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Writed by Randall Wallace score 918551 vote Mel Gibson genre War, Biography USA. Progressive, Simple and Accessible to all, S2 sets the standard for modern movie going. S2 is your destination for an incredible movie-going experience, in a setting that is truly inspirational. The interiors are chic with all screens equipped with state-of-the-art technology to provide you with an experience that cannot be replicated. WI-FI The space is Wi-Fi enabled so you can always stay connected. Guests at S2 may use the complimentary service by requesting staff for the Wi-Fi credentials. Food & Beverage Enjoy a wide selection of food and beverage at the snack counters at S2. Take your pick from a variety of popcorn, puffs, desserts, beverages and a whole lot more, freshly prepared to satisfy your movie time hunger pangs. Technology At S2 Cinemas, the screens are equipped with 3D technology, Harkness Clarus XC silver screens coupled with the prowess of 4k projection from Barco and the powerful sound of Dolby Atmos. We notice that you already have an order in progress for this session. Attention! Sorry, there are no seats available for this show right now. Please try again after a few minutes. Active Orders You cannot open more than three sessions. Please click on one of the active orders to continue.

Full cinema braveheart movies. He wasn' t right, in the head. Full cinema braveheart lyrics. Hands down best movie ever made in the modern era. Few movies touch your heart and make an impact the way Braveheart managed to. Full cinema braveheart 2. Full cinema braveheart download. Full cinema braveheart. Full cinema braveheart 2017. Braveheart is still an emotionally powerful movie over 20 years later. Mel Gibson's performance as Scottish freedom fighter William Wallace who believed in fighting for freedom against England's tyrannical rule. Mel Gibson can also proudly add another string to his bow by proving that he is not just a pretty face by showcasing his finest work as a director.
William Wallace's life has been plagued with tragedy. His father and brother died when he was a young child while battling the English and his newly wedded bride Murron (Catherine McCormack) is publicly executed by British soldiers after she tries to fight off an attempted rape. The British state that her public execution will be made an example to those who defy the British. Wallace returns to seek retribution of those British soldiers responsible, and with the help of several villagers slaughter the British soldiers. Swearing vengeance of the British and tiring of the tyrannical British rule under the thumb of King Edward Longshanks of England (Patrick McGoohan) William Wallace inspires most of Scotland to form an Army and lead an uprising against the British which culminates in many victorious yet brutal battles between the makeshift militia and the well disciplined British troops. Edward Longshanks tries to send French princess Isabelle (Sophie Marceau) to broker peace talks in place of his weak willed son however they fall on deaf ears. In a memorable scene before going into battle at the victorious Battle of Stirling Bridge, William Wallace fires up his troops with his inspirational speech that gives goosebumps to everyone including the audience "They may take our lives, but they'll never FREEDOM! The battle scenes are spectacularly brilliant. Without a doubt, besides the storyline, they form the most memorable scenes of the movie.
As an actor and director, Mel Gibson has never been finer. Randall Wallace's screenplay is brilliant and the late James Horner's score is tranquil during quiet moments and inspirationally brilliant during the frantic battle scenes.
Released in 1995, Braveheart has been one of those movies that has certainly held a special place with me for a long time. It is an emotionally powerful and an epic movie that well and truly deserved to be the winner of 5 Academy Awards including Best Picture and Best Director.
Mel Gibson, take a bow. You will always be remembered for your finest work for Braveheart.
10/10.

THIS WEBSITE IS UPDATED EVERY WEDNESDAY EVENING THIS WEEKS PRINTABLE PROGRAM 13th - 19th FEBRUARY THIS WEEK  NEXT WEEKS COMING SOON CLICK POSTER FOR  TRAILER FABULOUS NEW FILM OPENING SOON! IN AN AUSTRALIAN PREMIERE SCREENING COMMENCING 26th March One English couple. One battered English car. 5000 miles through the heart of India. Meet Rupert and Jan Grey, a free-spirited and eccentric upper-class English couple in their sixties, married for 35 years and on the verge of retirement. At a time when most couples are preparing for a simple life, this intrepid couple ignore everyones advice and embark on a remarkable journey – to drive Ruperts fathers 1936 Rolls Royce across India on their way to a photography festival in Dhaka, Bangladesh. Over six months and some 5000 miles, they fall into company with tea-wallahs and maharajahs, dodge tribal conflicts, negotiate with border officials and make constant mechanical repairs. Truly unique among adventure films, Oliver McGarveys stunning documentary is at once a rare glimpse into an inspired expedition, a study of a love story 35 years in the making and a reminder to never stop embracing the possibilities of life. MAKE SURE YOU ADD OUR EMAIL ADDRESS TO YOUR CONTACTS EXCITING NEW SERIES OF MUSICAL EVENTS FROM THE  PRODUCERS OF EXHIBITION ON SCREEN IN SEARCH OF MOZART 22nd & 23rd FEBRUARY AT 12:30PM (TIME TO BE CONFIRMED) ENCORE ANNOUNCED SATURDAY 22nd & SUNDAY 23rd ONLY NON REFUNDABLE BOOKING FEES APPLY NO FREE TICKETS NO MEMBER DISCOUNTS CLICK POSTER FOR  PRINTABLE FLYER BOX OFFICE NOW OPEN 11 12 & 13 APRIL  11 12 & 13 JULY 29 30 & 31 AUG 10 11 & 12 OCT CLICK POSTER FOR PRINTABLE FLYER NON REFUNDABLE BOOKING FEES APPLY NO MEMBER DISCOUNTS GET ROASTED. GO FUND ME FOR CINEMA 3 IF YOU  WOULD LIKE TO HELP  PLEASE CLICK THE LINK ON THE BUTTON BELO W ANY AMOUNT OPEN 7 DAYS FROM 1/2 HOUR PRIOR TO FIRST SESSION TRADING HOURS 3 HOURS FREE UNDERCOVER PARKING FOR CUSTOMERS CAR PARKING FULL ACCESS   WHEELCHAIR ACCESS LIFT FROM CAR PARK PICTURE MENU GUIDE & COMPANION DOGS WELCOME CONTACT CINEMAX CINEMAS SINCE JUNE  2009.

Press alt. to open this menu. Full cinema braveheart series. Our Darkest Hour but also Our Finest. Full cinema braveheart free. YouTube Shoja Del. When i was watching this I discovered a movie mistake go to 1:51 and quickly pause look in the left corner there is a car didn't know cars were around then very interesting Lol. Full cinema braveheart film. 1. Urban N, Drescher C. Potential and Limitations in Early Diagnosis of Ovarian Cancer. Adv. Exp. Med. Biol. 2008; 622:3–14. PubMed. Google Scholar] 2. Bast RC, Hennessy B, Mills GB. The Biology of Ovarian Cancer: New Opportunities for Translation. Nat. Rev. Cancer. 2009; 9:415–428. PMC free article. PubMed. Google Scholar] 3. Zhu CS, Pinsky PF, Cramer DW, Ransohoff DF, Hartge P, Pfeiffer RM, Urban N, Mor G, Bast RC, Jr, Moore LE. A Framework for Evaluating Biomarkers for Early Detection: Validation of Biomarker Panels for Ovarian Cancer. Can. Prev. Res. 2011; 4:375–383. PMC free article. PubMed. Google Scholar] 4. Clarke-Pearson DL. 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The same emotion in december 2019. YouTube. Full cinema braveheart online. Full cinema braveheart torrent. Raman scattering plays an increasingly important role in chemical and biological analysis. (1-3) Compared with the most commonly used flourescence that often suffers from photobleaching, self-quenching, autofluorescence, and broad emission bands, 4, 5) Raman scattering shows the following advantages. First, Raman scattering is resistant to photobleaching, which makes Raman signals highly stable and can be used for long-time analysis. Second, the peak width of Raman signals is narrow, allowing it facile for multiplex detection. Third, the rich molecular information on Raman signals can reveal the fingerprint behavior of biomolecules. (6, 7) However, the sensitivity of Raman spectroscopy is rather low because of its very small cross sections (∼10 –30 cm 2 per molecule. 8-10) Fortunately, the surface enhanced Raman effect as well as the resonance enhancement effect can significantly improve the sensitivity of this technique as high as single-molecule (SM) level. (11-13) This finding encouraged ones to fabricate surface-enhanced Raman scattering (SERS) tags for biomolecule detection. (14, 15) The initial attempts in using single metal nanoparticles appended with Raman dyes can only provide picomolar sensitivity. (16-18) To further improve sensitivity, it is highly beneficial to fabricate SERS tags with hot spots. (19) These tags have attracted much attention due to their super brightness. Although a lot of efforts have been made to fabricate SERS substrate with uniformly distributed hot spots, 20-23) it is still urgently needed to achieve the SERS detection of single biomolecules, especially biomarkers linked with clinical diagnosis. Reporter-labeled SERS tags have been widely studied for biomolecule detection owing to their high specificity and reproducibility. (6) Unlike conventional single-molecule SERS that probes the Raman scattering of analytes located at hot spots, 24) SERS tags allow biomolecule detection by recording the overall Raman signals of the tags. (25, 26) However, the average enhancement factor of a SERS tag has been predicted to be 10 4 times smaller than that at the hot spots. (27) Hence, the brightness of conventional SERS tags is generally insufficient for single-biomolecule detection. The most effective strategy to improve brightness is increasing hot spots in SERS tags. For instance, anisotropic nanoparticles and nanoclusters have been fabricated, 28-34) but their ability in single-biomolecule detection has not been demonstrated most likely due to their limited hot spots. Large aggregate tags with rich hot spots could be a solution to this issue, but it remains a challenge in creating uniform large aggregates. Moreover, the direct use of large sized tags can principally reduce the kinetic properties of recognition ligands that are tethered on the tags and lead to strong nonspecific adsorption on substrates. (35) In this report, we propose a general strategy for single-biomolecule detection based on the controllable assembly of dimeric silver nanoparticles (AgNPs) into large aggregates. The in situ hot-spot assembly is triggered through a cascade hybridization chain reaction (HCR) by which numerous hot spots were produced in single aggregates, thus greatly enhancing the brightness of the tags and ultimately achieving single-biomolecule detection. As a proof-of-concept, the expression levels of microRNA-21 (miRNA-21) in cancer cells and normal cells were quantitatively detected and differentiated at the single-cell level. Furthermore, single proteins have also been detected by this strategy. To create the multiple hot spot-embedded SERS tags for single-biomolecule detection, 4, 4′-biphenyldithiol (DBDT) was employed as both a Raman reporter and a linker to cause the formation of AgNP dimers with a roughly 1 nm gap, thus producing typical hot spots in the gapped dimers. The fabrication details are described in the Supporting Information, Figure S1, and several characterizations including transmission electron microscopy (TEM) dynamic light scattering (DLS) and UV–vis spectroscopy are shown in the Supporting Information, Figures S3–S5. Next, the AgNP dimers were subsequently functionalized with two complementary hairpin pairs and were assigned as probe 1 and probe 2, respectively ( Figure 1. Figure 1 Figure 1. Schematic of the DBDT-encoded AgNP dimer probes and the protocol for miRNA detection with the in situ hot-spot assembly strategy. With the probes in hand, we would like to detect single biomolecules on a chip. MiRNA-21 was employed as the model target since it has been identified as a potential biomarker overexpressed in various cancers. (36-38) To do this, a glass chip was coated with a polydimethylsiloxane (PDMS) film with array-holes (5 mm in diameter) to load the samples. We first bolted the locked nucleic acid (LNA) capture sequences on the chip surface, which recognize the partial sequence of target miRNA-21 specifically. The rest of the sequence of the immobilized miRNA-21 could hybridize with the LNA trigger sequence. The employment of LNA probes not only enhances the binding affinity to target miRNA-21 but also improves hybridization discrimination among closely related miRNAs. (39, 40) Then, the free single-stranded LNA trigger sequence opened up the hairpin structure on probe 1 specifically and thus captured this probe. Subsequently, the immobilized probe 1 can capture probe 2 via the specific hybridization between the hairpin pairs. Through the high-efficiency cascade HCR, the DBDT-coded AgNP dimers were assembled into a large aggregate. As a result, an extremely bright SERS tag can be yielded with numerous embedded hot spots. Figure 2 a displays the representative Raman spectrum of the hot-spot assembly tags for miRNA-21 detection with an average 1 biomolecule in each scattering point, which is compared with those of dimer tags and a control (in the absence of miRNA-21. By measuring their Raman intensity at 1587 cm –1, it is clearly that the intensity of the aggregate tags is much higher than that with dimers. SEM images for the corresponding substrates directly show the typical dimer structures ( Figure 2 d) and the in situ assembled AgNP aggregates ( Figure 2 e. Raman mapping images further demonstrate the ultrahigh brightness of the aggregate tags ( Figure 2 h. It is reasonable that the single aggregates containing plenty of hot spots can provide much higher Raman intensity than the dimer tags with limited hot spots. Moreover, the aggregates with different incubation times were prepared in solutions and their Raman intensities were also measured (Figures S6 and S7 in the Supporting Information. Similarly, the Raman intensity of DBDT in aggregates was significantly stronger than that in dimers. The appearance of corresponding characteristic Raman peaks of DNA phosphoric acid skeleton further demonstrates the formation of AgNP aggregates where both Raman reporters and hairpin pairs are embedded in the hot spots. Figure 2 Figure 2. (a) Raman spectra and (b) corresponding Raman intensities at 1587 cm –1 for the arrayed holes where miRNA-21 (1 pM) was detected with the AgNP dimer probe 1 and the hot-spot assembly strategy (the absence of miRNA-21 was used as a control. Error bars represent five signal points randomly selected from the substrates. (c–e) SEM images and (f–h) Raman mapping images of the substrates correspond to the above three cases. For the Raman mapping experimental conditions: λ ex = 532 nm, P ex ≈ 18 mW, acquisition time = 1 s, and laser point size ≈ 1 μm 2. In order to illustrate the single-molecule performance of the hot-spot assembly tags, 50 μL of PBS solution containing different concentrations of miRNA-21 (0, 1, 10, 100, and 1000 pM) were added to the array holes immobilized with a layer of LNA capture sequences. The capture efficiency of the LNA probes toward miRNA-21 was calculated to be 73. 6% by measuring the free miRNA-21 using RT-PCR before and after being pulled down onto the substrate (details are shown in the Supporting Information, Figure S8. On the basis of the capture efficiency and the area of the array holes (0. 2 cm 2) the corresponding densities of the captured miRNA-21 were estimated to be roughly 0, 1, 10, 100, and 1000 molecules/μm 2. Since the size of the confocal laser beam is approximately 1 μm 2, which means that the SERS signals from single scattering points are recorded at the single-molecule levels when the concentrations of the target miRNA-21 are less than 10 pM (i. e. 10 molecules in each scattering point. The Raman peak heights at 1587 cm –1 for each sample were measured from 360 different scattering points ( Figure 3 a. It is obviously that negligible signals can be observed in the absence of target miRNA-21. With the increase of miRNA-21, both the intensity and number of the Raman signals are elevated gradually. It should be noted that we set the threshold for signal collection to 88 counts, which is 3 times the standard variation in the mean background signal. Figure 3 Figure 3. (a) SERS signals measured at 1587 cm –1 for the detection of different amounts of miRNA-21 in each scattering point: from bottom to top are 0, 1, 10, 100, and 1000 pM, respectively. 360 Raman spectra were collected for each sample randomly. (b) Analyte number-dependent SERS activity and (c) profile of the relationship between the amount of miRNA-21 and the normalized Raman intensity. We further undertook a statistical analysis of the SERS activity in the 360 scattering points to verify the single-biomolecule detection performance. Figure 3 b shows the Raman intensity of the hot-spot assembly tags as a function of miRNA-21 numbers in each scattering point. By recording the SERS signals above 88 counts (signal-to-background threshold) the mean intensities of each probed point were determined to be 553, 2151, 3276, and 4887 counts for the samples of 1, 10, 100, and 1000 molecules/scattering point, respectively. We next devided the Raman intensity into five regions: 88–553, 553–2151, 2151–3276, 3276–4887, and >4887 counts, which represent the probability to detect 0–1, 1–10, 10–100, 100–1000, and >1000 miRNA-21 molecules in each scattering point, respectively. The results clearly reveal that, with the decrease of miRNA-21 concentration, the Raman signals distributed mainly in the regions with lower intensity. When decreasing the miRNA-21 concentration to a level where single miRNA-21 molecules (0–10 miRNA-21 molecules) are captured in each scattering point, a majority of the signals appear in the regions of 88–553 and 553–2151 counts. From statistical analysis point of view, signals falling into these two regions reflect the ability of single-biomolecule detection. The curve of the normalized Raman intensities against the concentration of miRNA-21 for the above samples were also plotted ( Figure 3 c. With the increase amount of miRNA-21, the normalized Raman intensities increase and reach a plateau eventually. This is reasonable because high-density miRNA-21 may lead to steric hindrance effect on the hot-spot assembly among the adjacent aggregate tags. Next, we compared the detection limits of the in situ hot-spot assembly strategy and the dimer probes. The hot-spot assembly method provides much higher Raman intensities than that of the dimer probes across 3 orders of dynamic range, with a limit of 1 pM (i. e., 1 molecule/scattering point) while the detection limit of the dimer probes is 1 nM (Figure S9 in the Supporting Information. The significantly improved sensitivity of the in situ hot-spot assembly strategy is primarily due to the large amounts of hot spots in single scattering points. In addition, the increased size of the aggregates may enable them to be probed more easily than the dimer probes. Furthermore, the in situ hot-spot assembly tags show negligible nonspecific adsorption as often observed in directly using large sized SERS tags. The combination of the ultrahigh brightness and low background interference enables single biomarkers, not limited to miRNA-21, to be detected. Reproducibility is another key issue to SERS. Unlike conventional single-molecule SERS that detects the Raman scattering of analytes residing at hot spots, we recorded the overall Raman signals of the assembled SERS tags which are bound to their corresponding analytes on the chip. Therefore, the signal reproducibility of the assembly strategy relies on the binding capability and the assembly activity of the SERS tags. With respect to binding capability, we employed LNA sequences to maximize the hybridization efficiency and specificity of the assembly tags toward the target miRNA-21. With the purpose of enhancing assembly activity, we utilized a high-efficiency cascade HCR to trigger the dimer probes to assemble into aggregate tags with relatively uniform size and distribution (Figure S10 in the Supporting Information. Whats more, the concentration-dependent average numbers of aggregates (0. 89, 7. 3, 29. 31, and 43. 34 per μm 2) are in good agreement with the increased trend of Raman intensity for different concentration of miRNA-21. The integration of LNA and HCR allows the SERS signals to be collected with relatively high reproducibility. To evaluate the specificity of this approach, miRNA-21 with one-point mutation (M1) and two-point mutation (M2) were detected under identical experimental conditions and compared with that of fully matched miRNA-21 (Figure S11 in the Supporting Information. It can be seen that the targets with mutations induced a substantial decrease in both signal intensity and number. Here, we defined the number of detectable signal points for fully matched miRNA-21 as 100% and the signal points corresponding to M1 and M2 are estimated to be 12. 4% and 4. 6% respectively, showing the excellent mismatch discrimination ability of the strategy. Such high specificity was most likely attributed to the use of LNAs, which simultaneously provide high binding affinity toward their complementary sequences. (41, 42) To investigate the feasibility of our proposed hot-spot assembly strategy for probing miRNA-21 in complex biological matrixes, we analyzed single-cell lysate samples prepared from three cancer cell lines (MCF-7, HepG 2, and HeLa) and one normal cell line (3T3. To maximize hybridization efficiency, magnetic beads were employed to capture the miRNA-21 lysed from the single cells and allowed the reaction to proceed in homogeneous solutions. After completing the hot-spot assembly, the beads served as the substrates for Raman mapping directly ( Figure 4 a. Figure 4 b displays the statistical analysis of the normalized Raman intensities of miRNA-21 for the four different types of single cells. Through collection of the signals from 4 single cells for each cell line, it is convinced that the levels of miRNA-21 in cancer cell lines are higher than that in normal 3T3 cell. On the basis of a calibration curve (Figure S12 in the Supporting Information) the average amounts of miRNA-21 are calculated to be 873, 5087, 12870, and 29859 in each 3T3, HeLa, MCF-7, and HepG 2 cell, respectively, which is in agreement with the results obtained with RT-PCR (Figure S13 in the Supporting Information) as well as those previously reported. (43) Since miRNA-21 is a cancer biomarker overexpressed in diverse cancer cells, it is reasonable that the levels of miRNA-21 in cancer cells could be higher than that in normal cells. It should be noted that the amounts of miRNA-21 in single cells are unable to be directly detected by RT-PCR (Figure S14 in the Supporting Information. 7) Instead, RT-PCR can only provide information at population levels, while our hot-spot assembly strategy allows the quantification and discrimination of miRNA-21 expression at the single-cell level. In comparison with the biomedical detection performed at the population level, probing biomolecules at the single-cell level is of great interest due to the heterogeneity among cell populations. Figure 4 Figure 4. Detection of miRNA-21 lysed from single 3T3, HeLa, MCF-7, and HepG 2 cells. (a) Representative Raman imaging of magnetic microbeads (8–9 μm in diameter) that were incubated with the lysates collected from different single cells and then performed with hot-spot assembly strategy. λ ex = 532 nm, P ex ≈ 0. 9 mW, acquisition time = 1 s, and laser point size ≈ 1 μm 2. (b) Normalized Raman intensities for the measurement of different types of single cells. The absence of cell lysates were employed as a control. For each cell, 500 points were recorded from 8 randomly distributed microbeads. Results are mean s. e. m. Asterisks denote significance compared with normal cell and the indicated cancer cell groups, n = 4 cells per group. p < 0. 05. p < 0. 01. At present, protein biomarkers are widely used for in vitro diagnostics in clinical practice. Improving sensitivity for protein detection could provide invaluable information for early diagnosis of disease, monitoring therapeutic responses, and guiding drug discovery. Single-protein detection (human IgG was employed as a model protein in this study) was achieved by this in situ hot-spot assembly approach (details are shown in the Supporting Information. The detection limit can reach 1 ng/mL (i. e., 9. 1 molecules per scattering point) and over 4 orders of dynamic range can be successfully obtained (Figures S15 and S16 in the Supporting Information. The results are impressive and will motivate us to develop protein arrays for multiple cancer biomarkers detection that is highly required in clinical diagnostics. Whats more, the platform shows more advantages when compared with other HCR-based techniques (Table S1 in the Supporting Information. In conclusion, we have developed an in situ hot-spot assembly strategy for single-biomolecule detection. On the basis of the novel design, this strategy provides several advantages as tags: i) single biomolecules in each scattering point can be detected by mapping the assembled aggregate tags; ii) the HCR-triggered hot-spot assembly relies on accumulation of dimeric AgNPs in situ, and the involvement of small sized AgNP dimers eliminates the nonspecific adsorption of the tags as usually observed in directly using large sized clusters; iii) owing to their very narrow emission bands, Raman tags outperform commonly used fluorescent labels with respect to parallel multiplexing and analytes rationing. By means of the hot-spot assembly strategy, the expression of miRNA-21 in single cancer cells and normal cells can be unambiguously quantified and differentiated. Although this work focused on miRNA-21 detection, we further demonstrated the feasibility of single proteins detection using the hot-spot assembly tags. We therefore believe that the application of the in situ hot-spot assembly strategy can be readily extended to other nucleic acids or proteins detection by replacing only the LNAs with specific nucleotide sequences or antibody pairs. Future study will focus on integration of the single-biomolecule sensitivity, specificity, and broad dynamic range with the multiplexing capability of SERS tags and enable simultaneous detection of multiple low-abundance analytes in clinically relevant samples. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10. 1021/acs. analchem. 7b00461. Additional experimental details, synthetic scheme for AgNP dimer probes, SEM and TEM images, DLS data, UV–vis spectra, Raman data, PCR amplification curves, SERS spectra, comparisons of some HCR-based techniques, sequences of DNA and RNA used, and primers used for RT-PCR ( PDF) The authors declare no competing financial interest. 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This study was supported by the National Natural Science Foundation of China (Grants 21475066 and 81401463) the Natural Science Foundation of Tianjin City (Grant 15JCZDJC65700) the Fundamental Research Funds for Central Universities (China) and the Thousand Youth Talents Plan of China. This article references 43 other publications. 1 Pieczonka, N. P. Aroca, R. F. Chem. Soc. Rev. 2008, 37, 946 – 954 DOI: 10. 1039/b709739p [ Crossref. PubMed. CAS]   Google Scholar 1 Single molecule analysis by surfaced-enhanced Raman scattering Pieczonka, Nicholas P. W. Aroca, Ricardo F. Chemical Society Reviews ( 2008) 37 ( 5) 946-954 CODEN: CSRVBR; ISSN: 0306-0012. ( Royal Society of Chemistry) A review. The authors' main objective in this tutorial review is to provide insight into some of the questions surrounding single mol. detection (SMD) using surface-enhanced Raman scattering (SERS) and surface-enhanced resonance Raman scattering (SERRS. Discovered thirty years ago, SERS is now a powerful anal. tool, strongly tied to plasmonics, a field that encompasses and profits from the optical enhancement found in nanostructures that support localized plasmon excitations. The spectrum of the single mol. carries the quantum fingerprints of the system modulated by the mol. -nanostructure interactions and the electronic resonances that may result under laser excitation. This information is embedded in vibrational band parameters. The dynamics and the mol. environment will affect the bandwidth of the obsd. Raman bands. The localized surface plasmon resonances (LSPR) empower the nanostructure with a no. of optical properties that will also leave their mark on the obsd. inelastic scattering process. Therefore, controlling size, shape and the formation of the aggregation state (or fractality) of certain metallic nanostructures becomes a main task for exptl. SERS/SERRS. This mol. -nanostructure coupling may, inevitably, lead to spectral fluctuations, increase photobleaching or photochem. An attempt is made here to guide the interpretation of this wealth of information when approaching the single mol. regime. 2 Qian, X. M. Nie, S. 2008, 37, 912 – 920 DOI: 10. 1039/b708839f [ Crossref. PubMed. CAS]   Google Scholar 2 Single-molecule and single-nanoparticle SERS: from fundamental mechanisms to biomedical applications Qian, X. -M. Chemical Society Reviews 912-920 CODEN: This tutorial review discusses a new class of colloidal metal nanoparticles that is able to enhance the efficiencies of surface-enhanced Raman scattering (SERS) by as much as 1014-1015 fold. This enormous enhancement allows spectroscopic detection and identification of single mols. located on the nanoparticle surface or at the junction of two particles under ambient conditions. Considerable progress has been made in understanding the enhancement mechanisms, including definitive evidence for the single-mol. origin of fluctuating SERS signals. For applications, SERS nanoparticle tags have been developed based on the use of embedded reporter mols. and a silica or polymer encapsulation layer. The SERS nanoparticle tags are capable of providing detailed spectroscopic information and are much brighter than semiconductor quantum dots in the near-IR spectral window. These properties have raised new opportunities for multiplexed mol. diagnosis and in vivo Raman spectroscopy and imaging. 3 Kneipp, K. Kneipp, H. Appl. Spectrosc. 2006, 60, 322 – 334 DOI: 10. 1366/000370206779321418 [ Crossref. PubMed. CAS]   Google Scholar 3 Single molecule Raman scattering Kneipp, Katrin; Kneipp, Harald Applied Spectroscopy ( 2006) 60 ( 12) 322A CODEN: APSPA4; ISSN: 0003-7028. ( Society for Applied Spectroscopy) A review. First, the physics behind surface-enhanced Raman scattering (SERS) is discussed for an extremely high enhancement level. Such an enhancement level is mainly based on strongly enhanced and spatially highly confined local optical fields in the vicinity of Au and Ag nanostructures. Then, single-mol. SERS expts. are described taking into account 2 different configurations. The 1st one deals with single-mol. Raman spectroscopy in soln. using Au and Ag nanoclusters as the SERS active substrates, and the 2nd one describes the single-mol. detection on a dry fractal Ag surface. Next, single-mol. SERS spectroscopy at the anti-Stokes side of the excitation laser is treated. Due to the extremely high effective SERS cross section, spontaneous anti-Stokes Raman scattering becomes a 2-photon process. Finally, potential applications of single-mol. Raman spectroscopy are addressed. The perspectives and limitations of single-mol. 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Surface enhanced Raman spectroscopy (SERS) is a powerful anal. technique that has been proposed as a substitute for fluorescence for biol. imaging and detection but is not yet com. utilized. The reason lies primarily in the lower intensity and poor reproducibility of most metal nanoparticle-based tags as compared to their fluorescence-based counterparts. Here, using a technique that scrupulously preserves the same no. of dye mols. in both the SERS and fluorescence measurements, we show that SERS-based biotags (SBTs) with highly reproducible optical properties can be nanoengineered such that their brightness is at least equal to that of fluorescence-based tags. 6 Li, J. Huang, Y. Ding, Y. Yang, Z. L. Li, S. Zhou, X. S. Fan, F. R. Zhang, W. Zhou, Z. Y. Wu, D. Ren, B. Wang, Z. Tian, Z. Q. Nature 2010, 464, 392 – 395 DOI: 10. 1038/nature08907 [ Crossref. PubMed. 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A no. of approaches have extended the technique to non-traditional substrates, most notably tip-enhanced Raman spectroscopy (TERS) where the probed substance (mol. or material surface) can be on a generic substrate and where a nanoscale gold tip above the substrate acts as the Raman signal amplifier. The drawback is that the total Raman scattering signal from the tip area is rather weak, thus limiting TERS studies to mols. with large Raman cross-sections. Here, we report an approach, which we name shell-isolated nanoparticle-enhanced Raman spectroscopy, in which the Raman signal amplification is provided by gold nanoparticles with an ultrathin silica or alumina shell. A monolayer of such nanoparticles is spread as 'smart dust' over the surface that is to be probed. The ultrathin coating keeps the nanoparticles from agglomerating, separates them from direct contact with the probed material and allows the nanoparticles to conform to different contours of substrates. High-quality Raman spectra were obtained on various mols. adsorbed at Pt and Au single-crystal surfaces and from Si surfaces with hydrogen monolayers. These measurements and our studies on yeast cells and citrus fruits with pesticide residues illustrate that our method significantly expands the flexibility of SERS for useful applications in the materials and life sciences, as well as for the inspection of food safety, drugs, explosives and environment pollutants. 7 Cao, Y. Jin, R. Mirkin, C. A. Science 2002, 297, 1536 DOI: 10. 1126/science. 297. 5586. 1536 [ Crossref. PubMed. CAS]   Google Scholar 7 Nanoparticles with Raman spectroscopic fingerprints for DNA and RNA detection Cao, YunWei Charles; Jin, Rongchao; Mirkin, Chad A. Science (Washington, DC, United States) 2002) 297 ( 5586) 1536-1540 CODEN: SCIEAS; ISSN: 0036-8075. ( American Association for the Advancement of Science) Multiplexed detection of oligonucleotide targets has been performed with gold nanoparticle probes labeled with oligonucleotides and Raman-active dyes. The gold nanoparticles facilitate the formation of a silver coating that acts as a surface-enhanced Raman scattering promoter for the dye-labeled particles that have been captured by target mols. and an underlying chip in microarray format. The strategy provides the high-sensitivity and high-selectivity attributes of gray-scale scanometric detection but adds multiplexing and ratioing capabilities because a very large no. of probes can be designed based on the concept of using a Raman tag as a narrow-band spectroscopic fingerprint. Six dissimilar DNA targets with six Raman-labeled nanoparticle probes were distinguished, as well as two RNA targets with single nucleotide polymorphisms. The current unoptimized detection limit of this method is 20 femtomolar. 8 Braun, G. Lee, S. J. 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These species can include a Raman label (a mol. with a very large Raman cross-section) that dominates the spectrum and generates highly reproducible signals. The self-assembly process does not take place in the absence of the target species. Consequently, a strong SERS signal is obsd. only in the presence of the target. The SERS signal was also absent in the presence of noncomplementary species. AFM anal. indicates that strong SERS signal intensity arises from only a few surface-bound nanoparticles which generate an enhancement factor ∼105-106 greater than the metal film alone. Notably, this nanoparticle-film DNA detection method does not require any chem. deposition of silver to read out the SERS spectrum from the surface-bound labels. 9 Fabris, L. O. Bazan, G. C. 2007, 129, 6086 – 6087 DOI: 10. 1021/ja0705184 [ ACS Full Text. CAS]   Google Scholar 9 A Heterogeneous PNA-Based SERS Method for DNA Detection Fabris, Laura; Dante, Mark; Braun, Gary; Lee, Seung Joon; Reich, Norbert O. 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The accurate exptl. detn. of single mol. enhancement factors is tackled with 2 recently developed techniques, namely: bi-analyte SERS (BiASERS) and temp. -dependent SERS vibrational pumping. SERS EFs ≥107, as opposed to the figure of 1014 often claimed in the literature, are sufficient for the observation of single mol. SERS signals, with max. single mol. EFs typically on the order of ∼1010. A significant amt. of review material is included. 12 Kneipp, K. Wang, Y. Perelman, L. T. Itzkan, I. Dasari, R. Feld, M. Lett. 1997, 78, 1667 – 1670 DOI: 10. 1103/PhysRevLett. 78. 1667 [ Crossref. CAS]   Google Scholar 12 Single molecule detection using surface-enhanced Raman scattering (SERS) Kneipp, Katrin; Wang, Yang; Kneipp, Harald; Perelman, Lev T. Itzkan, Irving; Dasari, Ramachandra R. Feld, Michael S. Physical Review Letters ( 1997) 78 ( 9) 1667-1670 CODEN: PRLTAO; ISSN: 0031-9007. ( American Physical Society) By exploiting the extremely large effective cross sections (10-17-10-16 cm2/mol. available from surface-enhanced Raman scattering (SERS) the authors achieved the 1st observation of single mol. Raman scattering. Measured spectra of a single crystal violet mol. in aq. colloidal Ag soln. using one 2nd collection time and ∼2 × 105 W/cm2 nonresonant near-IR excitation show a clear fingerprint of its Raman features between 700 and 1700 cm-1. Spectra obsd. in a time sequence for an av. of 0. 6 dye mol. in the probed vol. exhibited the expected Poisson distribution for actually measuring 0, 1, 2, or 3 mols. 13 Nie, S. Emory, S. Science 1997, 275, 1102 – 1106 DOI: 10. 275. 5303. 1102 [ Crossref. PubMed. CAS]   Google Scholar 13 Probing single molecules and single nanoparticles by surface-enhanced Raman scattering Nie, Shuming; Emory, Steven R. 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( National Academy of Sciences) Raman spectroscopy is a newly developed, noninvasive preclin. imaging technique that offers picomolar sensitivity and multiplexing capabilities to the field of mol. imaging. In this study, we demonstrate the ability of Raman spectroscopy to sep. the spectral fingerprints of up to 10 different types of surface enhanced Raman scattering (SERS) nanoparticles in a living mouse after s. c. injection. Based on these spectral results, we simultaneously injected the five most intense and spectrally unique SERS nanoparticles i. v. to image their natural accumulation in the liver. All five types of SERS nanoparticles were successfully identified and spectrally sepd. using our optimized noninvasive Raman imaging system. In addn., we were able to linearly correlate Raman signal with SERS concn. after injecting four spectrally unique SERS nanoparticles either s. (R2 = 0. 998) or i. 992. These results show great potential for multiplexed imaging in living subjects in cases in which several targeted SERS probes could offer better detection of multiple biomarkers assocd. with a specific disease. 16 Li, M. Kang, J. Sukumar, S. Barman, I. 2015, 6, 3906 – 3914 DOI: 10. 1039/C5SC01054C [ Crossref. PubMed. CAS]   Google Scholar 16 Multiplexed detection of serological cancer markers with plasmon-enhanced Raman spectro-immunoassay Li, Ming; Kang, Jeon Woong; Sukumar, Saraswati; Dasari, Ramachandra Rao; Barman, Ishan Chemical Science 6 ( 7) 3906-3914 CODEN: CSHCCN; ISSN: 2041-6520. Circulating biomarkers have emerged as promising non-invasive, real-time surrogates for cancer diagnosis, prognostication and monitoring of therapeutic response. Emerging data, however, suggest that single markers are inadequate in describing complex pathol. transformations. Architecting assays capable of parallel measurements of multiple biomarkers can help achieve the desired clin. sensitivity and specificity while conserving patient specimen and reducing turn-around time. Here we describe a plasmon-enhanced Raman spectroscopic assay featuring nanostructured biomol. probes and spectroscopic imaging for multiplexed detection of disseminated breast cancer markers cancer antigen (CA) 15-3, CA 27-29 and cancer embryonic antigen (CEA. In the developed SERS assay, both the assay chip and surface-enhanced Raman spectroscopy (SERS) tags are functionalized with monoclonal antibodies against CA15-3, CA27-29 and CEA, resp. Sequential addn. of biomarkers and functionalized SERS tags onto the functionalized assay chip enable the specific recognition of these biomarkers through the antibody-antigen interactions, leading to a sandwich spectro-immunoassay. In addn. to offering extensive multiplexing capability, our method provides higher sensitivity than conventional immunoassays and demonstrates exquisite specificity owing to selective formation of conjugated complexes and fingerprint spectra of the Raman reporter. We envision that clin. translation of this assay may further enable asymptomatic surveillance of cancer survivors and speedy assessment of treatment benefit through a simple blood test. 17 Dasary, S. Singh, A. K. Senapati, D. Yu, H. Ray, P. 2009, 131, 13806 – 13812 DOI: 10. 1021/ja905134d [ ACS Full Text. CAS]   Google Scholar 17 Gold Nanoparticle Based Label-Free SERS Probe for Ultrasensitive and Selective Detection of Trinitrotoluene Dasary, Samuel S. Singh, Anant Kumar; Senapati, Dulal; Yu, Hongtao; Ray, Paresh Chandra Journal of the American Chemical Society 131 ( 38) 13806-13812 CODEN: TNT is one of the most commonly used nitro arom. explosives used for landmine and military purpose. Due to the significant detrimental effects, contamination of soil and groundwater with TNT is the major concern. Driven by the need to detect trace amts. of TNT from environmental samples, this article demonstrates for the first time a highly selective and ultra sensitive, cysteine modified gold nanoparticle based label-free surface enhanced Raman spectroscopy (SERS) probe, for TNT recognition in 2 pico molar (pM) level in aq. soln. Due to the formation of Meisenheimer complex between TNT and cysteine, gold nanoparticles undergo aggregation in the presence of TNT via electrostatic interaction between Meisenheimer complex bound gold nanoparticle and cysteine modified gold nanoparticle. As a result, it formed several hot spots and provided a significant enhancement of the Raman signal intensity by 9 orders of magnitude through electromagnetic field enhancements. A detailed mechanism for termendous SERS intensity change has been discussed. Our exptl. results show that TNT can be detected quickly and accurately without any dye tagging in lower pM level with excellent discrimination against other nitro compds. and heavy metals. 18 Bishnoi, S. Lin, Y. Tibudan, M. Nakaema, M. Swarup, V. Keiderling, T. Anal. 2011, 83, 4053 – 4060 DOI: 10. 1021/ac103195e [ ACS Full Text. CAS]   Google Scholar 18 SERS Biodetection Using Gold-Silica Nanoshells and Nitrocellulose Membranes Bishnoi, Sandra Whaley; Lin, Yu-jen; Tibudan, Martin; Huang, Yiming; Nakaema, Marcelo; Swarup, Vimal; Keiderling, Timothy A. Analytical Chemistry (Washington, DC, United States) 2011) 83 ( 11) 4053-4060 CODEN: ANCHAM; ISSN: 0003-2700. We have developed a rapid, reproducible, easy to execute, surface enhanced Raman scattering (SERS) method for detection of low vols. and total amts. of biol. antigens using an analyte capture system derived from methods commonly used in Western blotting. Our method is a "half-sandwich" assay with an antigen detection scheme that employs a nitrocellulose (NC) membrane with 200 nm pore size to capture subnanograms of analyte and conc. them in a small area from applied vols. as low as one microliter. The SERS probes used for detection consist of gold-silica nanoshells modified with a two-component mixed monolayer system. One component consists of a poly(ethylene glycol) PEG) modified Raman-active chromophore bound to the gold surface which allows for SERS detection and imparts particle stability. The second component uses (ortho-pyridyl) disulfide-PEG-succinimidyl ester to couple the recognition antibody to the particle surface. By controlling the reaction time and concn. of thiols, a mixed monolayer is prepd. on the nanoshell surface with the ability to recognize low concns. of analyte and generate reproducible SERS signals. Using this strategy, we have achieved SERS signals that are proportional to antigen present on the membrane allowing detection of total antigen amts. as low as 1. 25 ng for some cases. The performance of this new SERS bioassay has been tested with a variety of potential antigens, demonstrating the potential for multiplexed detection of analytes. 19 Harmsen, S. Bedics, M. Wall, M. Huang, R. Detty, M. Kircher, M. Nat. Commun. 2015, 6, 6570 DOI: 10. 1038/ncomms7570 [ Crossref. PubMed. CAS]   Google Scholar 19 Rational design of a chalcogenopyrylium-based surface-enhanced resonance Raman scattering nanoprobe with attomolar sensitivity Harmsen, Stefan; Bedics, Matthew A. Wall, Matthew A. Huang, Ruimin; Detty, Michael R. Kircher, Moritz F. Nature Communications ( 6570 CODEN: NCAOBW; ISSN: 2041-1723. High sensitivity and specificity are two desirable features in biomedical imaging. Raman imaging has surfaced as a promising optical modality that offers both. Here we report the design and synthesis of a group of near-IR absorbing 2-thienyl-substituted chalcogenopyrylium dyes tailored to have high affinity for gold. When adsorbed onto gold nanoparticles, these dyes produce biocompatible SERRS nanoprobes with attomolar limits of detection amenable to ultrasensitive in vivo multiplexed tumor and disease marker detection. 20 Lim, D. Jeon, K. -S. Kim, H. Nam, J. Suh, Y. D. Mater. 2010, 9, 60 – 67 DOI: 10. 1038/nmat2596 [ Crossref. PubMed. CAS]   Google Scholar 20 Nanogap-engineerable Raman-active nanodumbbells for single-molecule detection Lim, Dong-Kwon; Jeon, Ki-Seok; Kim, Hyung Min; Nam, Jwa-Min; Suh, Yung Doug Nature Materials 9 ( 1) 60-67 CODEN: NMAACR; ISSN: 1476-1122. Surface-enhanced Raman scattering (SERS) based signal amplification and detection methods using plasmonic nanostructures have been widely investigated for imaging and sensing applications. However, SERS-based mol. detection strategies have not been practically useful because there is no straightforward method to synthesize and characterize highly sensitive SERS-active nanostructures with sufficiently high yield and efficiency, which results in an extremely low cross-section area in Raman sensing. Here, the authors report a high-yield synthetic method for SERS-active gold-silver core-shell nanodumbbells, where the gap between two nanoparticles and the Raman-dye position and environment can be engineered on the nanoscale. Atomic-force-microscope-correlated nano-Raman measurements of individual dumbbell structures demonstrate that Raman signals can be repeatedly detected from single-DNA-tethered nanodumbbells. These programmed nanostructure fabrication and single-DNA detection strategies open avenues for the high-yield synthesis of optically active smart nanoparticles and structurally reproducible nanostructure-based single-mol. detection and bioassays. 21 Lim, D. Hwang, J. H. Kwon, S. Nanotechnol. 2011, 6, 452 – 460 DOI: 10. 1038/nnano. 2011. 79 [ Crossref. PubMed. CAS]   Google Scholar 21 Highly uniform and reproducible surface-enhanced Raman scattering from DNA-tailorable nanoparticles with 1-nm interior gap Lim, Dong-Kwon; Jeon, Ki-Seok; Hwang, Jae-Ho; Kim, Hyoki; Kwon, Sunghoon; Suh, Yung Doug; Nam, Jwa-Min Nature Nanotechnology 452-460 CODEN: NNAABX; ISSN: 1748-3387. An ideal surface-enhanced Raman scattering (SERS) nanostructure for sensing and imaging applications should induce a high signal enhancement, generate a reproducible and uniform response, and should be easy to synthesize. Many SERS-active nanostructures have been investigated, but they suffer from poor reproducibility of the SERS-active sites, and the wide distribution of their enhancement factor values results in an unquantifiable SERS signal. Here, we show that DNA on gold nanoparticles facilitates the formation of well-defined gold nanobridged nanogap particles (Au-NNP) that generate a highly stable and reproducible SERS signal. The uniform and hollow gap (∼1 nm) between the gold core and gold shell can be precisely loaded with a quantifiable amt. of Raman dyes. SERS signals generated by Au-NNPs showed a linear dependence on probe concn. (R2 > 0. 98) and were sensitive down to 10fM concns. Single-particle nano-Raman mapping anal. revealed that >90% of Au-NNPs had enhancement factors greater than 1. 0 × 108, which is sufficient for single-mol. detection, and the values were narrowly distributed between 1. 0 × 108 and 5. 0 × 109. 22 Feng, Y. Wang, H. Chen, T. Tay, Y. Yao, L. Yan, Q. Chen, H. Small 2012, 8, 246 – 251 DOI: 10. 1002/smll. 201102215 [ Crossref. PubMed. CAS]   Google Scholar 22 Engineering "Hot" Nanoparticles for Surface-Enhanced Raman Scattering by Embedding Reporter Molecules in Metal Layers Feng, Yuhua; Wang, Yong; Wang, Hong; Chen, Tao; Tay, Yee Yan; Yao, Lin; Yan, Qingyu; Li, Shuzhou; Chen, Hongyu Small ( 2012) 8 ( 2) 246-251 CODEN: SMALBC; ISSN: 1613-6810. The authors carried out colloidal deposition of Ag on Au nanoparticles (NPs) functionalized with 4-mercaptobenzoic acid (MBA) thus forming a core-shell structure with embedded MBA. The uniform nanostructures are correlated to their colloidal Raman spectra, which show at least 20 times stronger SERS signals than the already enhanced SERS signal of MBA on Ag NPs of similar size. Polymer encapsulation in polystyrene-block-poly(acrylic acid) is used to ensure that few aggregates exist in the resulting NPs, and thus, the authors are able to present unambiguous evidence for the large SERS enhancement arising from the embedded MBA. Exploiting this clear SERS-nanostructure correlation, the authors carried out detailed control expts. that supported the embedding of org. mols. during Ag deposition. 23 Shen, W. Lin, X. Jiang, C. Li, C. Lin, H. Huang, J. Wang, S. Liu, G. Yan, X. Zhong, Q. 2015, 54, 7308 – 7312 DOI: 10. 201502171 [ Crossref. PubMed. CAS]   Google Scholar 23 Reliable Quantitative SERS Analysis Facilitated by Core-Shell Nanoparticles with Embedded Internal Standards Shen, Wei; Lin, Xuan; Jiang, Chaoyang; Li, Chaoyu; Lin, Haixin; Huang, Jingtao; Wang, Shuo; Liu, Guokun; Yan, Xiaomei; Zhong, Qiling; Ren, Bin Angewandte Chemie, International Edition 54 ( 25) 7308-7312 CODEN: Quant. anal. is a great challenge in surface-enhanced Raman scattering (SERS. Core-mol. -shell nanoparticles with two components in the mol. layer, a framework mol. to form the shell, and a probe mol. as a Raman internal std., were rationally designed for quant. SERS anal. The signal of the embedded Raman probe provides effective feedback to correct the fluctuation of samples and measuring conditions. Meanwhile, target mols. with different affinities can be adsorbed onto the shell. The quant. of target mols. over a large concn. range was demonstrated with a linear response of the relative SERS intensity vs. the surface coverage, which was not achieved by conventional SERS methods. 24 Lee, J. Lim, D. Lee, H. ACS Nano 2012, 6, 9574 – 9584 DOI: 10. 1021/nn3028216 [ ACS Full Text. CAS]   Google Scholar 24 Tuning and Maximizing the Single-Molecule Surface-Enhanced Raman Scattering from DNA-Tethered Nanodumbbells Lee, Jung-Hoon; Nam, Jwa-Min; Jeon, Ki-Seok; Lim, Dong-Kwon; Kim, Hyoki; Kwon, Sunghoon; Lee, Haemi; Suh, Yung Doug ACS Nano 9574-9584 CODEN: ANCAC3; ISSN: 1936-0851. The authors extensively study the relations between single-mol. surface-enhanced Raman scattering (SMSERS) intensity, enhancement factor (EF) distribution over many particles, interparticle distance, particle size/shape/compn. and excitation laser wavelength using the single-particle AFM-correlated Raman measurement method and theor. calcns. Two different single-DNA-tethered Au-Ag core-shell nanodumbbell (GSND) designs with an engineerable nanogap were used in this study: the GSND-I with various interparticle nanogaps from ∼4. 8 nm to <1 nm or with no gap and the GSND-II with the fixed interparticle gap size and varying particle size from a 23-30 nm pair to a 50-60 nm pair. From the GSND-I, synthesizing a <1 nm gap is a key to obtain strong SMSERS signals with a narrow EF value distribution. Importantly, in the case of the GSND-I with <1 nm interparticle gap, an EF value of ≤5. 9 × 1013 (av. value = 1. 8 × 1013) was obtained and the EF values of analyzed particles were narrowly distributed between 1. 9 × 1012 and 5. 9 × 1013. In the case of the GSND-II probes, a combination of >50 nm Au cores and 514. 5 nm laser wavelength that matches well with Ag shell generated stronger SMSERS signals with a more narrow EF distribution than <50 nm Au cores with 514. 5 nm laser or the GSND-II structures with 632. 8 nm laser. Results show the usefulness and flexibility of these GSND structures in studying and obtaining SMSERS structures with a narrow distribution of high EF values and that the GSNDs with < 1 nm are promising SERS probes with highly sensitive and quant. detection capability when optimally designed. 25 Guarrotxena, N. Adv. 2014, 26, 1941 – 1946 DOI: 10. 1002/adma. 201304107 [ Crossref. PubMed. CAS]   Google Scholar 25 Antitags: SERS-Encoded Nanoparticle Assemblies that Enable Single-Spot Multiplex Protein Detection Guarrotxena, Nekane; Bazan, Guillermo C. Advanced Materials (Weinheim, Germany) 26 1941-1946 CODEN: ADVMEW; ISSN: 0935-9648. Antitags, namely multi-nanoparticle assemblies with SERS reporting linkers and surface functionalities capable of specific recognition and minimizing non-specific interactions, can be used as the basis for developing versatile protein detection protocols. The assay overcomes conventional limitations on simultaneous multiple detection in complex mixts. and attests the efficiency and selectivity of the individual antitags for single-spot simultaneous recognition of specific targets with sensitivities of 100 pM. The effective SERS-based multivariate deconvolution approach provides accurate and distinguishable identification of single and multiple proteins in complex samples. These features are desirable for developing innovative early disease diagnosis and high-throughput biomols. screening methods. 26 Driskell, J. Kwarta, K. Lipert, R. Porter, M. Neill, J. Ridpath, J. 2005, 77, 6147 – 6154 DOI: 10. 1021/ac0504159 [ ACS Full Text. CAS]   Google Scholar 26 Low-Level Detection of Viral Pathogens by a Surface-Enhanced Raman Scattering Based Immunoassay Driskell, Jeremy D. Kwarta, Karen M. Lipert, Robert J. Porter, Marc D. Neill, John D. Ridpath, Julia F. Analytical Chemistry ( 2005) 77 6147-6154 CODEN: The need for rapid, highly sensitive, and versatile diagnostic tests for viral pathogens spans from human and veterinary medicine to bioterrorism prevention. As an approach to meet these demands, a diagnostic test employing monoclonal antibodies (mAbs) for the selective extn. of viral pathogens from a sample in a chip-scale, sandwich immunoassay format has been developed using surface-enhanced Raman scattering (SERS) as a readout method. The strengths of SERS-based detection include its inherent high sensitivity and facility for multiplexing. The capability of this approach is demonstrated by the capture of feline calicivirus (FCV) from cell culture media that is exposed to a gold substrate modified with a covalently immobilized layer of anti-FCV mAbs. The surface-bound FCVs are subsequently coupled with an extrinsic Raman label (ERL) for identification and quantification. The ERLs consist of 60-nm gold nanoparticles coated first with a layer of Raman reporter mols. and then a layer of mAbs. The Raman reporter mol. is strategically designed to chemisorb as a thiolate adlayer on the gold nanoparticle, to provide a strong and unique spectral signature, and to covalently link a layer of mAbs to the gold nanoparticle. The last feature provides a means to selectively tag substrate-bound FCV. This paper describes the development of the assay, which uses cell culture media as a sample matrix and has a linear dynamic range of 1 × 106-2. 5 × 108 viruses/mL and a limit of detection of 1 × 106 viruses/mL. These results reflect the findings from a detailed series of investigations on the effects of several exptl. parameters (e. g., salt concn., ERL binding buffer, and sample agitation) all of which were aimed at minimizing nonspecific binding and maximizing FCV binding efficiency. The performance of the assay is correlated with the no. of captured FCV, detd. by at. force microscopy, as a means of method validation. 27 Camden, J. Dieringer, J. Zhao, J. Van Duyne, R. Acc. Res. 2008, 41, 1653 – 1661 DOI: 10. 1021/ar800041s [ ACS Full Text. CAS]   Google Scholar 27 Controlled Plasmonic Nanostructures for Surface-Enhanced Spectroscopy and Sensing Camden, Jon P. Dieringer, Jon A. Zhao, Jing; Van Duyne, Richard P. Accounts of Chemical Research 41 1653-1661 CODEN: ACHRE4; ISSN: 0001-4842. After its discovery more than 30 years ago, surface-enhanced Raman spectroscopy (SERS) was expected to have major impact as a sensitive anal. technique and tool for fundamental studies of surface species. Unfortunately, the lack of reliable and reproducible fabrication methods limited its applicability. In recent years, SERS has enjoyed a renaissance, and there is renewed interest in both the fundamentals and applications of SERS. New techniques for nanofabrication, the design of substrates that maximize the electromagnetic enhancement, and the discovery of single-mol. SERS are driving the resurgence of this field. This Account highlights our group's recent work on SERS. Initially, we discuss SERS substrates that have shown proven reproducibility, stability, and large field enhancement. These substrates enable many anal. applications, such as anthrax detection, chem. warfare agent stimulant detection, and in vitro and in vivo glucose sensing. We then turn to a detailed study of the wavelength and distance dependence of SERS, which further illustrate predictions obtained from the electromagnetic enhancement mechanism. Last, an isotopic labeling technique applied to the rhodamine 6G (R6G) silver system serves as an addnl. proof of the existence of single-mol. SERS and explores the dynamical features of this process. This work, in conjunction with theor. calcns., allows us to comment on the possible role of charge transfer in the R6G/silver system. 28 Indrasekara, A. Paladini, B. Naczynski, D. Starovoytov, V. Moghe, P. V. Fabris, L. Healthcare Mater. 2013, 2, 1370 – 1376 DOI: 10. 1002/adhm. 201200370 [ Crossref. PubMed. CAS]   Google Scholar 28 Dimeric Gold Nanoparticle Assemblies as Tags for SERS-Based Cancer Detection Indrasekara, A. Swarnapali D. Paladini, Bryan J. Naczynski, Dominik J. Starovoytov, Valentin; Moghe, Prabhas V. Fabris, Laura Advanced Healthcare Materials 2 1370-1376 CODEN: AHMDBJ; ISSN: 2192-2640. Herein, a new class of multifunctional materials combining a clustered nanoparticle-based probe is presented for surface enhanced Raman scattering (SERS) based microscopy and surface functionalization for tissue targeting. Controlled assembly of spherical gold nanoparticles into dimers (DNP-REP) is engineered using a small, rigid Raman-active dithiolated linking reporter (REP) to yield narrow internanoparticle gaps and to strategically generate the "hot spot" while concurrently placing the reporter within the region of highest SERS enhancement. Peptide functionalized DNP-REP materials are highly stable even upon incubation with living cells and show controlled levels of binding and intracellular endocytosis. To demonstrate the functionality of such probes for disease detection, differentially targeted DNP-REPs are incubated over various time points with cultured human glioblastoma cells. Using human glioblastoma cells, the SERS maps of targeted tumor cells show the markedly enhanced signals of the DNP-REP, compared to conventional confocal fluorescence based approaches, esp. at low incubation times. Even with as few as 40 internalized DNP-REP, a relatively intense SERS signal is measured, demonstrating the high signal to noise ratio and inherent biocompatibility of the materials. Thus, these Raman reporter-based nanoparticle cluster probes present a promising and versatile optical imaging tool for fast, reliable, selective, and ultrasensitive tissue targeting and disease detection and screening. 29 Shaw, C. Fan, M. Lane, C. Barry, G. Jirasek, A. I. Brolo, A. C 2013, 117, 16596 – 16605 DOI: 10. 1021/jp404250q [ ACS Full Text. CAS]   Google Scholar 29 Statistical Correlation Between SERS Intensity and Nanoparticle Cluster Size Shaw, Conor P. Fan, Meikun; Lane, Chelsey; Barry, Garrett; Jirasek, Andrew I. Brolo, Alexandre G. Journal of Physical Chemistry C 117 16596-16605 CODEN: Surface enhanced Raman scattering (SERS) mapping of biomarkers has shown great promise in detg. the distribution of proteins of interest in cells and tissues. Metallic nanoparticle (NP) probes are generally used in such mapping. Since SERS intensities from NPs are dependent on size, shape, and interparticle distance/distribution, it is unclear if this method can provide reliable biomarker quantification. To address this problem, we investigated a statistical approach to the quantification of SERS from SERS probe clusters. The investigation began by considering multiple biotinylated surfaces that had been exposed to pegylated NPs (designed for biol. SERS mapping) functionalized with streptavidin (defined as SERS probes. The surfaces were imaged with a scanning electron microscope and SERS-mapped with a Raman microscope. Statistical distributions of the SERS probe clusters and mapped SERS intensities on the surfaces were developed. It was found that there was a smooth polynomial relationship between SERS intensity and probe cluster size. Our result is in contrast to the sharp, highly variable intensity increases obsd. in studies of unmodified NPs. Based on the polynomial relationship found, it is clear that pegylated NP SERS probes might be useful for quantification in the SERS mapping of biol. material, as the SERS intensity can be potentially related back to the no. of probes at the acquisition point. 30 Song, C. Zhang, R. Yang, J. Tan, X. Cui, Y. Biosens. Bioelectron. 2009, 25, 826 – 831 DOI: 10. 1016. Crossref. PubMed. CAS]   Google Scholar 30 Highly sensitive immunoassay based on Raman reporter-labeled immuno-Au aggregates and SERS-active immune substrate Song, Chunyuan; Wang, Zhuyuan; Zhang, Ruohu; Yang, Jing; Tan, Xuebin; Cui, Yiping Biosensors & Bioelectronics 25 ( 4) 826-831 CODEN: BBIOE4; ISSN: 0956-5663. ( Elsevier B. ) A highly sensitive immunoassay based on surface enhanced Raman scattering (SERS) was developed with a novel immune marker named as Raman reporter-labeled immuno-Au aggregates on a SERS-active immune substrate. The features of those immune aggregates were characterized by UV-vis extinction spectra, TEM images, SEM pictures and SERS spectra. It is found that stable gold aggregates in appropriate morphologies can be induced by mixing proper amts. of reporter mols. with gold nanoparticles. Based on those reporter-labeled Au aggregates, the immune aggregates with high stability can be prepd. successfully by immobilizing antibody to the surface of the aggregates. Using those easily prepd. aggregates showing strong SERS activity and high bio-specificity, a highly effective SERS-based immunoassay was performed. Moreover, a SERS-active immune substrate was introduced to replace a typical immune substrate without any SERS activity. The authors' exptl. results indicate that the SERS-active immune substrate makes a certain contribution to the highly sensitive immunoassay. As a result, with this proposed immunoassay structure, the concn. detection of human-IgG was performed and a calibration curve was obtained in the range from 100 ng mL-1 to 100 fg mL-1. This work opens a new avenue for sensitive immunoassay and other biochem. based on SERS. 31 Rodríguez-Lorenzo, L. Álvarez-Puebla, R. Pastoriza-Santos, I. Mazzucco, S. Stéphan, O. Kociak, M. Liz-Marzán, L. García de Abajo, F. 2009, 131, 4616 – 4618 DOI: 10. 1021/ja809418t [ ACS Full Text. CAS]   Google Scholar 31 Zeptomol detection through controlled ultrasensitive surface-enhanced Raman scattering Rodriguez-Lorenzo, Laura; Alvarez-Puebla, Ramon A. Pastoriza-Santos, Isabel; Mazzucco, Stefano; Stephan, Odile; Kociak, Mathieu; Liz-Marzan, Luis M. Garcia de Abajo, F. Javier Journal of the American Chemical Society ( 13) 4616-4618 CODEN: SERS permits identifying the nature of mols. in extremely low concns., but it is hindered by poor enhancement or low reproducibility. The authors demonstrate controllable ∼1010 signal amplification reaching the zeptomol detection limit for a nonresonant mol. by sandwiching the analyte between the tips of star-shaped gold nanoparticles and a planar gold surface using a simple synthetic procedure. This unprecedented control over light-intensity amplification opens a new avenue toward high-yield, fully reproducible, SERS-based, zeptomol detection and holds promise for nonlinear optics applications at the single-particle level. 32 Shiohara, A. Photochem. Photobiol., C 2014, 21, 2 – 25 DOI: 10. 1016/hotochemrev. 2014. 09. 001 [ Crossref. CAS]   Google Scholar 32 Recent approaches toward creation of hot spots for SERS detection Shiohara, Amane; Wang, Yusong; Liz-Marzan, Luis M. Journal of Photochemistry and Photobiology, C: Photochemistry Reviews 21 2-25 CODEN: JPPCAF; ISSN: 1389-5567. A review. Surface enhanced Raman scattering (SERS) spectroscopy is a powerful technique that provides mol. information through greatly enhanced Raman scattering from minute amts. of substance near nanostructured metallic surfaces. SERS is thus a promising technique for ultrasensitive sensing applications. Plasmonic nanostructures including metal nanoparticles and lithog. prepd. nanostructures are ideal substrates to produce enhanced Raman signals. Numerous studies have been published on the prodn. of SERS-active substrates for SERS measurements including soln. phase methods and solid supports. In SERS applications, hot spots where the electromagnetic field is particularly intense, play a key role. In this review, we provide an overview of techniques designed for the creation of SERS hot spots both in soln. and on solid supports. We first introduce the self-assembly of spherical and anisotropic nanoparticles in soln., to then focus on a wide variety of techniques to assemble nanoparticles onto solid supports. We also describe top-down approaches typically based on lithog. techniques. Finally, we provide our own view on the current state of the field and the aspects where further development is expected. 33 Gonzá lez-Rubio, G. González-Izquierdo, J. Bañares, L. Tardajos, G. Rivera, A. Altantzis, T. Bals, S. Peña-Rodríguez, O. Guerrero-Martínez, A. 2015, 15, 8282 – 8288 DOI: 10. 5b03844 34 Rodríguez-Fernández, D. Langer, J. Henriksen-Lacey, M. 2015, 27, 2540 – 2545 DOI: 10. 1021/emmater. 5b00128 [ ACS Full Text. CAS]   Google Scholar 34 Hybrid Au-SiO2 Core-Satellite Colloids as Switchable SERS Tags Rodriguez-Fernandez, Denis; Langer, Judith; Henriksen-Lacey, Malou; Liz-Marzan, Luis M. Chemistry of Materials 27 2540-2545 CODEN: CMATEX; ISSN: 0897-4756. Gold-silica self-assembled nanostructures are reported as multiplex surface enhanced Raman scattering (SERS) tags for bioimaging applications. These hybrid colloidal particles were obtained by heteroassembly of Au-SiO2 Janus particles with smaller Au spheres and two Raman-active mols. that can be independently imaged by varying the wavelength of the excitation laser. The Janus structure of Au-SiO2 not only directs the assembly but also provides phys. sepn. of the Raman tags and colloidal stability thereby facilitating complete silica encapsulation. The bioimaging capabilities of this system are demonstrated through SERS mapping of individual cells. 35 Laing, S. Gracie, K. Faulds, K. 2016, 45, 1901 – 1918 DOI: 10. 1039/C5CS00644A [ Crossref. PubMed. CAS]   Google Scholar 35 Multiplex in vitro detection using SERS Laing, Stacey; Gracie, Kirsten; Faulds, Karen Chemical Society Reviews ( 2016) 45 1901-1918 CODEN: The ability to detect multiple disease-related targets from a single biol. sample in a quick and reliable manner is of high importance in diagnosing and monitoring disease. The technique known as surface enhanced Raman scattering (SERS) has been developed for the simultaneous detection of multiple targets present in biol. samples. Advances in the SERS method have allowed for the sensitive and specific detection of biol. relevant targets, such as DNA and proteins, which could be useful for the detection and control of disease. This review focuses on the strengths of SERS for the detection of target mols. from complex mixts. and the clin. relevance of recent work combining SERS with multiplexed detection of biol. targets. 36 Bertoli, G. Cava, C. Castiglioni, I. Theranostics 2015, 5, 1122 – 1143 DOI: 10. 7150/thno. 11543 [ Crossref. PubMed. CAS]   Google Scholar 36 MicroRNAs: new biomarkers for diagnosis, prognosis, therapy prediction and therapeutic tools for breast cancer Bertoli, Gloria; Cava, Claudia; Castiglioni, Isabella Theranostics 5 1122-1143 CODEN: THERDS; ISSN: 1838-7640. ( Ivyspring International Publisher) Dysregulation of microRNAs (miRNAs) is involved in the initiation and progression of several human cancers, including breast cancer (BC) as strong evidence has been found that miRNAs can act as oncogenes or tumor suppressor genes. This review presents the state of the art on the role of miRNAs in the diagnosis, prognosis, and therapy of BC. Based on the results obtained in the last decade, some miRNAs are emerging as biomarkers of BC for diagnosis (i. e., miR-9, miR-10b, and miR-17-5p) prognosis (i. e., miR-148a and miR-335) and prediction of therapeutic outcomes (i. e., miR-30c, miR-187, and miR-339-5p) and have important roles in the control of BC hallmark functions such as invasion, metastasis, proliferation, resting death, apoptosis, and genomic instability. Other miRNAs are of interest as new, easily accessible, affordable, non-invasive tools for the personalized management of patients with BC because they are circulating in body fluids (e. g., miR-155 and miR-210. In particular, circulating multiple miRNA profiles are showing better diagnostic and prognostic performance as well as better sensitivity than individual miRNAs in BC. New miRNA-based drugs are also promising therapy for BC (e. g., miR-9, miR-21, miR34a, miR145, and miR150) and other miRNAs are showing a fundamental role in modulation of the response to other non-miRNA treatments, being able to increase their efficacy (e. g., miR-21, miR34a, miR195, miR200c, and miR203 in combination with chemotherapy. 37 Dong, H. Lei, J. Ding, L. Wen, Y. Ju, H. Zhang, X. 2013, 113, 6207 – 6233 DOI: 10. 1021/cr300362f [ ACS Full Text. CAS]   Google Scholar 37 MicroRNA: Function, Detection, and Bioanalysis Dong, Haifeng; Lei, Jianping; Ding, Lin; Wen, Yongqiang; Ju, Huangxian; Zhang, Xueji Chemical Reviews (Washington, DC, United States) 8) 6207-6233 CODEN: A review. 38 Duan, R. Zuo, X. Quan, X. Chen, D. Chen, Z. Jiang, L. Fan, C. Xia, F. 2013, 135, 4604 – 4607 DOI: 10. 1021/ja311313b [ ACS Full Text. CAS]   Google Scholar 38 Lab in a tube: Ultrasensitive detection of microRNAs at the single-cell level and in breast cancer patients using quadratic isothermal amplification Duan, Ruixue; Zuo, Xiaolei; Wang, Shutao; Quan, Xiyun; Chen, Dongliang; Chen, Zhifei; Jiang, Lei; Fan, Chunhai; Xia, Fan Journal of the American Chemical Society 135 4604-4607 CODEN: Through rational design of a functional mol. probe with high sequence specificity that takes advantage of sensitive isothermal amplification with simple operation, we developed a one-pot hairpin-mediated quadratic enzymic amplification strategy for microRNA (miRNA) detection. Our method exhibits ultrahigh sensitivity toward miR-21 with detection limits of 10 fM at 37 C and 1 aM at 4 C, which corresponds to nine strands of miR-21 in a 15 μL sample, and it is capable of distinguishing among miRNA family members. More importantly, the proposed approach is also sensitive and selective when applied to crude extns. from MCF-7 and PC3 cell lines and even patient tissues from intraductal carcinoma and invasive ductal carcinoma of the breast. 39 Wark, A. Corn, R. 2008, 47, 644 – 652 DOI: 10. 200702450 [ Crossref. PubMed. CAS]   Google Scholar 39 Multiplexed detection methods for profiling microRNA expression in biological samples Wark, Alastair W. Lee, Hye Jin; Corn, Robert M. Angewandte Chemie, International Edition 47 644-652 CODEN: A review. The recent discovery of short, non-protein coding RNA mols., such as microRNA mols. (miRNAs) that can control gene expression has unveiled a whole new layer of complexity in the regulation of cell function. Since 2001, there has been a surge of interest in understanding the regulatory role of the hundreds to thousands of miRNAs expressed in both plants and animals. Significant progress in this area requires the development of quant. bioanal. methods for the rapid, multiplexed detection of all miRNAs that are present in a particular cell or tissue sample. In this Minireview, we discuss some of the latest methods for high-throughput miRNA profiling and the unique technol. challenges that must be surmounted in this endeavor. 40 Valoczi, A. Hornyik, C. Varga, N. Burgyan, J. Kauppinen, S. Havelda, Z. Nucleic Acids Res. 2004, 32, e175 DOI: 10. 1093/nar/gnh171 [ Crossref. PubMed. CAS]   Google Scholar 40 Sensitive and specific detection of microRNAs by northern blot analysis using LNA-modified oligonucleotide probes Valoczi, Anna; Hornyik, Csaba; Varga, Nora; Burgyan, Jozsef; Kauppinen, Sakari; Havelda, Zoltan Nucleic Acids Research ( 2004) 32 ( 22) e175/1-e175/7 CODEN: NARHAD; ISSN: 0305-1048. ( Oxford University Press) We describe here a new method for highly efficient detection of microRNAs by northern blot anal. using LNA (locked nucleic acid) modified oligonucleotides. In order to exploit the improved hybridization properties of LNA with their target RNA mols., the authors designed several LNA-modified oligonucleotide probes for detection of different microRNAs in animals and plants. By modifying DNA oligonucleotides with LNAs using a design, in which every third nucleotide position was substituted by LNA, the authors could use the probes in northern blot anal. employing std. end-labeling techniques and hybridization conditions. The sensitivity in detecting mature microRNAs by northern blots was increased by at least 10-fold compared to DNA probes, while simultaneously being highly specific, as demonstrated by the use of different single and double mismatched LNA probes. Besides being highly efficient as northern probes, the same LNA-modified oligonucleotide probes would also be useful for miRNA in situ hybridization and miRNA expression profiling by LNA oligonucleotide microarrays. 41 Yin, F. Liu, H. Li, Q. Gao, X. Yin, Y. Liu, D. 2016, 88, 4600 – 4604 DOI: 10. 6b00772 [ ACS Full Text. CAS]   Google Scholar 41 Trace MicroRNA Quantification by Means of Plasmon-Enhanced Hybridization Chain Reaction Yin, Fangfei; Liu, Huiqiao; Li, Qiang; Gao, Xia; Yin, Yongmei; Liu, Dingbin Analytical Chemistry (Washington, DC, United States) 88 4600-4604 CODEN: Quantifying trace microRNAs (miRNAs) is extremely important in a no. of biomedical applications but remains a great challenge. Here we present an enzyme-free amplification strategy called plasmon-enhanced hybridization chain reaction (PE-HCR) for quantifying trace miRNAs with an outstanding linear range from 1 fM to 1 pM (r2 = 0. 991) along with a detection limit of 0. 043 fM (1300 mols. in 50 μL of sample. The merits of the PE-HCR assay, including high sensitivity and specificity, quant. detection, no enzyme involvement, low false positives, and easy-to-operate procedures, have been demonstrated for high-confidence quantification of the contents of miRNAs in even single cancer cells. The PE-HCR assay may open up new avenues for highly sensitive quantification of biomarkers and thus should hold great potentials in clin. diagnosis and prognosis. 42 Fang, S. Wark, A. 2006, 128, 14044 – 14046 DOI: 10. 1021/ja065223p [ ACS Full Text. CAS]   Google Scholar 42 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 128 ( 43) 14044-14046 CODEN: 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. 43 Dodgson, B. Mazouchi, A. Wegman, D. Gradinaru, C. Krylov, S. N. 2012, 84, 5470 – 5474 DOI: 10. 1021/ac301546p [ ACS Full Text. CAS]   Google Scholar 43 Detection of a Thousand Copies of miRNA without Enrichment or Modification Dodgson, Bryan J. Mazouchi, Amir; Wegman, David W. Gradinaru, Claudiu C. Krylov, Sergey N. Analytical Chemistry (Washington, DC, United States) 84 5470-5474 CODEN: The authors report direct quant. of multiple miRNAs with a detection limit of 1000 copies without miRNA enrichment or modification. A 300-fold improvement over the previously published detection limit was achieved by combining capillary electrophoresis with confocal time-resolved fluorescence detection through an embedded capillary interface. The method was used to det. levels of three miRNA biomarkers of breast cancer (miRNA 21, 125b, 145) in a human breast cancer cell line (MCF-7. A 30 pL vol. of the cell lysate with approx. a material content of a single cell was sampled for the anal. MiRNA 21, which is up-regulated in breast cancer, was detected at a level of ∼12 thousand copies per cells. MiRNAs 125b and 145, which are down-regulated in breast cancer, were below the 1000-copy detection limit. This sensitive method may facilitate the anal. of miRNA in fine-needle-biopsy samples and even in single cells without enrichment or modification of miRNA. Advantageously, the instrumental setup developed here can be reproduced by others as it requires no sophisticated custom-made parts. This article is cited by 15 publications. Fei Hu, Juan Li, Zengming Zhang, Ming Li, Shuhao Zhao, Zhipeng Li, Niancai Peng. Smartphone-Based Droplet Digital LAMP Device with Rapid Nucleic Acid Isolation for Highly Sensitive Point-of-Care Detection. Analytical Chemistry 2020, 92 (2) 2258-2265. DOI: 10. 9b04967. Jin-Xuan Chen, Ying Zhuo, Xin Peng, Ya-Qin Chai, Ruo Yuan, Wen-Bin Liang. A Dynamic DNA Machine via Free Walker Movement on Lipid Bilayer for Ultrasensitive Electrochemiluminescent Bioassay. Analytical Chemistry 2019, 91 (21) 14125-14132. DOI: 10. 9b03999. Qi Zhang, Jia Liu, Yueru Dong, Wei Li, Rongrong Xing, Yanyan Ma, Zhen Liu. Gold Nanoparticle-Decorated [email protected] Nanocomposite-Based Plasmonic Affinity Sandwich Assay of Circulating MicroRNAs in Human Serum. ACS Applied Nano Materials 2019, 2 (6) 3960-3970. DOI: 10. 1021/acsanm. 9b00855. Wei Liu, Anyi Chen, Shengkai Li, Kanfu Peng, Yaqin Chai, Ruo Yuan. Perylene Derivative/Luminol Nanocomposite as a Strong Electrochemiluminescence Emitter for Construction of an Ultrasensitive MicroRNA Biosensor. Analytical Chemistry 2019, 91 (2) 1516-1523. DOI: 10. 8b04638. Cuicui Fu, Sila Jin, Wenbing Shi, Joohee Oh, Haiyan Cao, Young Mee Jung. Catalyzed Deposition of Signal Reporter for Highly Sensitive Surface-Enhanced Raman Spectroscopy Immunoassay Based on Tyramine Signal Amplification Strategy. Analytical Chemistry 2018, 90 (22) 13159-13162. DOI: 10. 8b02419. Ruiyan Guo, Fangfei Yin, Yudie Sun, Lan Mi, Lin Shi, Zhijin Tian, Tao Li. Ultrasensitive Simultaneous Detection of Multiplex Disease-Related Nucleic Acids Using Double-Enhanced Surface-Enhanced Raman Scattering Nanosensors. ACS Applied Materials & Interfaces 2018, 10 (30) 25770-25778. DOI: 10. 1021/acsami. 8b06757. Ying Zhang, Zhenhua Shuai, Hao Zhou, Zhimin Luo, Bing Liu, Yinan Zhang, Lei Zhang, Shufen Chen, Jie Chao, Lixing Weng, Quli Fan, Chunhai Fan, Wei Huang, Lianhui Wang. Single-Molecule Analysis of MicroRNA and Logic Operations Using a Smart Plasmonic Nanobiosensor. Journal of the American Chemical Society 2018, 140 (11) 3988-3993. DOI: 10. 1021/jacs. 7b12772. Carlos Diego L. de Albuquerque, Regivaldo G. Sobral-Filho, Ronei J. Poppi, and Alexandre G. Brolo. Digital Protocol for Chemical Analysis at Ultralow Concentrations by Surface-Enhanced Raman Scattering. Analytical Chemistry 2018, 90 (2) 1248-1254. DOI: 10. 7b03968. Priya Dharmalingam, Krishnan Venkatakrishnan, Bo Tan. An atomic-defect enhanced Raman scattering (DERS) quantum probe for molecular level detection – Breaking the SERS barrier. Applied Materials Today 2019, 16, 28-41. DOI: 10. 1016/ Fengquan Zhang, Hongjie Si, Chaodong Xue, Songfan Wang, Yixiang Xu, Chenjie Gu, Tao Jiang, Jun Zhou. A space-tunable gold nanoparticle arrays and investigation on their SERS performance. Materials Research Express 2019, 6 (8) 0850h7. DOI: 10. 1088/2053-1591/ab29bf. Xin Gu, Michael J. Trujillo, Jacob E. Olson, Jon P. Camden. SERS Sensors: Recent Developments and a Generalized Classification Scheme Based on the Signal Origin. Annual Review of Analytical Chemistry 2018, 11 (1) 147-169. DOI: 10. 1146/annurev-anchem-061417-125724. Despina P. Kalogianni, Panagiota M. Kalligosfyri, Iraklis K. Kyriakou, Theodore K. Christopoulos. Advances in microRNA analysis. Analytical and Bioanalytical Chemistry 2018, 410 (3) 695-713. DOI: 10. 1007/s00216-017-0632-z. Jing Xue, Feng Chen, Min Bai, Xu Yu, Jing Wei, Ping Huang, Yongxi Zhao. DNA-Mediated Assembly of Gold Nanoparticles and Applications in Bioanalysis. ChemNanoMat 2017, 3 (10) 725-735. DOI: 10. 1002/cnma. 201700177. Lu Zhang, Baozhi Ding, Qinghua Chen, Qiang Feng, Ling Lin, Jiashu Sun. Point-of-care-testing of nucleic acids by microfluidics. TrAC Trends in Analytical Chemistry 2017, 94, 106-116. DOI: 10. 1016/ Huiyuan Guo, Lili He, Baoshan Xing. Applications of surface-enhanced Raman spectroscopy in the analysis of nanoparticles in the environment. Environmental Science: Nano 2017, 4 (11) 2093-2107. DOI: 10. 1039/C7EN00653E.

Edit Release Dates USA 18 May 1995 (Seattle International Film Festival) 19 May 1995 (Los Angeles, California) premiere) 24 May 1995 Australia 1 June 1995 South Korea 17 June 1995 Taiwan 8 July 1995 Brazil 14 July 1995 Philippines 19 July 1995 Uruguay 4 August 1995 Argentina 10 August 1995 Denmark 11 August 1995 Norway 18 August 1995 Czech Republic 31 August 1995 Finland 1 September 1995 Sweden Netherlands 7 September 1995 UK 8 September 1995 Ireland Poland Turkey 22 September 1995 Spain 29 September 1995 France 4 October 1995 Germany 5 October 1995 Romania 13 October 1995 Japan 14 October 1995 Hungary 26 October 1995 Greece 24 November 1995 Italy 1 December 1995 Portugal 15 December 1995 Serbia 14 March 1996 (Belgrade) 11 November 2016 (re-release) Also Known As (AKA) original title) Braveheart Corazón valiente Coração Valente Bulgaria (Bulgarian title) Canada (French title) Coeur vaillant Canada (English title) Croatia Hrabro srce Statečné srdce Estonia Kartmatu Finland (Swedish title) Braveheart - Den okuvlige Braveheart - Taipumaton A rettenthetetlen Rettenthetetlen India (Hindi title) Iran (Persian title) Shojadel Israel (Hebrew title) Lev Amitz Braveheart - Cuore impavido Japan (English title) Japan (Japanese title) ブレイブハート Latvia Drošsirdis Lithuania Narsioji širdis Mexico Peru Braveheart - Waleczne serce Braveheart: O Desafio do Guerreiro Inima neînfricata Russia Slovakia Statočné Srdce Slovenia Pogumno srce Sweden (video box title) 梅爾吉勃遜之英雄本色 Turkey (Turkish title) Cesur Yürek Ukraine (transliterated title) Khorobre sertse Ukraine Venezuela Vietnam Trái Tim Dũng Cảm.

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Full cinema braveheart scene. Surface-enhanced Raman scattering (SERS) as an ultrasensitive vibrational spectroscopic technique, has been applied to many analyses, especially in biochemistry and life sciences in the past decade. (1, 2) To pursue optimum quality of SERS tags, various metal nanoparticles (NPs) and nanocluster-based SERS substrates have been reported. (3, 4) However, another major influence factor, the reporter molecule, has not been studied as extensively as SERS substrates; rational design, screening, and systematic characteristic investigation of novel reporters have been rarely addressed until now. (5, 6) One key problem is that most researchers prefer to apply the ready-made SERS tags. However, the thoughtless use of SERS reporter is problematic for complex detection system, because the overlap of Raman bands between reporters and interfering molecules generates significant impact on the accuracy of the results. Demand on multiplex targeted and simultaneous sensing detection, the complexity of screening, and systematic characteristic investigation of novel reporters will gradually become more obvious. (7, 8) To address this issue, a large number of customized SERS reporters are urgently needed for interference-free multiplex detection at this stage. The alkyne moiety shows a unique Raman emission at ∼2120 cm –1, where most of interfering substances have no Raman response, so a variety of alkyne derivatized metabolic labeling reagents have been used for studying DNA, RNA, proteins, lipids, and other biomolecules. (9, 10) In addition, the Raman shift and intensity of alkynyls can be tuned by changing the substitution pattern, even editing alkyne-carbons with 13 C, 11, 12) which portends that the alkynyl-containing molecules should be very suitable for multiplex detection without optical interferences. Nevertheless, the wide application of alkyne-tag is still fraught with difficulties due to the extremely weak spontaneous Raman scattering of alkynyls. In order to detect the weak signal, relying on long exposure time and nondestructive hi-power excitation, the sensitivity of Raman mapping utilizing stimulated Raman scattering (SRS) technique has been fundamentally improved but still not enough for routine biological imaging. (10, 12) Considering the 10 6 –10 14 -fold of enhancement in Raman signal intensity, the SERS effect based technique seems to be more accessible. Herein, we developed a narrow emission, band shift-tunable, and ultrasensitive alkyne-modulated SERS palette through combining the advantages of alkyne-tags and SERS effect, which was successfully applied to triplex-targeted imaging on both plant and mammalian cells without interference. Because of its super sensitivity and minimal optical interferences, the alkynyl-modulated SERS-palette is of great significance for multiplex sensing and multicolor biological imaging when the hyperspectral and fairly intense optical noises originating from lower wavenumber region (<1800 cm –1) are inevitable under complex ambient conditions. Even more important, our results give birth to a new type of highly sensitive Raman tags for optical interference-free multiplex cellular imaging and multitarget assay. Considering the representative types of Raman reporters, thiol-containing small molecules have the advantages of cheap, strong binding affinity to metal and few Raman peaks (beneficial for multiplexing) compared to nitrogen-containing cationic dyes and thiol-containing dyes. (13) In addition, previous research has indicated that conjugation of alkyne to an aromatic ring greatly increases the Raman intensity of the alkyne and conjugated diynes show much higher intensity compared to simple alkynes. (14) So consulting the chemical structure of thiol-containing small molecules, such as 4-mercaptobenzoic acid, the 4-ethynylbenzenethiol and 4- buta-1, 3-diynyl) benzenethiol are considered as the parent structures of potential alkynes of SERS-palette ( Figure 1. Then the Raman shifts of alkynyl can be tuned by means of linking different substituent groups at the benzene ring or terminal alkynes. In order to verify which alkyne-containing reporter is more suitable as the parent structure and where the substituent groups link on can more obviously adjust the alkyne-Raman-shift, we evaluated and speculated the Raman shifts of alkynyl with the aid of density functional theory (DFT) calculations. The calculated spectra usually are not exactly the same with the experimental results; however, the basic changing trends of alkynyls Raman shifts can be estimated correctly, which is quickly and easily compared to molecular synthetic and actual tests. (15, 16) Figure 1 Figure 1. Computational Raman shifts of 16 alkyne-derivatives, and B1 and C1 are parent structures of alkyne-modulated SERS tagging molecules. Focusing on the above-mentioned ideas, the Raman shifts of alkynyl in 16 alkynes were calculated and shown in Figure 1 (for details, the calculated spectra are shown in Figures S6 and S7. These alkynes were divided into three groups based on the position of substituents and the parent structures. For the typical shift of alkynyl in 4-ethynylbenzenethiol (ν ∼ 2118 cm –1) even different substituents at the benzene ring are incapable of altering the Raman shifts of alkynyl to a great extent, which is just ranging from 2118 to 2126 cm –1 (group A1–A6. On the other hand, adding substituents on the terminal alkyne, however, can readily tune the Raman shifts hugely ranging from 2118 to 2238 cm –1 (group B1–B5) due to the inductive effect of adjacent groups. These results are in agreement with description of previous reports: the Raman shifts of terminal alkynes were generally observed at lower wavenumber (2080–2120 cm –1) those of internal alkynyls were observed at higher wavenumbers (2200 cm –1 or higher. 11, 12) For the B group of alkynes, the increasing electron donation of the alkynes compounds (B1 < B2 < B3 < B5 < B4) makes the charge density of the C≡C bond increase gradually, which leads to bond energy enhancement, and finally the Raman shift of alkynyl is tuned to higher wavenumbers. In addition, the conjugated diynes showed much higher intensity compared to the simple alkyne as expected (see Figures S6 and S7, group C > B ≈ A. However, it is noteworthy that the 4- buta-1, 3-diynyl) benzenethiol-derivatives present two alkynyl Raman peaks if there is great difference between the chemical environments of two alkynes, such as C1–C3. On the basis of the computational results of Raman shifts of 16 alkynes, to substitute different residues at the terminal alkyne of 4-ethynylbenzenethiol is an ideal option for modulating Raman emission of alkynyls. Because in the proposed SERS palettes of alkyne-tags, to select a suitable Raman reporter for multiplex labeling should obey the following two principles: 1) single narrow band emission is more essential and (2) the long interval among different Raman emissions of alkynyls. Considering these, three molecules including B1, B2, and B3 are therefore selected as the reporters in our experiments (synthesis and characterization are shown in the Supporting Information. 17) The sensitivity and reproducibility of SERS intensity are directly related to the surface coverage with reporter molecules and their uniform orientation. It is indispensable that the modification of three aforementioned molecules (B1, B2, B3 recorded as OPE0, OPE1, and OPE2, Figure 2 a) should be investigated and optimized before its application in cellular imaging. The corresponding results and discussion about the adjustment of surface modification and SERS emission of alkyne molecules were shown in Figures S8–S10 and “The optimization for the signal of alkyne molecule” in the Supporting Information. Afterward, three 4-ethynylbenzenethiol-derivatives modified [email protected] NPs show discernible SERS peaks at 2105, 2159, and 2212 cm –1 with excitation at 532 nm ( Figure 2 b. Figure 2 Figure 2. (a) Molecular structures of 4-ethynylbenzenethiol-derivatives. (b) SERS spectra of 4-ethynylbenzenethiol-derivatives with excitation at 532 nm; the colors of the spectra and molecular structures are one-to-one correspondence. Generally, fluorescent and even traditional SERS imaging techniques are applied to plant issues, and there are complications associated with the optical interferences of fairly strong auto fluorescence or resonance-enhanced Raman scattering from plant cell components, such as cell walls, chloroplasts, and pigments. (18, 19) For example, Figure 3 a presents the SEM image of lilys pollen, in which the obvious network structure can be observed for its exine. After tentative Raman measurements, a Raman mapping was performed on the surface of pollen using a green laser at 532 nm. From the rectangular region of Figure 3 a, the fine network structure is also described by a Raman shift at 1580 cm –1, which perfectly matches the fine line in the SEM image. In contrast, the Raman image in Figure S11a is less consistent with SEM characterization if selecting the Raman band at 1525 cm –1 as a marker. The reason is that the network structure of exine is made up of sporopollenin including fatty acid, phenylpropanoids, phenols, and a small amount of pigment molecules (e. g., carotenoids. The Raman band at 1580 cm –1 should be assigned to C–C vibrations of benzene-containing molecules, which mainly contributes the skeletons exine. However, the characteristic vibrational band of carotenoids at 1525 cm –1 is incapable of describing the fine lines of the exine of lilys pollen. In this case, the advantage on minimal optical interferences of alkyne-coded SERS probe would be embodied completely. In our experiments, what we expected that the traditional SERS probes will be disturbed in the fingerprint region because pollen itself also contributes to the fairly strong intrinsic Raman signals in the same spectral region. Also in fact is the case in our studies, in which the OPE2-coded SERS probes were absorbed randomly on the surface of pollen. In Figure 3 b, the pollen and SERS probes both display strong Raman band at 1580 cm –1, so the red area marked by this channel is the sum of spontaneous Raman scattering of exine and SERS signals of probes. On the other hand, there are no Raman responses at 2212 cm –1 for pollen, so the green area marked by this channel just can reveal the real distribution of SERS probes, which has been shown in Figure 3 c. The Raman spectra of positions 1 and 2 in Figure 3 b, c can be addressed in Figure S11b. Figure 3 Figure 3. (a) SEM image of lilys pollen: the Raman image (red) is drawn in a rectangular region relying on Raman shift at 1580 cm –1. (b) After the OPE2 coded-SERS probe absorbed on the surface of pollen, the Raman image is drawn still relying on Raman shift at 1580 cm –1 in the same region, but in part c the alkynyls SERS band at 2212 cm –1 describes the real distribution of the SERS probe. Besides the advantage of optical interference-free, the alkyne-modulated SERS probes are more accessible for multiplex cellular imaging with vibrant colors because the marker channels of these reporters are always relying on single narrow Raman emission. Corresponding to three alkyne-containing molecules, three kinds of functional NPs with each reporter were coated with HS-PEG (MW 5000) as the SERS probes. To test the orthogonality of SERS probes, HeLa cells treated with 200 μL of each SERS probes for 12 h, then fixed, were analyzed using a Raman microspectrometer. The high-resolution SERS imaging of each cell was presented according to the intensity of the following four different Raman shifts: 2000 (off) 2105 (OPE0) 2159 (OPE1) and 2212 cm –1 (OPE2. To obtain the cell image, we used an excitation of 532 nm wavelength, 1. 0 mW incident laser power density and 1 s integration time per pixel (0. 8 μm × 0. 8 μm) which allowed us to obtain an approximately 70 × 60 pixel (depending on cell size) single cell image within 2 h considering the mechanical moving time of the x and y directions. As shown in Figure 4, the OPE0-NPs exhibits intense Raman signal at 2105 cm –1 as expected, depicting the morphology of cells. In contrast, the intensity of Raman bands at 2000, 2159, and 2212 cm –1 is almost not visible. Similarly, when using OPE1-NPs or OPE2-NPs, SERS responses are only detected at the corresponding Raman shift at 2159 and 2212 cm –1. These SERS images, through the spectroscopic data, unambiguously prove that the adjustable Raman peak of alkynyl is suitable for interference-free SERS imaging as a series of novel biomarkers. Figure 4 Figure 4. SERS imaging of HeLa cells incubated with the SERS probes for 12 h. For each sample incubated with OPE0, OPE1, or OPE2, imaging is drawn according to the intensity of four different Raman shifts, respectively: 2000 (off) 2105 (OPE0) 2159 (OPE1) and 2212 cm –1 (OPE2. After verification of the superiority of alkyne-coded SERS tags for label detection in living cells, we attempted to study the triplex cellular imaging in the same single cell. For the preparation of three kinds of targeting SERS probes, we choose three well-documented target molecules of HeLa cells for our demonstration: recent studies have revealed that folate (FA) and luteinizing hormone-releasing hormone (LHRH) can be used as cancer target molecules, since FA-Rs (FA-receptors) and LHRH-Rs (LHRH-receptors) are overexpressed in the tumor cellular membrane, 20, 21) CALNNR 8, a polypeptide chain containing multiple arginine, can help the nanoparticles to enter cells quickly. (22) As shown in Figure 5 a, the alkyne-reporters were successfully encapsulated in the polyallylamine (PAH, MW = 17 000) shell through the Ag–S bond between the reporters and the NPs surface. In addition, the abundant −NH 2 groups of PAHylation are beneficial for the following modification with FA, LHRH, or CALNNR 8 by EDC reaction. (23) The successful conjugation to PAH, CALNNR 8 or FA, LHRH ligands were confirmed through the changes in hydrodynamic diameter and zeta potential measurements obtained via dynamic light scattering ( Table S1. After the completion of the SERS probes preparation (see the Supporting Information for synthetic details) HeLa cells were treated with three kinds of SERS probes simultaneously for 12 h before being imaged by a Raman microspectrometer. Figure 5 Figure 5. Three-color SERS imaging using SERS probes of alkyne SERS palette. (a) The schematic diagram for the PAHylation and peptide/small-molecules modifications on the Raman dye-coded [email protected] b) Normalized Raman spectra of SERS probes marked with OPE0 (red) OPE1 (green) and OPE2 (blue. c) Three-color SERS imaging using SERS probes of alkyne SERS palette in the same live HeLa cells. The Raman spectrum in Figure 5 b was acquired from position “1” (arrow in Figure 5 c) on the HeLa cell, which illustrates that three kinds of labeled particles were existing at this position. The peaks at 2105, 2158, 2212 cm –1 within the cellular Raman-silent region come from the labeled OPE0, OPE1, and OPE2 coated SERS probes, respectively; and the peak at 1580 cm –1 is their mutual peak. The efficiency of the alkyne-modulated SERS palette for triplex cellular imaging was also clearly demonstrated in Figure 5 c. In the 2105 and 2158 cm –1 channel, the OPE0 and OPE1 signal show the distribution of FA-Rs and LHRH-Rs proteins on the membrane. However, because cell height is insufficient and the longitudinal resolution of laser confocal Raman microscopy is not enough, the OPE0 and OPE1 signal are distributed throughout the whole cell. In the 2212 cm –1 channel, the signal is derived from OPE2 that exhibits cytosolic localization except the nucleus because the [email protected] NPs of 50 nm diameter are not easy to enter the nucleus. In conclusion, we present a predictive study on the molecular structure-Raman shift relationship of 16 alkynes using the density functional theory (DFT) calculation; the rational design scheme of the alkyne-modulated SERS-palette based on the optimization and theoretical explanation of the alkynyls SERS emissions of 4-ethynylbenzenethiol-derivatives and their application in optical interference-free cellular imaging on pigment-rich plant cells (e. g., lilys pollen) and single narrow emission-based triplex cellular imaging on mammalian cells. However, the narrow band of alkynyls SERS emissions are not only emerging at 2105, 2158, and 2212 cm –1 in this paper; actually, they can be proper tuned in a wide range of 2000–2300 cm –1 depending on specific application needs. Whats more, the proposed SERS palette provides a more effective solution for multicolor SERS imaging and multiplex sensing when the hyperspectral and fairly intense optical noises originating from lower wavenumber region (<1800 cm –1) are inevitable under complex ambient conditions. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10. 1021/acs. analchem. 6b01374. Experimental details, TEM images, Raman spectra, and NMR ( PDF) § Author Contributions Y. C. and J. -Q. R. contributed equally. The authors declare no competing financial interest. Terms & Conditions Electronic Supporting Information files are available without a subscription to ACS Web Editions. The American Chemical Society holds a copyright ownership interest in any copyrightable Supporting Information. Files available from the ACS website may be downloaded for personal use only. Users are not otherwise permitted to reproduce, republish, redistribute, or sell any Supporting Information from the ACS website, either in whole or in part, in either machine-readable form or any other form without permission from the American Chemical Society. For permission to reproduce, republish and redistribute this material, requesters must process their own requests via the RightsLink permission system. Information about how to use the RightsLink permission system can be found at. This work was supported by the National Natural Science Foundation of China (Grants 81471696, 21475100, 41273093, 21533006, and 21175101) Natural Science Foundation of Hubei Province of China (Grant No. 2014CFA002) National Major Scientific Instruments and Device Development Project (Grant 2012YQ16000701) and Foundation of China Geological Survey (Grant 12120113015200. This article references 23 other publications. 1 Jarvis, R. M. Goodacre, R. Chem. Soc. Rev. 2008, 37, 931 – 936 DOI: 10. 1039/b705973f [ Crossref. PubMed. CAS]   Google Scholar 1 Characterisation and identification of bacteria using SERS Jarvis, Roger M. Goodacre, Royston Chemical Society Reviews ( 2008) 37 ( 5) 931-936 CODEN: CSRVBR; ISSN: 0306-0012. ( Royal Society of Chemistry) A review. Within microbiol. Raman spectroscopy is considered as a very important whole-organism fingerprinting technique, which is used to characterize, discriminate and identify microorganisms and assess how they respond to abiotic or biotic stress. Enhancing the sensitivity of Raman spectroscopy is very beneficial for the rapid anal. of bacteria (and indeed biol. systems in general) where the ultimate goal is to achieve this without the need for lengthy cell culture. Bypassing this step would provide significant benefits in many areas such as medical, environmental and industrial microbiol., microbial systems biol., biol. warfare countermeasures and bioprocess monitoring. In this tutorial review we will report on the advances made in bacterial studies, a relatively new and exciting application area for SERS. 2 Xu, H. Li, Q. Wang, L. He, Y. Shi, J. Tang, B. Fan, C. 2014, 43, 2650 – 2661 DOI: 10. 1039/c3cs60309a [ Crossref. PubMed. CAS]   Google Scholar 2 Nanoscale optical probes for cellular imaging Xu, Hui; Li, Qian; Wang, Lihua; He, Yao; Shi, Jiye; Tang, Bo; Fan, Chunhai Chemical Society Reviews ( 2014) 43 ( 8) 2650-2661 CODEN: A review. Nanomaterials with unique optical properties have shown great promise as probes for cellular imaging. Based on these properties, a wide range of plasmonic, fluorescent and Raman probes have been designed and prepd. Nanomaterials of different sizes and shapes have also been functionalized with various types of biomols., such as antibodies, DNA or RNA, which are actively exploited to realize targeted imaging. In this review, we will summarize recent advances in using functional nanomaterials for imaging, primarily cellular imaging. These nanomaterials are categorized based on their conducting properties, i. e. conductors, semiconductors and insulators. 3 Huang, X. El-Sayed, I. H. Qian, W. El-Sayed, M. A. J. Am. 2006, 128, 2115 – 2120 DOI: 10. 1021/ja057254a [ ACS Full Text. CAS]   Google Scholar 3 Cancer Cell Imaging and Photothermal Therapy in the Near-Infrared Region by Using Gold Nanorods Huang, Xiaohua; El-Sayed, Ivan H. Qian, Wei; El-Sayed, Mostafa A. Journal of the American Chemical Society ( 2006) 128 ( 6) 2115-2120 CODEN: JACSAT; ISSN: 0002-7863. ( American Chemical Society) Due to strong elec. fields at the surface, the absorption and scattering of electromagnetic radiation by noble metal nanoparticles are strongly enhanced. These unique properties provide the potential of designing novel optically active reagents for simultaneous mol. imaging and photothermal cancer therapy. It is desirable to use agents that are active in the near-IR (NIR) region of the radiation spectrum to minimize the light extinction by intrinsic chromophores in native tissue. Gold nanorods with suitable aspect ratios (length divided by width) can absorb and scatter strongly in the NIR region (650-900 nm. In the present work, we provide an in vitro demonstration of gold nanorods as novel contrast agents for both mol. Nanorods are synthesized and conjugated to anti-epidermal growth factor receptor (anti-EGFR) monoclonal antibodies and incubated in cell cultures with a nonmalignant epithelial cell line (HaCat) and two malignant oral epithelial cell lines (HOC 313 clone 8 and HSC 3. The anti-EGFR antibody-conjugated nanorods bind specifically to the surface of the malignant-type cells with a much higher affinity due to the overexpressed EGFR on the cytoplasmic membrane of the malignant cells. As a result of the strongly scattered red light from gold nanorods in dark field, obsd. using a lab. microscope, the malignant cells are clearly visualized and diagnosed from the nonmalignant cells. It is found that, after exposure to continuous red laser at 800 nm, malignant cells require about half the laser energy to be photothermally destroyed than the nonmalignant cells. Thus, both efficient cancer cell diagnostics and selective photothermal therapy are realized at the same time. 4 Xie, J. Zhang, Q. Lee, J. Y. Wang, D. I. ACS Nano 2008, 2, 2473 – 2480 DOI: 10. 1021/nn800442q [ ACS Full Text. CAS]   Google Scholar 4 The Synthesis of SERS-Active Gold Nanoflower Tags for In Vivo Applications Xie, Jianping; Zhang, Qingbo; Lee, Jim Yang; Wang, Daniel I. ACS Nano 2 ( 12) 2473-2480 CODEN: ANCAC3; ISSN: 1936-0851. This paper reports a simple, one-pot, template-free synthesis of flower-like Au nanoparticles (three-dimensional branched nanoparticles with more than 10 tips) with high yield and good size monodispersity at room temp. The size of the Au nanoflowers could be tuned by controlling the compn. of the starting reaction mixt. The key synthesis strategy was to use a common Good's buffer, HEPES, as a weak reducing and particle stabilizing agent to confine the growth of the Au nanocrystals in the special reaction region of limited ligand protection (LLP. Time-course measurements by UV-vis spectroscopy and TEM were used to follow the reaction progress and the evolution of the flower-like shape. The Au nanoflowers exhibited strong surface-enhanced effects which were utilized in the design of an efficient, stable, and nontoxic Raman-active tag for in vivo applications. 5 Samanta, A. Maiti, K. K. Soh, K. S. Liao, X. Vendrell, M. Dinish, U. Yun, S. W. Bhuvaneswari, R. Kim, H. Rautela, S. Angew. Chem., Int. Ed. 2011, 50, 6089 – 6092 DOI: 10. 1002/anie. 201007841 [ Crossref. PubMed. CAS]   Google Scholar 5 Ultrasensitive Near-Infrared Raman Reporters for SERS-Based In Vivo Cancer Detection Samanta, Animesh; Maiti, Kaustabh Kumar; Soh, Kiat-Seng; Liao, Xiaojun; Vendrell, Marc; Dinish, U. Yun, Seong-Wook; Bhuvaneswari, Ramaswamy; Kim, Hyori; Rautela, Shashi; Chung, Junho; Olivo, Malini; Chang, Young-Tae Angewandte Chemie, International Edition ( 2011) 50 ( 27) 6089-6092, S6089/1-S6089/23 CODEN: ACIEF5; ISSN: 1433-7851. ( Wiley-VCH Verlag GmbH & Co. KGaA) The authors have prepd. a lipoic acid-contg. NIR-active tricarbocyanine library (CyNAMLA) and screened the SERS properties after chemisorption in gold nanoparticles (AuNPs. CyNAMLA compds. exhibited strong SERS intensities, and the authors identified CyNAMLA-381 as a highly sensitive NIR SERS reporter mol. with excellent signal stability and 12-fold higher sensitivity than the current std. DTITC. The authors further applied CyNAMLA-381 to the prepn. of ultrasensitive SERS probes for in vivo cancermaging by conjugating CyNAMLA-381-AuNPs to scFv anti-HER2 antibodies. These nanotags displayed very good SERS intensity and selectivity towards HER2-pos. cancer cells under both Raman and dark-field microscopes. Furthermore, the authors confirmed their in vivo application in HER2-pos. and -neg. xenograft models. The high sensitivity and tumor specificity of scFv-conjugated CyNAMLA-381 SERS nanotags proves their excellent potential as noninvasive diagnostic tools and opens up a new window for the development of SERS probes for cancer bioimaging. 6 Narayanan, N. Karunakaran, V. Paul, W. Venugopal, K. Sujathan, K. Biosens. Bioelectron. 2015, 70, 145 – 152 DOI: 10. 1016. Crossref. PubMed. CAS]   Google Scholar 6 Aggregation induced Raman scattering of squaraine dye: Implementation in diagnosis of cervical cancer dysplasia by SERS imaging Narayanan, Nisha; Karunakaran, Varsha; Paul, Willi; Venugopal, Karunakaran; Sujathan, K. Kumar Maiti, Kaustabh Biosensors & Bioelectronics ( 2015) 70 ( 145-152 CODEN: BBIOE4; ISSN: 0956-5663. ( Elsevier B. V. ) The extent of squaraine dye aggregation that reflects on surface enhanced Raman signal scattering (SERS) intensity upon adsorption on nano-roughened gold surface has been investigated. Here we have synthesized a serious of six squaraine dyes consisting of two different electron donor moiety i. 1, 1, 2-trimethyl-1H-benzo[e]indole and 2-methylbenzo[d]thiazole which modulates the chemisorptions and hydrophobicity being designated as SQ1, SQ2, SQ3, SQ4, SQ5 and SQ6. Interestingly, SQ2 (mono lipoic acid appended) SQ5 and SQ6 (conjugated with hexyl and dodecyl side chain) squaraine derivs. having more tendency of aggregation in DMSO-water mixed solvent showed significant increase of Raman scattering in the fingerprint region when chemisorbed on spherical gold nanoparticles. Two sets of SERS nanotags were prepd. with colloidal gold nanoparticle (Au-NPs size: 40 nm) by incorporating Raman reporters SQ2 and SQ5 followed by thiolated PEG encapsulation (SH-PEG, SH-PEG-COOH) denoted as AuNPs-SQ2-PEG and AuNPs-SQ5-PEG. Further conjugation of these nanotag with monoclonal antibodies specific to over expressed receptors, EGFR and p16/Ki-67 in cervical cancer cell, HeLa showed prominent SERS mapping intensity and selectivity towards cell surface and nucleus. The fast and accurate recognition obtained by antibody triggered SERS-nanotag has been compared with conventional time consuming immunocytochem. technique which prompted us to extend further investigation using real patient cervical smear sample for a non-invasive, ultrafast and accurate diagnosis. 7 Maiti, K. Samanta, A. Park, S. Olivo, M. Chang, Y. T. Nano Today 2012, 7, 85 – 93 DOI: 10. 1016. Crossref. CAS]   Google Scholar 7 Multiplex targeted in vivo cancer detection using sensitive near-infrared SERS nanotags Maiti, Kaustabh Kumar; Dinish, U. Samanta, Animesh; Vendrell, Marc; Soh, Kiat-Seng; Park, Sung-Jin; Olivo, Malini; Chang, Young-Tae Nano Today ( 2012) 7 ( 2) 85-93 CODEN: NTAOCG; ISSN: 1748-0132. ( Elsevier Ltd. ) One of the most promising advantages of surface-enhanced Raman scattering (SERS) technique for in vivo biosensing is the multiplexing potential, which is under explored due to the limited availability of near-IR Raman reporters. Here, we report the synthesis of multiplexing capable and biocompatible SERS nanotags using highly sensitive novel NIR Raman reporters. Two new NIR Raman reporter mols., Cy7LA, Cy7. 5LA are developed to partner with recently synthesized CyNAMLA-381 reporter to construct SERS nanotags with multiplexing capability. These nanotags possess excellent SERS signal stability over a period of one month. As a proof of concept for multiplex targeted in vivo detection, we successfully demonstrated the simultaneous sensing of cancer in living mouse using these three bioconjugated nanotags. To the best of our knowledge, this is the first real demonstration of in vivo multiplex targeted detection using SERS nanotags. Further, in vivo kinetic study of these nanotags in tumor revealed their excellent sensitivity, stability and tumor specificity. These probes also show rapid clearance from the liver indicating their possible excretion. This validation renders our SERS nanotags as an ultrasensitive in vivo nanoprobe for the detection and imaging of multiple biomarkers for early diagnosis of diseases. 8 Zavaleta, C. L. Smith, B. Walton, I. Doering, W. Davis, G. Shojaei, B. Natan, M. Gambhir, S. Proc. Natl. Acad. Sci. U. 2009, 106, 13511 – 13516 DOI: 10. 1073/pnas. 0813327106 [ Crossref. PubMed. CAS]   Google Scholar 8 Multiplexed imaging of surface enhanced Raman scattering nanotags in living mice using noninvasive Raman spectroscopy Zavaleta, Cristina L. Smith, Bryan R. Walton, Ian; Doering, William; Davis, Glenn; Shojael, Borzoyeh; Natan, Michael J. Gambhir, Sanjiv S. Proceedings of the National Academy of Sciences of the United States of America ( 2009) 106 ( 32) 13511-13516, S13511/1-S13511/4 CODEN: PNASA6; ISSN: 0027-8424. ( National Academy of Sciences) Raman spectroscopy is a newly developed, noninvasive preclin. imaging technique that offers picomolar sensitivity and multiplexing capabilities to the field of mol. imaging. In this study, we demonstrate the ability of Raman spectroscopy to sep. the spectral fingerprints of up to 10 different types of surface enhanced Raman scattering (SERS) nanoparticles in a living mouse after s. c. injection. Based on these spectral results, we simultaneously injected the five most intense and spectrally unique SERS nanoparticles i. v. to image their natural accumulation in the liver. All five types of SERS nanoparticles were successfully identified and spectrally sepd. using our optimized noninvasive Raman imaging system. In addn., we were able to linearly correlate Raman signal with SERS concn. after injecting four spectrally unique SERS nanoparticles either s. (R2 = 0. 998) or i. 992. These results show great potential for multiplexed imaging in living subjects in cases in which several targeted SERS probes could offer better detection of multiple biomarkers assocd. with a specific disease. 9 Yamakoshi, H. Dodo, K. Okada, M. Ando, J. Palonpon, A. Fujita, K. Kawata, S. Sodeoka, M. 2011, 133, 6102 – 6105 DOI: 10. 1021/ja108404p [ ACS Full Text. CAS]   Google Scholar 9 Imaging of EdU, an Alkyne-Tagged Cell Proliferation Probe, by Raman Microscopy Yamakoshi, Hiroyuki; Dodo, Kosuke; Okada, Masaya; Ando, Jun; Palonpon, Almar; Fujita, Katsumasa; Kawata, Satoshi; Sodeoka, Mikiko Journal of the American Chemical Society 133 ( 16) 6102-6105 CODEN: Click-free imaging of the nuclear localization of an alkyne-tagged cell proliferation probe, EdU, in living cells was achieved for the first time by Raman microscopy. The alkyne tag shows an intense Raman band in a cellular Raman-silent region that is free of interference from endogenous mols. This approach may eliminate the need for click reactions in the detection of alkyne-labeled mols. 10 Wei, L. Hu, F. Shen, Y. Chen, Z. X. Yu, Y. Lin, C. Wang, M. Min, W. Nat. Methods 2014, 11, 410 – 412 DOI: 10. 1038/nmeth. 2878 [ Crossref. PubMed. CAS]   Google Scholar 10 Live-cell imaging of alkyne-tagged small biomolecules by stimulated Raman scattering Wei, Lu; Hu, Fanghao; Shen, Yihui; Chen, Zhixing; Yu, Yong; Lin, Chih-Chun; Wang, Meng C. Min, Wei Nature Methods 11 ( 4) 410-412 CODEN: NMAEA3; ISSN: 1548-7091. ( Nature Publishing Group) Sensitive and specific visualization of small biomols. in living systems is highly challenging. We report stimulated Raman-scattering imaging of alkyne tags as a general strategy for studying a broad spectrum of small biomols. in live cells and animals. We demonstrate this technique by tracking alkyne-bearing drugs in mouse tissues and visualizing de novo synthesis of DNA, RNA, proteins, phospholipids and triglycerides through metabolic incorporation of alkyne-tagged small precursors. 11 Yamakoshi, H. 2012, 134, 20681 – 20689 DOI: 10. 1021/ja308529n [ ACS Full Text. CAS]   Google Scholar 11 Alkyne-Tag Raman Imaging for Visualization of Mobile Small Molecules in Live Cells Yamakoshi, Hiroyuki; Dodo, Kosuke; Palonpon, Almar; Ando, Jun; Fujita, Katsumasa; Kawata, Satoshi; Sodeoka, Mikiko Journal of the American Chemical Society 134 ( 51) 20681-20689 CODEN: Alkyne has a unique Raman band that does not overlap with Raman scattering from any endogenous mol. in live cells. Here, we show that alkyne-tag Raman imaging (ATRI) is a promising approach for visualizing nonimmobilized small mols. An examn. of structure-Raman shift/intensity relationships revealed that alkynes conjugated to an arom. ring and/or to a second alkyne (conjugated diynes) have strong Raman signals in the cellular silent region and can be excellent tags. Using these design guidelines, we synthesized and imaged a series of alkyne-tagged coenzyme Q (CoQ) analogs in live cells. Cellular concns. of diyne-tagged CoQ analogs could be semiquant. estd. Finally, simultaneous imaging of two small mols., 5-ethynyl-2' deoxyuridine (EdU) and a CoQ analog, with distinct Raman tags was demonstrated. 12 Chen, Z. Paley, D. Wei, L. Weisman, A. Friesner, R. Nuckolls, C. 2014, 136, 8027 – 8033 DOI: 10. 1021/ja502706q [ ACS Full Text. CAS]   Google Scholar 12 Multicolor Live-Cell Chemical Imaging by Isotopically Edited Alkyne Vibrational Palette Chen, Zhixing; Paley, Daniel W. Wei, Lu; Weisman, Andrew L. Friesner, Richard A. Nuckolls, Colin; Min, Wei Journal of the American Chemical Society 136 ( 22) 8027-8033 CODEN: Vibrational imaging such as Raman microscopy is a powerful technique for visualizing a variety of mols. in live cells and tissues with chem. contrast. Going beyond the conventional label-free modality, recent advance of coupling alkyne vibrational tags with stimulated Raman scattering microscopy paves the way for imaging a wide spectrum of alkyne-labeled small biomols. with superb sensitivity, specificity, resoln., biocompatibility, and minimal perturbation. Unfortunately, the currently available alkyne tag only processes a single vibrational "color" which prohibits multiplex chem. imaging of small mols. in a way that is being routinely practiced in fluorescence microscopy. Herein the authors develop a three-color vibrational palette of alkyne tags using a 13C-based isotopic editing strategy. The authors first synthesized 13C isotopologues of EdU, a DNA metabolic reporter, by using the newly developed alkyne cross-metathesis reaction. Consistent with theor. predictions, the mono-13C (13C≡12C) and bis-13C (13C≡13C) labeled alkyne isotopologues display Raman peaks that are red shifted and spectrally resolved from the originally unlabeled (12C≡12C) alkynyl probe. The authors further demonstrated three-color chem. imaging of nascent DNA, RNA, and newly uptaken fatty-acid in live mammalian cells with a simultaneous treatment of three different isotopically edited alkynyl metabolic reporters. The alkyne vibrational palette presented here thus opens up multicolor imaging of small biomols., enlightening a new dimension of chem. 13 Wang, Y. Q. Yan, B. Chen, L. 2013, 113, 1391 – 1428 DOI: 10. 1021/cr300120g [ ACS Full Text. 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The conjugation of the C≡C and C:C bonds decreases the v(C≡C) frequency and increases the Raman intensity 4 times in comparison with the 1-alkynes; the ir intensity remains approx. const. The wave no. decrease is less and the Raman intensity decreases in PhC≡CH and p-ClC6H4C≡CH in comparison with 1-alkynes. The intensity is strongly dependent on the para substituents. The OH group of α-acetylenic alcs. has only a slight effect on the polarity and polarizability of the C:C bond. In α-acetylenic ketones, the v(C≡C) frequency is lowered in comparison with 1-alkynes; the intensity increase is higher in the ir than in the Raman spectra. Hence, the C:O group increases the C≡C bond polarity more than itspolarizability. In the compds. contg. the Ph-C:O-C≡C grouping, the Raman intensity increases less than in those with the C:O-C≡C-Ph grouping; their intensity changes very slightly. The v(C≡C) band intensity of monosubstituted acetylenic compds. is less than those of the disubstituted C≡C bonds. 15 Etchegoin, P. G. Ru, E. Meyer, M. 2009, 131, 2713 – 2716 DOI: 10. 1021/ja808934d [ ACS Full Text. CAS]   Google Scholar 15 Evidence of Natural Isotopic Distribution from Single-Molecule SERS Etchegoin, Pablo G. Le Ru, Eric C. Meyer, Matthias Journal of the American Chemical Society 131 ( 7) 2713-2716 CODEN: The authors report on the observation of the natural isotopic spread of C from single-mol. surface-enhanced Raman spectroscopy (SM-SERS. By choosing a dye mol. with a very localized Raman-active vibration in a cyano bond (C-N triple bond) the authors observe (in a SERS colloidal liq. a small fraction of SM-SERS events where the frequency of the cyano mode is softened and in agreement with the effect of substituting 12C by the next most abundant isotope, 13C. This example adds another demonstration of single-mol. sensitivity in SERS through isotopic editing, which in this case is done not by artificial isotopic editing but rather by nature itself. It also highlights SERS as a unique spectroscopic tool that is capable of detecting an isotopic change in one atom of a single mol. 16 Wang, A. Huang, Y. F. Sur, U. Wu, D. Ren, B. Rondinini, S. Amatore, C. Tian, Z. 2010, 132, 9534 – 9536 DOI: 10. 1021/ja1024639 [ ACS Full Text. 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N. 2014, 136, 5656 – 5663 DOI: 10. 1021/ja411046j [ ACS Full Text. CAS]   Google Scholar 17 Revealing CD38 cellular localization using a cell permeable, mechanism-based fluorescent small-molecule probe Shrimp, Jonathan H. Hu, Jing; Dong, Min; Wang, Brian S. MacDonald, Robert; Jiang, Hong; Hao, Quan; Yen, Andrew; Lin, Hening Journal of the American Chemical Society ( 15) 5656-5663 CODEN: NAD is increasingly recognized as an important signaling mol. that affects numerous biol. pathways. Thus, enzymes that metabolize NAD can have important biol. functions. One NAD-metabolizing enzyme in mammals is CD38, a type II transmembrane protein that converts NAD primarily to ADP ribose (ADPR) and a small amt. of cyclic ADP ribose (cADPR. Localization of CD38 was originally thought to be only on the plasma membrane, but later reports showed either significant or solely, intracellular CD38. With the efficient NAD-hydrolysis activity, the intracellular CD38 may lead to depletion of cellular NAD, thus producing harmful effects. Therefore, the intracellular localization of CD38 needs to be carefully validated. Here, we report the synthesis and application of a cell permeable, fluorescent small mol. (SR101-F-araNMN) that can covalently label enzymically active CD38 with minimal perturbation of live cells. Using this fluorescent probe, we revealed that CD38 is predominately on the plasma membrane of Raji and retinoic acid (RA) treated HL-60 cells. Addnl., the probe revealed no CD38 expression in K562 cells, which was previously reported to have solely intracellular CD38. The finding that very little intracellular CD38 exists in these cell lines suggests that the major enzymic function of CD38 is to hydrolyze extracellular rather than intracellular NAD. The fluorescent activity-based probes that we developed allow the localization of CD38 in different cells to be detd., thus enabling a better understanding of the physiol. function. 18 Xu, X. D. Soutto, M. Xie, Q. Servick, S. Subramanian, C. von Arnim, A. Johnson, H. 2007, 104, 10264 – 10269 DOI: 10. 0701987104 [ Crossref. PubMed. CAS]   Google Scholar 18 Imaging protein interactions with bioluminescence resonance energy transfer (BRET) in plant and mammalian cells and tissues Xu, Xiaodong; Soutto, Mohammed; Xie, Qiguang; Servick, Stein; Subramanian, Chitra; von Arnim, Albrecht G. Johnson, Carl Hirschie Proceedings of the National Academy of Sciences of the United States of America ( 2007) 104 ( 24) 10264-10269 CODEN: FRET is a well established method for cellular and subcellular imaging of protein interactions. However, FRET obligatorily necessitates fluorescence excitation with its concomitant problems of photobleaching, autofluorescence, phototoxicity, and undesirable stimulation of photobiol. processes. A sister technique, bioluminescence resonance energy transfer (BRET) avoids these problems because it uses enzyme-catalyzed luminescence; however, BRET signals usually have been too dim to image effectively in the past. Using a new generation electron bombardment-charge-coupled device camera coupled to an image splitter, the authors demonstrate that BRET can be used to image protein interactions in plant and animal cells and in tissues; even subcellular imaging is possible. The authors have applied this technol. to image two different protein interactions: i) dimerization of the developmental regulator, COP1, in plant seedlings; and (ii) CCAAT/enhancer binding protein α (C/EBPα) in the mammalian nucleus. This advance heralds a host of applications for imaging without fluorescent excitation and its consequent limitations. 19 Shen, A. Guo, J. Z. Xie, W. Sun, M. Richards, R. Raman Spectrosc. 2011, 42, 879 – 884 DOI: 10. 1002/jrs. 2812 [ Crossref. CAS]   Google Scholar 19 Surface-enhanced Raman spectroscopy in living plant using triplex Au-Ag-C core-shell nanoparticles Shen, Aiguo; Guo, Jingzhe; Xie, Wei; Sun, Mengxiang; Richards, Ryan; Hu, Jiming Journal of Raman Spectroscopy 42 879-884 CODEN: JRSPAF; ISSN: 0377-0486. ( John Wiley & Sons Ltd. ) This paper presents, for the first time, noninvasive imaging of a living plant using biocompatible carbon-encapsulated Au-Ag nanoparticles (NPs) using micro-Raman spectroscopy (MRS. A convenient and controllable hydrothermal synthetic route was developed to synthesize the layer-by-layer triplex Au-Ag-C core-shell NPs, which can incorporate the reporter mol. 4-mercapto benzoic acid (4-MBA. A unique approach was devised to deliver the carbon-encapsulated surface-enhanced Raman scattering (SERS) tags into the leaf of Nicotiana benthamiana. In vivo SERS mapping was subsequently performed to monitor the distribution of tags inside the leaf, which successfully avoided interference of autofluorescence from plant tissue. The imaging modality reported here and further the bio-functionalized carbon-encapsulated SERS NP showed significant potential as a strategy for biochem. imaging in living plants in a noninvasive and nontoxic manner, which might open up exciting opportunities for plant sciences. Copyright 2010 John Wiley & Sons, Ltd. 20 Hu, C. Liu, Y. Qin, J. Nie, G. Lei, B. Xiao, Y. Zheng, M. Rong, J. ACS Appl. Mater. Interfaces 2013, 5, 4760 – 4768 DOI: 10. 1021/am4000485 [ ACS Full Text. CAS]   Google Scholar 20 Fabrication of Reduced Graphene Oxide and Sliver Nanoparticle Hybrids for Raman Detection of Absorbed Folic Acid: A Potential Cancer Diagnostic Probe Hu, Chaofan; Liu, Yingliang; Qin, Jinlan; Nie, Guangting; Lei, Bingfu; Xiao, Yong; Zheng, Mingtao; Rong, Jianhua ACS Applied Materials & Interfaces 5 ( 11) 4760-4768 CODEN: AAMICK; ISSN: 1944-8244. Reduced graphene oxide (RGO) and silver nanoparticle (AgNP) hybrids (RGO-AgNP) were prepd. by a facile one-pot method using Poly (N-vinyl-2-pyrrolidone) as reductant and stabilizer. Folic acid (FA) mols. were attached to the RGO-AgNP by physisorption for targeting specific cancer cells with folate receptors (FRs) and using as Raman reporter mols. The internalization of the FA loaded RGO-AgNP (RGO-AgNP-FA) inside the FRs-pos. cancer cell was confirmed by confocal laser scanning and TEM. The Raman signals of the FA in live cancer cells were detected by confocal Raman spectroscope at 514 nm excitation, indicating that the RGO-AgNP-FA material has great potential as a Raman probe for cancer diagnosis in vitro. 21 Zhang, J. Wu, G. Song, C. Li, Y. Qiao, H. Zhu, P. Hinterdorfer, P. Zhang, B. Tang, J. Phys. B 2012, 116, 13331 – 13337 DOI: 10. 1021/jp306882r [ ACS Full Text. CAS]   Google Scholar 21 Single Molecular Recognition Force Spectroscopy Study of a Luteinizing Hormone-Releasing Hormone Analogue as a Carcinoma Target Drug Zhang, Jing; Wu, Guangmou; Song, Chunli; Li, Yongjun; Qiao, Haiyan; Zhu, Ping; Hinterdorfer, Peter; Zhang, Bailin; Tang, Jilin Journal of Physical Chemistry B 116 ( 45) 13331-13337 CODEN: JPCBFK; ISSN: 1520-5207. The LH-releasing hormone-Pseudomonas aeruginosa exotoxin 40 (LHRH-PE40) is a candidate target drug assocd. with elevated LHRH receptor (LHRH-R) expression in malignant tumor tissue. The capability of LHRH-PE40 to recognize LHRH-Rs on a living cell membrane was studied with single mol. recognition force spectroscopy (SMFS) based on at. force microscopy (AFM. The recognition force of LHRH-PE40/LHRH-R was compared with that of LHRH/LHRH-R by dynamic force spectroscopy. Meanwhile, cell growth inhibition assay and fluorescence imaging were presented as complementary characterization. The results show that LHRH moiety keeps its capability to recognize LHRH-R specifically, which implies that recombinant protein LHRH-PE40 can be a promising target drug. 22 Dykman, L. Khlebtsov, N. 2014, 114, 1258 – 1288 DOI: 10. 1021/cr300441a [ ACS Full Text. CAS]   Google Scholar 22 Uptake of Engineered Gold Nanoparticles into Mammalian Cells Dykman, Lev A. Khlebtsov, Nikolai G. Chemical Reviews (Washington, DC, United States) 114 1258-1288 CODEN: A review. Topics reviewed include dependence of intracellular uptake on gold nanoparticle shape and size; effects of gold nanoparticle functionalization on intracellular uptake; use of penetrating peptides for the delivery of gold nanoparticles into cells; intracellular uptake of oligonucleotide-coated gold nanoparticles; selective internalization of engineered gold nanoparticles into cancer cells; and interaction of gold nanoparticles with immune cell. 23 Staros, J. Wright, R. Swingle, D. Anal. Biochem. 1986, 156, 220 – 222 DOI: 10. 1016/0003-2697(86)90176-4 [ Crossref. PubMed. CAS]   Google Scholar 23 Enhancement by N-hydroxysulfosuccinimide of water-soluble carbodiimide-mediated coupling reactions Staros, James V. Wright, Rick W. Swingle, Deborah M. Analytical Biochemistry ( 1986) 156 ( 1) 220-2 CODEN: ANBCA2; ISSN: 0003-2697. Water-sol. carbodiimides are frequently employed in coupling or conjugation reactions, e. g., to link a peptide immunogen to a carrier protein. However, their utility is limited by low coupling yields obtained under some conditions. In a model aq. reaction system consisting of [14C]glycine (3. 1 mM) keyhole limpet hemocyanin (7. 0 mg/mL) and 1-ethyl-3- 3-dimethylaminopropyl)carbodiimide HCl, the yield of [14C]glycine-hemocyanin was improved ∼15-fold by the inclusion of 5 mM N-hydroxysulfosuccinimide in the reaction mixt. Comparable enhancements were obsd. over a 10-fold range of glycine concn. (1-10 mM. Replacing hemocyanin with bovine serum albumin gave similar results. This article is cited by 42 publications. Rongchao Mei, Yunqing Wang, Qian Yu, Yingchao Yin, Rongfang Zhao, Lingxin Chen. Gold Nanorod Array-Bridged Internal-Standard SERS Tags: From Ultrasensitivity to Multifunctionality. ACS Applied Materials & Interfaces 2020, 12 (2) 2059-2066. DOI: 10. 1021/acsami. 9b18292. Meng-Yue Gao, Qiao Chen, Wei Li, Ai-Guo Shen, Ji-Ming Hu. Combined Surface-Enhanced Raman Scattering Emissions for High-Throughput Optical Labels on Micrometer-Scale Objects. Analytical Chemistry 2019, 91 (21) 13866-13873. DOI: 10. 9b03357. Jing Wang, Duanwei Liang, Jie Feng, Xinjing Tang. Multicolor Cocktail for Breast Cancer Multiplex Phenotype Targeting and Diagnosis Using Bioorthogonal Surface-Enhanced Raman Scattering Nanoprobes. Analytical Chemistry 2019, 91 (17) 11045-11054. DOI: 10. 9b01382. Xiang-Ru Bai, Li-Hua Wang, Jia-Qiang Ren, Xiang-Wei Bai, Ling-Wen Zeng, Ai-Guo Shen, Ji-Ming Hu. Accurate Clinical Diagnosis of Liver Cancer Based on Simultaneous Detection of Ternary Specific Antigens by Magnetic Induced Mixing Surface-Enhanced Raman Scattering Emissions. Analytical Chemistry 2019, 91 (4) 2955-2963. DOI: 10. 8b05153. Chamari S. Wijesooriya, Charles K. Nyamekye, Emily A. Smith. Optical Imaging of the Nanoscale Structure and Dynamics of Biological Membranes. Analytical Chemistry 2019, 91 (1) 425-440. DOI: 10. 8b04755. 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Rapid and Reliable Detection of Alkaline Phosphatase by a Hot Spots Amplification Strategy Based on Well-Controlled Assembly on Single Nanoparticle. ACS Applied Materials & Interfaces 2017, 9 (35) 29547-29553. DOI: 10. 7b09336. Zhuyuan Wang, Shenfei Zong, Lei Wu, Dan Zhu, and Yiping Cui. SERS-Activated Platforms for Immunoassay: Probes, Encoding Methods, and Applications. Chemical Reviews 2017, 117 (12) 7910-7963. DOI: 10. 7b00027. Huixia Di, Huiqiao Liu, Mingmin Li, Jin Li, and Dingbin Liu. High-Precision Profiling of Sialic Acid Expression in Cancer Cells and Tissues Using Background-Free Surface-Enhanced Raman Scattering Tags. Analytical Chemistry 2017, 89 (11) 5874-5881. DOI: 10. 7b00199. Wen Zhou, Qiang Li, Huiqiao Liu, Jie Yang, and Dingbin Liu. Building Electromagnetic Hot Spots in Living Cells via Target-Triggered Nanoparticle Dimerization. ACS Nano 2017, 11 (4) 3532-3541. DOI: 10. 1021/acsnano. 7b00531. Mingmin Li, Jin Li, Huixia Di, Huiqiao Liu, and Dingbin Liu. Live-Cell Pyrophosphate Imaging by in Situ Hot-Spot Generation. Analytical Chemistry 2017, 89 (6) 3532-3537. DOI: 10. 6b04786. Yi Zeng, Jiaqiang Ren, Aiguo Shen, and Jiming Hu. Field and Pretreatment-Free Detection of Heavy-Metal Ions in Organic Polluted Water through an Alkyne-Coded SERS Test Kit. ACS Applied Materials & Interfaces 2016, 8 (41) 27772-27778. DOI: 10. 6b09722. Joshua Phelan, Ali Altharawi, K. Andrew Chan. Tracking glycosylation in live cells using FTIR spectroscopy. Talanta 2020, 211, 120737. DOI: 10. 1016/j. talanta. 2020. 120737. Xuehui Liu, Xiaoman Liu, Pengfei Rong, Dingbin Liu. Recent advances in background-free Raman scattering for bioanalysis. TrAC Trends in Analytical Chemistry 2020, 123, 115765. DOI: 10. 1016/ Jie Wang, Kaiyuan Liu, Shangzhong Jin, Li Jiang, Pei Liang. A Review of Chinese Raman Spectroscopy Research Over the Past Twenty Years. Applied Spectroscopy 2020, 74 (2) 130-159. DOI: 10. 1177/0003702819828360. Liang Chen, Lei Wang, Yangyang Wan, Ying Zhang, Zeming Qi, Xiaojun Wu, Hangxun Xu. Acetylene and Diacetylene Functionalized Covalent Triazine Frameworks as Metal‐Free Photocatalysts for Hydrogen Peroxide Production: A New Two‐Electron Water Oxidation Pathway. Advanced Materials 2020, 32 (2) 1904433. DOI: 10. 1002/adma. 201904433. Yue Sun, Zhihua Li, Xiaowei Huang, Di Zhang, Xiaobo Zou, Jiyong Shi, Xiaodong Zhai, Caiping Jiang, Xiaoou Wei, Tingting Liu. A nitrile-mediated aptasensor for optical anti-interference detection of acetamiprid in apple juice by surface-enhanced Raman scattering. Biosensors and Bioelectronics 2019, 145, 111672. DOI: 10. 1016/ Xueliang Lin, Yunyi Wang, Lingna Wang, Yudong Lu, Jin Li, Dechan Lu, Ting Zhou, Zufang Huang, Jun Huang, Huifang Huang, Sufang Qiu, Rong Chen, Duo Lin, Shangyuan Feng. 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