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8GAP-ENHANCED RAMAN TAGS FOR ANALYTICAL AND IMAGING APPLICATIONS
BORIS KHLEBTSOV1, ANDREY BUROV1, DANIIL BRATASHOV2, NIKOLAI KHLEBTSOV12
1 Institute of biochemistry and physiology ofplants and microorganisms RAS, 410049 Saratov, Russia 2 Saratov State University, 410012 Saratov, Russia Khlebtsov_b@ibppm.ru
ABSTRACT
Gap-enhanced Raman tags (GERTs) are new emerging probes of the surface-enhanced Raman scattering (SERS) spectroscopy that have found promising analytical and bioimaging applications. Because of the internal localization of Raman reporter molecules, they are protected from unwanted external environments and particle aggregation and demonstrate superior SERS responses due to strongly enhanced electromagnetic fields in the gaps between metal core-shell structures. We discuss recent progress in the synthesis, design of optical properties, and biomedical applications of novel spherically symmetrical and anisotropic GERTs. Particular attention is paid to the use of nanoparticles as labels for immunoassays and for Raman bioimaging of cancer cells and tissues.
Plasmonic enhancement of local electromagnetic (EM) fields by metal nanostructures is the key physical process behind the surface-enhanced Raman spectroscopy (SERS). Raman spectra provide unique information about molecular vibration modes, thus ensuring analytical means for the detection of molecules and their local environment in condensed phases.
There are several ways to enhance the local EM fields through the EM coupling of excited modes, including the formation of a small nanogap between plasmonic particles or between particles and tips or flat metal surfaces. Although such nanostructures can be fabricated through modern controllable technologies with predicted geometrical parameters, the SERS response may suffer from possible unwanted interference effects of surrounding media. From this point of view, the nanostructures with Raman molecules (RMs) embedded into internal nanogaps seem very promising for biomedical applications. SERS core/shell tags with inner nanogaps (also called gap-enhanced Raman tags - GERTs [1]) have attracted considerable attention because of several advantage features: (1) RMs are protected from desorption and environmental conditions; (2) they are subjected to a uniform and strongly enhanced EM field in the gap; (3) GERT probes can be multiplexed by incorporating different RMs into two-layered or multilayered GERTs.
Our talk include our recent data on the synthesis and plasmonic properties of GERTs, and biomedical and theranostic topics such as analytical sensing, in vivo and in vitro bioimaging, and intra-operation diagnostic of cancer tumor margins.
The widely used Raman reporters for GERTs are thiolated aromatic molecules that possess a number of promising modalities. Because of the high chemical affinity of the thiol group to the metal surface, thiolated molecules can easily be conjugated to a wide range of NPs by simple mixing. Synthesis of such GERTs starts with the synthesis of CTAC stabilized spherical Au particles as cores, followed by conjugation with a monolayer of 1,4-BDT (Fig. 1B) and growth of Au shell on the cores by mixing Au salt (HAuCl4) with a capping agent (CTAC) and ascorbic acid as a mild reducing agent. The HRTEM images of resulted particles reveal a sub-nm gap between core and shell indicating the successful trapping of the Raman reporters between the core and shell. Note that 1,4-BDT embedded GERTs demonstrated the SERS response one order of magnitude higher compared other particles with surface adsorbed 1,4-BDT molecules. We have also demonstrated that not only 1,4-BDT, but also other thiolated aromatic molecules, such as 4-aminothiophenol (4-ATP) and 4-methylthiophenol (4-MTP), can be used to synthesize core/shell particles with embedded Raman reporters and distinct gaps [2, 3].
yellow arrows indicate the internal and external nanogaps, respectively. The panels (f) and (g) represent sample photos and extinction spectra, FDTD simulations and SERS spectra. The bottom panel (h) illustrates the synthetic scheme of P-GERTs.
GERTs with spherical symmetry and smooth outer shell demonstrate typically high but not extraordinary EFs. To increase the SERS performance of gap-enhanced tags, several attempts have been made to modify the outer shell morphology in a star-like [4] or irregular roughness manners [5]. Still, there is an urgent need in strong increasing the GERT efficiency down to single-particle level. Recently, we fabricated a new version of GERTs with spherical core, hollow gap, and a branched petal-like shell structures (for brevity, such particles were called P-GERTs; Fig. 3) [6]. Because of the generation of strong EM hot spots in both the internal gap and petal-like structures, the fundamental EF was as high as 5 x 109, thus enabling single-particle detection. Remarkably, the synthetic protocols of both conventional GERTs (S-GERTs) and P-GERTs are very similar and differ mainly in using 1,4-BDT and 4-NBT as spacers.
The quantitative determination of bioactive molecules, including proteins, nucleic acids, aptamers and small molecules, are the common goals in biodetection assays. In the SERS tag based biodetection procedures, a sandwich assay is widely adopted as the analyte is "sandwiched" between two targeting sites: typically, the targeting site (such as antibodies or complementary nucleic acids) are first immobilized onto the substrates; the target biomarkers are then captured by them; afterward, the biofunctionalized GERTs are attached through the recognition of biomarkers onto the substrate, contributing a distinct signal for quantitative analysis. GERTs are advantageous as optical labels in the biodetection assay owing to their super-high sensitivity (down to a single-NP level) and specificity, leading to an improved limit of detection (LOD) compared to common SERS tags.
Figure 2 The GERT-based LFIA for cTnI, the average SERS spectra in the test zones and the standard curve of different cTnI
concentrations
More importantly, the embedded Raman reporters can be considered as the internal standard for the calibration of Raman signal fluctuations induced by different measurement conditions and local states of the NPs, such as aggregation. This can greatly overcome the reproducibility issue in common quantitative SERS-based biodetection [7]. Recently, we have reported the successful application of GERTs-based lateral flow immunoassay (LFIA), either using anisotropic rodlike GERTs (Fig. 2) [8] or using bimetallic double-shell structured GERTs [Error! Bookmark not defined.]. The sensitivity of the former has highly surpassed that of colorimetric LFIA, with a LOD for cardiac troponin I (cTnI) to be 0.1 ng/mL, close to the diagnostic criteria for blood serum in the case of heart infarction. The latter has reached a LOD of 0.025 mIU human chorionic gonadotropin (HCG), which is three orders of magnitude better than the commercial strips.
Despite major advances in targeted drug and radiation therapies, surgery is still the most preferred and effective treatment for localized tumors. Precision cancer surgery guided by intraoperative optical imaging is of broad interest in engineering and medicine.
Figure 3 The SERS-guided detection and chemo-photothermal therapy of abdominal disseminated microtumors in mice using GERTs
loaded with cisplatin.
Over the past decade, many efforts have been made to optimize the performances of SERS-guided surgery from three strategies: developing superior SERS tags, achieving higher specificity in delivery, improving the speed by optimizing Raman system and imaging methodologies. When it comes to a more complete tumor resection of the primary tumor, draining lymph nodes and metastatic sites, a superior SERS is required to be of high brightness, good targeting capability and specificity [9]. From this point, GERTs can be the promising probes. We have reported on GERT-guided surgery and
cancer therapy. The 1,4-BDT embedded GERTs developed by us appeared to have a detection limit in liquid of 20 fM with the laser energy of 105 W/cm2 and an integration time of 1.86 s [10].
The high brightness makes it possible for the real-time intraoperative sensitive detection and treatment of the residual prostate tumors [10] as well as the diagnostics of disseminated ovarian cancers (Fig. 3). These tags can specifically identify and eliminate the tumor margin and micro satellite metastases, making it possible to have a complete tumor resection. Furthermore, by loading the chemotherapy drug cisplatin within the mesoporous silica layer of GERTs, the Raman-guided chemo-photothermal synergistic therapy can be achieved to kill tumor cells in a precise way (Fig. 3). These investigations unveil the attractiveness of GERTs as a robust platform for the intraoperative diagnosis and eradication of microtumors, which would push Raman technologies forward to deep theranostic and related biomedical applications.
We acknowledge the financial support from the Russian Scientific Foundation (project no. 18-14-00016) REFERENCES
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