. S.; Kim, C.; Li, X.; Rotello, V. M. Chem. Rev. 2012, 112, 2739– 2779
  27
  Gold nanoparticles in chemical and biological sensing
  Saha, Krishnendu; Agasti, Sarit S.; Kim, Chaekyu; Li, Xiaoning; Rotello, Vincent M.
  Chemical Reviews (Washington, DC, United States) (2012), 112 (5), 2739-2779CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)
  A review. Topics include synthesis ans surface functionalization; phys. properties; colorimetric and fluorimetric sensing; elec. al and electrochem. sensing; SERS; gold nanoparticles in quartz crystal microbalance-based sensing; application of gold nanoparticles in bio-barcode assays.
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28. 28
  Stewart, M. E.; Anderton, C. R.; Thompson, L. B.; Maria, J.; Gray, S. K.; Rogers, J. A.; Nuzzo, R. G. Chem. Rev. 2008, 108, 494– 521
  There is no corresponding record for this reference.
29. 29
  Beeram, S. R.; Zamborini, F. P. J. Am. Chem. Soc. 2009, 131, 11689– 11691
  There is no corresponding record for this reference.
30. 30
  Chen, S.; Svedendahl, M.; Duyne, R. P. V.; Käll, M. Nano Lett. 2011, 11, 1826– 1830
  There is no corresponding record for this reference.
31. 31
  Chen, Y.; Munechika, K.; Ginger, D. S. Nano Lett. 2007, 7, 690– 696
  There is no corresponding record for this reference.
32. 32
  Haes, A. J.; Hall, W. P.; Chang, L.; Klein, W. L.; Van Duyne, R. P. Nano Lett. 2004, 4, 1029– 1034
  There is no corresponding record for this reference.
33. 33
  Stranahan, S. M.; Willets, K. A. Nano Lett. 2010, 10, 3777– 3784
  33
  Super-resolution Optical Imaging of Single-Molecule SERS Hot Spots
  Stranahan, Sarah M.; Willets, Katherine A.
  Nano Letters (2010), 10 (9), 3777-3784CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)
  The authors present the 1st super-resoln. optical images of single-mol. surface-enhanced Raman scattering (SM-SERS) hot spots, using super-resoln. imaging as a powerful new tool for understanding the interaction between single mols. and nanoparticle hot spots. Using point spread function fitting, the authors map the centroid position of SM-SERS with ±10 nm resoln., revealing a spatial relation between the SM-SERS centroid position and the highest SERS intensity. The authors are also able to measure the unique position of the SM-SERS centroid relative to the centroid assocd. with nanoparticle photoluminescence, which allows the authors to speculate on the presence of multiple hot spots within a single diffraction-limited spot. These measurements allow the authors to follow dynamic movement of the SM-SERS centroid position over time as it samples different locations in space and explores regions larger than the expected size of a SM-SERS hot spot. Probably the movement of the SERS centroid is due to diffusion of a single mol. on the surface of the nanoparticle, which leads to changes in coupling between the scattering dipole and the optical near field of the nanoparticle.
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34. 34
  Elghanian, R.; Storhoff, J. J.; Mucic, R. C.; Letsinger, R. L.; Mirkin, C. A. Science 1997, 277, 1078– 1081
  34
  Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles
  Elghanian, Robert; Storhoff, James J.; Mucic, Robert C.; Letsinger, Robert L.; Mirkin, Chad A.
  Science (Washington, D. C.) (1997), 277 (5329), 1078-1080CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
  A highly selective, colorimetric polynucleotide detection method based on mercaptoalkyloligonucleotide-modified gold nanoparticle probes is reported. Introduction of a single-stranded target oligonucleotide (30 bases) into a soln. contg. the appropriate probes resulted in the formation of a polymeric network of nanoparticles with a concomitant red-to-pinkish/purple color change. Hybridization was facilitated by freezing and thawing of the solns., and the denaturation of these hybrid materials showed transition temps. over a narrow range that allowed differentiation of a variety of imperfect targets. Transfer of the hybridization mixt. to a reverse-phase silica plate resulted in a blue color upon drying that could be detected visually. The unoptimized system can detect about 10 fmol in oligonucleotide.
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35. 35
  Willets, K. A.; Van Duyne, R. P. Annu. Rev. Phys. Chem. 2007, 58, 267– 297
  35
  Localized surface plasmon resonance spectroscopy and sensing
  Willets, Katherine A.; Van Duyne, Richard P.
  Annual Review of Physical Chemistry (2007), 58 (), 267-297CODEN: ARPLAP; ISSN:0066-426X. (Annual Reviews Inc.)
  A review. Localized surface plasmon resonance (LSPR) spectroscopy of metallic nanoparticles is a powerful technique for chem. and biol. sensing expts. Also, the LSPR is responsible for the electromagnetic-field enhancement that leads to surface-enhanced Raman scattering (SERS) and other surface-enhanced spectroscopic processes. This review describes recent fundamental spectroscopic studies that reveal key relations governing the LSPR spectral location and its sensitivity to the local environment, including nanoparticle shape and size. The authors also describe studies on the distance dependence of the enhanced electromagnetic field and the relation between the plasmon resonance and the Raman excitation energy. Lastly, the authors introduce a new form of LSPR spectroscopy, involving the coupling between nanoparticle plasmon resonances and adsorbate mol. resonances. The results from these fundamental studies guide the design of new sensing expts., illustrated through applications in which researchers use both LSPR wavelength-shift sensing and SERS to detect mols. of chem. and biol. relevance.
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36. 36
  Malinsky, M. D.; Kelly, K. L.; Schatz, G. C.; Van Duyne, R. P. J. Am. Chem. Soc. 2001, 123, 1471– 1482
  There is no corresponding record for this reference.
37. 37
  Nusz, G. J.; Curry, A. C.; Marinakos, S. M.; Wax, A.; Chilkoti, A. ACS Nano 2009, 3, 795– 806
  There is no corresponding record for this reference.
38. 38
  Jackson, J. B.; Westcott, S. L.; Hirsch, L. R.; West, J. L.; Halas, N. J. Appl. Phys. Lett. 2003, 82, 257– 259
  There is no corresponding record for this reference.
39. 39
  Hao, E.; Schatz, G. C. J. Chem. Phys. 2004, 120, 357– 366
  39
  Electromagnetic fields around silver nanoparticles and dimers
  Hao, Encai; Schatz, George C.
  Journal of Chemical Physics (2004), 120 (1), 357-366CODEN: JCPSA6; ISSN:0021-9606. (American Institute of Physics)
  We use the discrete dipole approxn. to investigate the electromagnetic fields induced by optical excitation of localized surface plasmon resonances of silver nanoparticles, including monomers and dimers, with emphasis on what size, shape, and arrangement leads to the largest local elec. field (E-field) enhancement near the particle surfaces. The results are used to det. what conditions are most favorable for producing enhancements large enough to observe single mol. surface enhanced Raman spectroscopy. Most of the calcns. refer to triangular prisms, which exhibit distinct dipole and quadrupole resonances that can easily be controlled by varying particle size. In addn., for the dimer calcns. we study the influence of dimer sepn. and orientation, esp. for dimers that are sepd. by a few nanometers. We find that the largest |E|2 values for dimers are about a factor of 10 larger than those for all the monomers examd. For all particles and particle orientations, the plasmon resonances which lead to the largest E-fields are those with the longest wavelength dipolar excitation. The spacing of the particles in the dimer plays a crucial role, and we find that the spacing needed to achieve a given |E|2 is proportional to nanoparticle size for particles below 100 nm in size. Particle shape and curvature are of lesser importance, with a head to tail configuration of two triangles giving enhanced fields comparable to head to head, or rounded head to tail. The largest |E|2 values we have calcd. for spacings of 2 nm or more is ∼105.
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40. 40
  Sherry, L. J.; Jin, R.; Mirkin, C. A.; Schatz, G. C.; Van Duyne, R. P. Nano Lett. 2006, 6, 2060– 2065
  There is no corresponding record for this reference.
41. 41
  Chen, H.; Shao, L.; Woo, K. C.; Ming, T.; Lin, H.-Q.; Wang, J. J. Phys. Chem. C 2009, 113, 17691– 17697
  There is no corresponding record for this reference.
42. 42
  Joshi, G. K.; McClory, P. J.; Muhoberac, B. B.; Kumbhar, A.; Smith, K. A.; Sardar, R. J. Phys. Chem. C 2012, 116, 20990– 21000
  There is no corresponding record for this reference.
43. 43
  Joshi, G. K.; McClory, P. J.; Dolai, S.; Sardar, R. J. Mater. Chem. 2012, 22, 923– 931
  There is no corresponding record for this reference.
44. 44
  Haes, A. J.; Zou, S.; Zhao, J.; Schatz, G. C.; Van Duyne, R. P. J. Am. Chem. Soc. 2006, 128, 10905– 10914
  44
  Localized Surface Plasmon Resonance Spectroscopy near Molecular Resonances
  Haes, Amanda J.; Zou, Shengli; Zhao, Jing; Schatz, George C.; Van Duyne, Richard P.
  Journal of the American Chemical Society (2006), 128 (33), 10905-10914CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
  The peak location of the localized surface plasmon resonance (LSPR) of noble metal nanoparticles is highly dependent upon the refractive index of the nanoparticles' surrounding environment. New phenomena are revealed by exploring the influence of interacting mol. resonances and nanoparticle resonances. The LSPR peak shift and line shape induced by a resonant mol. vary with wavelength. In most instances, the oscillatory dependence of the peak shift on wavelength tracks with the wavelength dependence of the real part of the refractive index, as detd. by a Kramers-Kronig transformation of the mol. resonance absorption spectrum. A quant. assessment of this shift based on discrete dipole approxn. calcns. shows that the Kramers-Kronig index must be scaled to match expt.
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45. 45
  Hall, W. P.; Modica, J.; Anker, J.; Lin, Y.; Mrksich, M.; Van Duyne, R. P. Nano Lett. 2011, 11, 1098– 1105
  There is no corresponding record for this reference.
46. 46
  McFarland, A. D.; Van Duyne, R. P. Nano Lett. 2003, 3, 1057– 1062
  46
  Single Silver Nanoparticles as Real-Time Optical Sensors with Zeptomole Sensitivity
  McFarland, Adam D.; Van Duyne, Richard P.
  Nano Letters (2003), 3 (8), 1057-1062CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)
  This work utilizes dark-field optical microscopy to demonstrate the localized surface plasmon resonance λmax response of individual Ag nanoparticles to the formation of a monolayer of small-mol. adsorbates. The adsorption of fewer than 60 000 1-hexadecanethiol mols. on single Ag nanoparticles results in a localized surface plasmon resonance shift of 40.7 nm. Addnl., the kinetics of the single nanoparticle response was shown to be comparable to that of other real-time sensor technologies.
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47. 47
  Bain, C. D.; Whitesides, G. M. J. Am. Chem. Soc. 1989, 111, 7164– 7175
  47
  Formation of monolayers by the coadsorption of thiols on gold: variation in the length of the alkyl chain
  Bain, Colin D.; Whitesides, George M.
  Journal of the American Chemical Society (1989), 111 (18), 7164-75CODEN: JACSAT; ISSN:0002-7863.
  Mixts. of 2 long-chain alkanethiols, HS(CH2)nX and HS(CH2)mY (X, Y = CH3, OH; n > m), in which the alkyl chains have different lengths, adsorb from soln. on Au and form monolayers comprising a densely packed inner region adjacent to the Au surface and a disordered outer region in contact with the soln. When X = Y = Me (n ≠ m), this disordered phase makes the "mixed monolayer" more oleophilic than the ordered, pure (i.e., single-component) monolayers. When X = Y = OH, the pure monolayers are wetted by water, but the mixed monolayers are less hydrophilic because nonpolar polymethylene chains are exposed at the surface. When X = Me, Y = OH (n = 21, m = 11), a very sharp transition occurs from a monolayer composed largely of the longer, Me-terminated component to the shorter, OH-terminated component as the mol fraction of HS(CH2)11OH in the adsorption soln. is increased. From solns. contg. 2 thiols, adsorption of the thiol with the longer chain is preferred. This preference is greater when the monolayers are adsorbed from EtOH than from isooctane. The mixed monolayers do not act as ideal 2-dimensional solns. The adsorption isotherms suggest a pos. excess free energy of mixing of the 2 components in the monolayer. The compns. of the monolayers appear to be detd. largely by thermodn., although in some cases there is also a kinetic contribution. The 2 components in the mixed monolayers do not phase-segregate into macroscopic islands (greater than a few tens of angstroms across), but are probably not randomly dispersed within the monolayer. The wettability of mixed, Me-terminated monolayers can be rationalized partially by the geometric mean approxn., but a full description probably requires inclusion of the entropy of mixing at the monolayer-liq. interface. The hysteresis in the contact angle on these monolayers cannot be explained by theories of wetting based on macroscopic heterogeneity. Contact angles are more sensitive than optical ellipsometry or XPS to certain types of changes in the compn. and structure of these monolayers.
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48. 48
  Murphy, C. J.; Gole, A. M.; Stone, J. W.; Sisco, P. N.; Alkilany, A. M.; Goldsmith, E. C.; Baxter, S. C. Acc. Chem. Res. 2008, 41, 1721– 1730
  48
  Gold Nanoparticles in Biology: Beyond Toxicity to Cellular Imaging
  Murphy, Catherine J.; Gole, Anand M.; Stone, John W.; Sisco, Patrick N.; Alkilany, Alaaldin M.; Goldsmith, Edie C.; Baxter, Sarah C.
  Accounts of Chemical Research (2008), 41 (12), 1721-1730CODEN: ACHRE4; ISSN:0001-4842. (American Chemical Society)
  A review. Gold, enigmatically represented by the target-like design of its ancient alchem. symbol, has been considered a mystical material of great value for centuries. Nanoscale particles of gold now command a great deal of attention for biomedical applications. Depending on their size, shape, degree of aggregation, and local environment, gold nanoparticles can appear red, blue, or other colors. These visible colors reflect the underlying coherent oscillations of conduction-band electrons ("plasmons") upon irradn. with light of appropriate wavelengths. These plasmons underlie the intense absorption and elastic scattering of light, which in turn forms the basis for many biol. sensing and imaging applications of gold nanoparticles. The brilliant elastic light-scattering properties of gold nanoparticles are sufficient to detect individual nanoparticles in a visible light microscope with ∼102 nm spatial resoln. Despite the great excitement about the potential uses of gold nanoparticles for medical diagnostics, as tracers, and for other biol. applications, researchers are increasingly aware that potential nanoparticle toxicity must be investigated before any in vivo applications of gold nanoparticles can move forward. In this Account, we illustrate the importance of surface chem. and cell type for interpretation of nanoparticle cytotoxicity studies. We also describe a relatively unusual live cell application with gold nanorods. The light-scattering properties of gold nanoparticles, as imaged in dark-field optical microscopy, can be used to infer their positions in a living cell construct. Using this positional information, we can quant. measure the deformational mech. fields assocd. with living cells as they push and pull on their local environment. The local mech. environment experienced by cells is part of a complex feedback loop that influences cell metab., gene expression, and migration.
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49. 49
  Joshi, G. K.; Blodgett, K. N.; Muhoberac, B. B.; Johnson, M. A.; Smith, K. A.; Sardar, R. Nano Lett. 2014, 14, 532– 540
  There is no corresponding record for this reference.
50. 50
  Preis, M.; Gardner, T. B.; Gordon, S. R.; Pipas, J. M.; Mackenzie, T. A.; Klein, E. E.; Longnecker, D. S.; Gutmann, E. J.; Sempere, L. F.; Korc, M. Clin. Cancer Res. 2011, 17, 5812– 5821
  50
  MicroRNA-10b Expression Correlates with Response to Neoadjuvant Therapy and Survival in Pancreatic Ductal Adenocarcinoma
  Preis, Meir; Gardner, Timothy B.; Gordon, Stuart R.; Pipas, J. Marc; MacKenzie, Todd A.; Klein, Erin E.; Longnecker, Daniel S.; Gutmann, Edward J.; Sempere, Lorenzo F.; Korc, Murray
  Clinical Cancer Research (2011), 17 (17), 5812-5821CODEN: CCREF4; ISSN:1078-0432. (American Association for Cancer Research)
  INTRODUCTION: Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy. Diagnosis and management of PDAC are hampered by the absence of sensitive and specific disease biomarkers. MicroRNAs (miRNA) are noncoding regulatory RNAs involved in initiation and progression of human cancers. In this study, we sought to det. whether miR-10b could serve as a biomarker for PDAC. Exptl. Design: miRNA expression was characterized by fluorescence-based in situ hybridization using locked nucleic acid-modified DNA probes against miR-10b, miR-21, miR-155, miR-196a, and miR-210, followed by codetection of proteins by immunohistochem. on the same tissue sections. miRNA expression in surgically resected PDAC tissues and in endoscopic ultrasonog. (EUS)-guided fine-needle aspirate (EUS-FNA) samples was analyzed in cytokeratin 19 (CK19)-pos. epithelial cells using optical intensity anal. RESULTS: In 10 resected PDAC samples, miR-10b was the most frequently and consistently overexpressed miRNA among characterized miRNAs, exhibiting a four-fold increase in the cancer cells (P = 0.012). Given this preferential overexpression of miR-10b, we sought to det. whether miR-10b expression was clin. relevant. Accordingly, miR-10b expression was examd. in 106 EUS-FNA samples obtained from pancreatic lesions. miR-10b expression was increased in cancer cells compared with CK19-pos. epithelial cells in benign lesions (P = 0.0001). In patients with PDACs, lower levels of miR-10b were assocd. with improved response to multimodality neoadjuvant therapy, likelihood of surgical resection, delayed time to metastasis, and increased survival. CONCLUSION: miR-10b is a novel diagnostic biomarker for PDACs when assessing pancreatic lesions. Expression of miR-10b is predictive of response to neoadjuvant therapy and outcome in this disease. Clin Cancer Res; 17(17); 5812-21.
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51. 51
  Sempere, L. F.; Preis, M.; Yezefski, T.; Ouyang, H.; Suriawinata, A. A.; Silahtaroglu, A.; Conejo-Garcia, J. R.; Kauppinen, S.; Wells, W.; Korc, M. Clin. Cancer Res. 2010, 16, 4246– 4255
  51
  Fluorescence-Based Codetection with Protein Markers Reveals Distinct Cellular Compartments for Altered MicroRNA Expression in Solid Tumors
  Sempere, Lorenzo F.; Preis, Meir; Yezefski, Todd; Ouyang, Haoxu; Suriawinata, Arief A.; Silahtaroglu, Asli; Conejo-Garcia, Jose R.; Kauppinen, Sakari; Wells, Wendy; Korc, Murray
  Clinical Cancer Research (2010), 16 (16), 4246-4255CODEN: CCREF4; ISSN:1078-0432. (American Association for Cancer Research)
  PURPOSE: High-throughput profiling expts. have linked altered expression of microRNAs (miRNA) to different types of cancer. Tumor tissues are a heterogeneous mixt. of not only cancer cells, but also supportive and reactive tumor microenvironment elements. To clarify the clin. significance of altered miRNA expression in solid tumors, we developed a sensitive fluorescence-based in situ hybridization (ISH) method to visualize miRNA accumulation within individual cells in formalin-fixed, paraffin-embedded tissue specimens. This ISH method was implemented to be compatible with routine clin. immunohistochem. (IHC) assays to enable the detection of miRNAs and protein markers in the same tissue section for colocalization and functional studies. Exptl. Design: We used this combined ISH/IHC assay to study a subset of cancer-assocd. miRNAs, including miRNAs frequently detected at low (miR-34a and miR-126) and high (miR-21 and miR-155) levels, in a panel of breast, colorectal, lung, pancreas, and prostate carcinomas. RESULTS: Despite the distinct histopathol. alterations of each particular cancer type, general trends emerged that pinpointed distinct source cells of altered miRNA expression. Although altered expressions of miR-21 and miR-34a were manifested within cancer cells, those of miR-126 and miR-155 were predominantly confined to endothelial cells and immune cells, resp. These results suggest a heterogeneous participation of miRNAs in carcinogenesis by intrinsically affecting cancer cell biol. or by modulating stromal, vascular, and immune responses. CONCLUSIONS: We described a rapid and sensitive multicolor ISH/IHC assay and showed that it could be broadly applied as an investigational tool to better understand the etiol. relevance of altered miRNA expression in cancer.
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52. 52
  Elhadj, S.; Singh, G.; Saraf, R. F. Langmuir 2004, 20, 5539– 5543
  There is no corresponding record for this reference.
53. 53
  Greither, T.; Grochola, L. F.; Udelnow, A.; Lautenschläger, C.; Würl, P.; Taubert, H. Int. J. Cancer 2010, 126, 73– 80
  53
  Elevated expression of microRNAs 155, 203, 210 and 222 in pancreatic tumors is associated with poorer survival
  Greither, Thomas; Grochola, Lukasz F.; Udelnow, Andrej; Lautenschlaeger, Christine; Wuerl, Peter; Taubert, Helge
  International Journal of Cancer (2010), 126 (1), 73-80CODEN: IJCNAW; ISSN:0020-7136. (Wiley-Liss, Inc.)
  Pancreatic cancer is the eighth most common cancer and has an overall 5-yr survival rate lower than 10%. Because of their ability to regulate gene expression, microRNAs can act as oncogenes or tumor-suppressor genes and so have garnered interest as possible prognostic and therapeutic markers during the last decade. However, the prognostic value of microRNA expression in pancreatic cancer has not been thoroughly investigated. We measured the levels of miR-155, miR-203, miR-210, miR-216, miR-217 and miR-222 by quant. RT-PCR in a cohort of 56 microdissected pancreatic ductal adenocarcinomas (PDAC). These microRNAs were chosen as they had previously been shown to be differentially expressed in pancreatic tumors compared to normal tissues. The possible assocn. of microRNA expression and patients' survival was examd. using multivariate Cox's regression hazard analyses. Interestingly, significant correlations between elevated microRNA expression and overall survival were obsd. for miR-155 (RR = 2.50; p = 0.005), miR-203 (RR = 2.21; p = 0.017), miR-210 (RR = 2.48; p = 0.005) and miR-222 (RR = 2.05; p = 0.035). Furthermore, tumors from patients demonstrating elevated expression levels of all 4 microRNAs possessed a 6.2-fold increased risk of tumor-related death compared to patients whose tumors showed a lower expression of these microRNAs. This study provides the first evidence for an oncogenic activity of miR-155, miR-203, miR-210 and miR-222 in the development of pancreatic cancer as has been reported for other tumor types. Furthermore, the putative target genes for these microRNAs suggest a complex signaling network that can affect PDAC tumorigenesis and tumor progression.
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54. 54
  Stiles, P. L.; Dieringer, J. A.; Shah, N. C.; Van Duyne, R. P. Annu. Rev. Anal. Chem. 2008, 1, 601– 626
  54
  Surface-enhanced Raman spectroscopy
  Stiles, Paul L.; Dieringer, Jon A.; Shah, Nilam C.; Van Duyne, Richard P.
  Annual Review of Analytical Chemistry (2008), 1 (), 601-626CODEN: ARACFU; ISSN:1936-1327. (Annual Reviews Inc.)
  A review. The ability to control the size, shape, and material of a surface has reinvigorated the field of surface-enhanced Raman spectroscopy (SERS). Because excitation of the localized surface plasmon resonance of a nanostructured surface or nanoparticle lies at the heart of SERS, the ability to reliably control the surface characteristics has taken SERS from an interesting surface phenomenon to a rapidly developing anal. tool. This article first explains many fundamental features of SERS and then describes the use of nanosphere lithog. for the fabrication of highly reproducible and robust SERS substrates. In particular, we review metal film over nanosphere surfaces as excellent candidates for several expts. that were once impossible with more primitive SERS substrates (e.g., metal island films). The article also describes progress in applying SERS to the detection of chem. warfare agents and several biol. mols.
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55. 55
  Moskovits, M. J. Raman Spectrosc. 2005, 36, 485– 496
  55
  Surface-enhanced Raman spectroscopy: A brief retrospective
  Moskovits, Martin
  Journal of Raman Spectroscopy (2005), 36 (6/7), 485-496CODEN: JRSPAF; ISSN:0377-0486. (John Wiley & Sons Ltd.)
  A review. The electromagnetic theory of surface-enhanced Raman spectroscopy (SERS), despite its simplicity, can account for all major SERS observations, including: the need for a nanostructured material as the SERS-active system; the observation that some metals form good SERS-active systems while others do not; the observation that strongly interacting metal nanoparticles result in very much more effective SERS-active systems; the obsd. polarization sensitivity shown by nanoparticle aggregates; and the optical behavior of nanostructured metals in the absence of a mol. adsorbate. By extending the ideas inherent in the electromagnetic model one can also understand the seminal features reported for single-mol. SERS, including the puzzling observation that only a few Ag particles in an ensemble are hot (they are appropriately structured nanoparticle clusters) and that for a hot particle, once one is able to observe SERS, adding more adsorbate does not significantly alter the intensity (once the electromagnetic hot spot is occupied, adding adsorbate to other sites on the nanoparticle cluster will not add greatly to the obsd. intensity). However, the electromagnetic model does not account for all that is learned through SERS. Mol. resonances, charge-transfer transitions and other processes such as ballistic electrons transiently probing the region where the mol. resides and then modulating electronic processes of the metal as a result certainly contribute to the rich information SERS reports; and by virtue of the fact that these contributions will vary from mol. to mol., they will constitute the most interesting aspects reported by SERS. But, the overall reason why SERS produces such inordinate enhancements is largely an electromagnetic property of nanostructures.
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56. 56
  Singh, A. K.; Khan, S. A.; Fan, Z.; Demeritte, T.; Senapati, D.; Kanchanapally, R.; Ray, P. C. J. Am. Chem. Soc. 2012, 134, 8662– 8669
  56
  Development of a long-range surface-enhanced Raman spectroscopy ruler
  Singh, Anant Kumar; Khan, Sadia Afrin; Fan, Zhen; Demeritte, Teresa; Senapati, Dulal; Kanchanapally, Rajashekhar; Ray, Paresh Chandra
  Journal of the American Chemical Society (2012), 134 (20), 8662-8669CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
  Optical-ruler-based distance measurements are essential for tracking biomol. processes in a wide range of anal. biochem. applications. The normally used Forster resonance energy transfer (FRET) ruler is not useful for studying distance-dependent properties when distances are >10 nm. Driven by this limitation, the authors have developed a long-range surface-enhanced Raman spectroscopy (SERS) optical ruler using oval-shaped gold nanoparticles and Rh6G dye-modified rigid, variable-length double-strand DNAs. The bifunctional rigid dsDNA mol. serves as the SERS-active ruler. The authors' exptl. results show that one can tune the length of the SERS ruler between 8 and ∼18 nm by choosing the size of the oval-shaped gold nanoparticles. A possible mechanism for the authors' obsd. distance-dependent SERS phenomenon is discussed using the Gersten and Nitzan model. Ultimately, the authors' long-range SERS mol. rulers can be an important step toward understanding distance-dependent biol. processes.
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57. 57
  Jackson, J. B.; Halas, N. J. Proc. Nat. Acad. Sci. U.S.A. 2004, 101, 17930– 17935
  57
  Surface-enhanced Raman scattering on tunable plasmonic nanoparticle substrates
  Jackson, J. B.; Halas, N. J.
  Proceedings of the National Academy of Sciences of the United States of America (2004), 101 (52), 17930-17935CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
  Au and Ag nanoshells are studied as substrates for surface-enhanced Raman scattering (SERS). SERS enhancements on nanoshell films are dramatically different from those obsd. on colloidal aggregates, specifically that the Raman enhancement follows the plasmon resonance of the individual nanoparticles. Comparative finite difference time domain calcns. of fields at the surface of smooth and roughened nanoshells reveal that surface roughness contributes only slightly to the total enhancement. SERS enhancements as large as 2.5 × 1010 on Ag nanoshell films for the nonresonant mol. p-mercaptoaniline are measured.
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58. 58
  Otte, M. A.; Sepulveda, B.; Ni, W.; Juste, J. P.; Liz-Marzan, L. M.; Lechuga, L. M. ACS Nano 2009, 4, 349– 357
  There is no corresponding record for this reference.
59. 59
  Novo, C.; Funston, A. M.; Mulvaney, P. Nat. Nanotechnol. 2008, 3, 598– 602
  59
  Direct observation of chemical reactions on single gold nanocrystals using surface plasmon spectroscopy
  Novo, Carolina; Funston, Alison M.; Mulvaney, Paul
  Nature Nanotechnology (2008), 3 (10), 598-602CODEN: NNAABX; ISSN:1748-3387. (Nature Publishing Group)
  Heterogeneous catalysts were pivotal to the development of the modern chem. industry and are essential for catalyzing many industrial reactions. However, reaction rates are different for every individual catalyst particle and depend upon each particle's morphol. and size, crystal structure and compn. Measuring the rates of reaction on single nanocrystals will enable the role of catalyst structure to be quantified. Here, using surface plasmon spectroscopy, the authors have directly obsd. the kinetics of at. deposition onto a single gold nanocrystal and also monitored electron injection and extn. during a redox reaction involving the oxidn. of ascorbic acid on a gold nanocrystal surface. These results constitute the first direct measurement of the rates of redox catalysis on single nanocrystals.
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60. 60
  Livak, K. J.; Schmittgen, T. D. Methods 2001, 25, 402– 408
  60
  Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method
  Livak, Kenneth J.; Schmittgen, Thomas D.
  Methods (San Diego, CA, United States) (2001), 25 (4), 402-408CODEN: MTHDE9; ISSN:1046-2023. (Academic Press)
  The two most commonly used methods to analyze data from real-time, quant. PCR expts. are abs. quantification and relative quantification. Abs. quantification dets. the input copy no., usually by relating the PCR signal to a std. curve. Relative quantification relates the PCR signal of the target transcript in a treatment group to that of another sample such as an untreated control. The 2-ΔΔCT method is a convenient way to analyze the relative changes in gene expression from real-time quant. PCR expts. The purpose of this report is to present the derivation, assumptions, and applications of the 2-ΔΔCT method. In addn., we present the derivation and applications of two variations of the 2-ΔΔCT method that may be useful in the anal. of real-time, quant. PCR data. (c) 2001 Academic Press.
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Supporting Information
Supporting Information
Experimental detail of nanoprisms synthesis, fabrication of plasmonic biosensors, and additional UV–visible spectra, AFM images, and histograms. This material is available free of charge via the Internet at http://pubs.acs.org.
- nl503220s_si_001.pdf (1.12 MB)
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Highly Specific Plasmonic Biosensors for Ultrasensitive MicroRNA Detection in Plasma from Pancreatic Cancer PatientsClick to copy article linkArticle link copied!

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Fabrication of the Plasmonic Biosensor for miRs Detection

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Detection of miR Levels in in Plasma from Pancreatic Cancer Patients

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Fabrication of LSPR-Based miR Sensors and Detection

Total RNA Extraction and Quantification of MicroRNA by qRT-PCR

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Highly Specific Plasmonic Biosensors for Ultrasensitive MicroRNA Detection in Plasma from Pancreatic Cancer Patients
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