Chapter 6 Single molecule sensitivity in surface enhanced Raman scattering using surface plasmon

One of the most important obstacles for the application of surface enhanced Raman spectroscopy (SERS) is the poor reproducibility of SERS active substrates: SERS intensity can be varied from one substrate to another and moreover along the one substrate surface. Reproducible SERS substrate can be prepared through introduction of highly ordered metal array, where light focusing is achieved through excitation of surface plasmon-polaritons (SPPs). In this work, excimer laser patterning of poly(methyl methacrylate) followed by silver evaporation is proposed as an effective way for the creation of reproducible and effective surface plasmon-polaritons (SPP)-based SERS substrate. Detailed theoretical and experimental studies were performed to optimize structure parameter for effective SPP excitation. It was found that the narrow range of grating periodicity and metal thickness exist, where SPPs can be most efficiently excited. Despite the fact that SERS response was almost always achieved, the enhancement factor ...

Surface-enhanced Raman scattering (SERS) spectroscopy is a popular technique for detecting chemicals in small quantities. Rough metallic surfaces with nanofeatures are some of the most widespread and commercially successful substrates for efficient SERS measurements. A rough metallic surface creates a high-density random distribution of so-called "hot spots" with local optical field enhancement causing Raman signal to increase. In this Letter, we revisit the classic SERS experiment [Surf. Sci.158, 229 (1985)SUSCAS0039-602810.1016/0039-6028(85)90297-3] with rough metallic surfaces covered by a thin layer of copper phthalocyanine molecules. As a modification to the classic configuration, we apply an adaptive wavefront correction of a laser beam profile. As a result, we demonstrate an increase in brightness of local SERS hot spots and redistribution of Raman signal over the substrate area. We hypothesize that the improvement is due to optimal coupling of the shaped laser beam to the random plasmonic nanoantenna configurations. We show that the proposed adaptive-SERS modification is independent of the exact structure of the surface roughness and topography, works with many rough surfaces, and gives brighter Raman hot spots in comparison with conventional SERS measurements. We prove that the adaptive SERS is a powerful instrument for improving SERS sensitivity.

Surface enhanced Raman scattering (SERS) provides a label free method of analyzing molecules from diverse and complex signals, potentially with single molecule sensitivity. The chemical specificity inherent in the SERS spectrum can identify molecules; however signal variability arising from the diversity of plasmonic environments can limit quantification, particularly at low concentrations. Here we show that digitizing, or counting SERS events, can decrease the limit of detection in flowing solutions enabling quantification of single molecules. By using multivariate curve resolution and establishing a score threshold, each individual spectrum can be classified as containing an event or not. This binary “yes/no” can then be quantified, and a linear region can be established. This method was shown to lower the limit of detection to the lowest physical limit, and lowered the limit of detection by an order of magnitude from the traditional, intensity based LOD calculations.

<p>Surface enhanced Raman spectroscopy (SERS) is undoubtedly a powerful tool as it allows one to overcome the major disadvantage of Raman spectroscopy: the weakness of its signal. Enhancement factors (EF) of up to 1010 make it even possible to detect single molecules. However, using it as an analytical tool to make reproducible, quantitative measurements has so far been difficult as the enhancement of the signal is "bought" at the expense of reproducibility: The larger the EF the more the reproducibility of the substrate suffers. This has been dubbed informally the "SERS uncertainty principle" by Natan [1]. While currently a lot of research effort is taking place at the high-EF-side of the spectrum and ever more sophisticated SERS substrates are being explored, in this thesis we would like to make a shift in paradigm and revisit SERS on flat metallic surfaces, which arguably constitute the simplest substrates available. To this end we will show their usefulness in making quantitative measurements and how they are an ideal platform for a new hybrid technique that combines reproducibility and extreme sensitivity with substantial EFs. For making quantitativemeasurements two examples are explored in a systematic way: in the first example (Chapter 2) the determination of an unknown, resonant Raman cross-section is demonstrated on flat metallic films (possibly with some surface roughness) and confirmed with measurements done on more commonly used SERS substrates. Here the quantitative measurement is made possible by introducing a reference molecule as a standard and having statistics as our main ally: even though we do not know the exact EF that the individual molecules experience on the various substrates, we know that on average both, the unknown sample and the known reference, experience the same. In the second example (Chapter 3) we use commercially available flat films for which we verify experimentally that surface roughness is irrelevant. By themselves these substrates yield no enhancement – in fact they even quench the Raman signal. Yet they allow us to calculate and control the electric field on the surface which enables us to determine the orientation of adsorbed molecules by using surface selection rules (SSR). While the first example is mostly empirical, the second one allows us to test our theoretical understanding of plasmonic systems with proper numerical calculations that are in excellent agreement with the observed data. Finally, in Chapter 4, we use those flat films in a special configuration (called the Kretschmann configuration) to excite Surface Plasmon-Polaritons (SPP). This not only allows us to combine the spatial homogeneity of a flat surface with useful EFs easily predicted fromtheory but also to combine the extreme sensitivity of surface plasmon resonance spectroscopy (SPRS) with the analytical power of SERS. It is not our intention to claim that the work presented here is the first attempt to do analytical work with SERS. Rather the newmethods presented in this thesis will add new strategies and tools to the current research effort while the detailed analysis will provide the means to understand them theoretically and in their historical context.</p>

Η φασματοσκοπία Raman θεωρείται αξιόπιστη μέθοδος χαρακτηρισμού της μοριακής δομής της ύλης. Τελευταία δε καταβάλλεται μεγάλη προσπάθεια να αναδειχθεί και ως μη επεμβατική τεχνική ανίχνευσης ουσιών φαρμακευτικού και βιολογικού ενδιαφέροντος. Επειδή όμως το φαινόμενο Raman είναι ασθενές και τα όρια ανίχνευσης των ουσιών αυτών ιδιαίτερα χαμηλά, η συμβατική φασματοσκοπία Raman ανταποκρίνεται με μεγάλη δυσκολία στις απαιτήσεις μιας μη επεμβατικής τεχνικής. Τα τελευταία χρόνια με την ανακάλυψη της επιφανειακής ενίσχυσης της σκέδασης Raman (Surface Enhanced Raman Scattering) και την ανάπτυξη της ομώνυμης τεχνικής SERS αυξήθηκε η πιθανότητα του φαινομένου κατά τάξεις μεγέθους. Στο πλαίσιο αυτό, έχουν αναφερθεί και μελέτες SERS με ανίχνευση σκέδασης Raman ακόμη και από ένα μόνο μόριο (single molecule detection).Προϋπόθεση όμως ανάπτυξης φαινομένου SERS είναι η γειτνίαση της εξεταζόμενης ουσίας με νανοσωματίδια αργύρου (Ag) ή χρυσού (Au). Τα νανοσωματίδια αυτά χρησιμοποιούνται κυρίως είτε υπό μορφή κολλοειδών διαλυμάτων είτε υπό την μορφή νανοδομημένων επιφανειών. Μια επιπλέον παράμετρος που πρέπει να συνυπολογισθεί ώστε η μέθοδος αυτή να χαρακτηριστεί αναλυτική είναι η δυνατότητα πραγματοποίησης με SERS ποσοτικών μετρήσεων. Στην παρούσα εργασία ως “υπόστρωμα” εμφάνισης του φαινομένου SERS χρησιμοποιήθηκαν κολλοειδή διαλύματα Au και Ag (~2 mL), στα οποία προστίθεντο 25-100 μL διαλύματος της προς ανίχνευσης ουσίας. Η χρησιμοποίηση της γεωμετρίας σκέδασης 90ο, της πλέον ενδεδειγμένης για λήψη φασμάτων Raman από υγρά και κολλοειδή διαλύματα νανομετρικής κλίμακας (10-100 nm), έδωσε τη δυνατότητα ανίχνευσης συγκεντρώσεων ουσιών-στόχων ακόμη και της τάξης των fg/mL. Επιπλέον, η χρησιμοποίηση μιας κυψελίδας με συνεχή ταλάντωση σε κάθετη ως προς το επίπεδο σκέδασης διεύθυνση (oscillating or shaking cell), επέτρεψε την πραγματοποίηση ποσοτικών μετρήσεων SERS σε συγκεντρώσεις της τάξης των (sub)-ng & (sub)-pg/mL. Η διάταξη αυτή φωταψίας δείγματος και συλλογής σκεδαζόμενης ακτινοβολίας εφαρμόστηκε σε αντικαρκινικά φάρμακα (Novantrone® & Doxorubicin), αντιβιοτικό (Ciproxin®) και βάσεις του DNA. Στην περίπτωση του αντιβιοτικού με ενεργή ουσία το Ciprofloxacin ελήφθησαν ποσοτικά αποτελέσματα σε διαλύματα τεχνητών δακρύων της τάξης των ng/mL, κάτω από την συνολική κλίμακα της μέσης ανασταλτικής πυκνότητας (minimum inhibitory concentration range) του φαρμάκου. Στόχος και φιλοδοξία είναι η τεχνική αυτή να αναδειχθεί ως μια ταχεία μη επεμβατική αναλυτική μέθοδος μέτρησης φαρμακευτικών ή/και βιολογικών ουσιών σε σωματικά υγρά (π.χ. σίελος, δάκρυα, …), για την μελέτη της φαρμακοκινητικής, την έγκαιρη διάγνωση ασθενειών καθώς και τη μελέτη ελεγχόμενης αποδέσμευσης ενεργών ουσιών από πολυμερικές μήτρες σε πολύ μικρές συγκεντρώσεις. Επιπλέον, έγινε προσπάθεια μελέτης/ανίχνευσης μορίων Ε18 primary rat cortical cells με την χρήση του SERS σε συνθήκες προσομοίωσης ηλεκτροχημικού εμφυτεύματος. Για τον λόγο αυτό σχεδιάστηκε και αναπτύχθηκε ένας μικρο-επωαστής (micro-incubator) που επιτρέπει αφ’ ενός μεν τη συντήρηση των κυττάρων σε περιβάλλον κατάλληλο για τη μελέτη τους (layer of Neurobasal-culture medium with a 95% air & 5% CO₂ gas flow at 37°C), αφ’ ετέρου δε την προσαρμογή του σε χώρο υποδοχής δείγματος ενός φασματοφωτόμετρου μικρο-Raman για τη λήψη φασματοηλεκτροχημικών μετρήσεων Raman/SERS. Το κύριο σημείο ενδιαφέροντος στο οποίο εστιάσαμε στην παρούσα εργασία αναφέρεται στην διερεύνηση ανίχνευσης βασικών/θεμελιωδών μορίων των κυττάρων στo θρεπτικό υγρό του πειράματος για την ταχεία εκτίμηση ενδεχόμενης αποδέσμευσης DNA που θα αντικατόπτριζε την κατάσταση των κυττάρων.

<p>This thesis focusses on a number of topics in surface enhanced Raman scattering (SERS). The aim of the undertaken research was to deepen the general understanding of the SERS effect and, thereby, to clarify some of the disputed issues, among them: What is the origin of the enhancement? What is the physical or chemical effect of 'salt activation' in SERS systems? Can we observe single-molecules using SERS? Can we determine the absorbate's orientation on the surface? In part one (chapters 1-3), as a general introduction, I start with a short overview of the Raman effect and its relation to other molecular spectroscopic effects (such as fluorescence, Rayleigh scattering, etc... ). Following these basic remarks, the surface enhancementmechanisms underlying SERS are explained (as a largely electromagnetic field enhancement) and are investigated theoretically on the canonicalmodel of a nanoscopic dimer of silver spheres. The second part (chapter 4) reports on the experimental investigation (electron microscopy, in-situ Raman measurements) of a typical real SERS system: Lee & Meisel silver colloids. An emphasis is put on the self-limiting aggregation kinetics which is observed in such systems after salt addition. This is also investigated and rationalised by means of Monte-Carlo simulations which are footed on empiric theoretical considerations for the interaction potential. Part three (chapter 5) contains a discussion of the early attempts on singlemolecule SERS and points out the shortcomings of the previously used ultra-lowconcentration approach. In response, an improved andmore rigorous approach is presented: Bi-Analyte SERS. Examplary applications of the technique are discussed. Within these experiments the capability of the technique to prove/disprove (with statistical soundness) single-molecule sensitivity in any SERS system is demonstrated, and single-molecule enhancement factors are derived. The last part (chapter 6) presents computational studies based on densityfunctional theory and its use in the context of Raman spectroscopy and SERS. Of particular interest here were the Raman tensors, their visual representation appropriate in the SERS case, their relation to the relative intensities of Raman peaks, and their modification when the photon energy approaches the electronic resonance of the molecule. Last, but not least, a conclusion chapter is presented, where I highlight what has contributed by the thesis to the general understanding of the SERS effect.</p>

DNA origami nanostructures are a versatile tool to arrange metal nanostructures and other chemical entities with nanometer precision. In this way gold nanoparticle dimers with defined distance can be constructed, which can be exploited as novel substrates for surface enhanced Raman scattering (SERS). We have optimized the size, composition and arrangement of Au/Ag nanoparticles to create intense SERS hot spots, with Raman enhancement up to 10(10), which is sufficient to detect single molecules by Raman scattering. This is demonstrated using single dye molecules (TAMRA and Cy3) placed into the center of the nanoparticle dimers. In conjunction with the DNA origami nanostructures novel SERS substrates are created, which can in the future be applied to the SERS analysis of more complex biomolecular targets, whose position and conformation within the SERS hot spot can be precisely controlled.

This paper presents an in-depth study of Surface Enhanced Raman Scattering (SERS) enhancement factors (EFs) and cross-sections, including several issues often overlooked. In particular, various possible rigorous definitions of the SERS EFs are introduced and discussed in the context of SERS applications, such as analytical chemistry and single molecule SERS. These definitions highlight the importance of a careful characterization of the non-SERS cross-sections of the probes under consideration. This aspect is illustrated by experimental results for the non-SERS cross-sections of representative SERS probes along with average SERS EFs for the same probes. In addition, the accurate experimental determination of single molecule enhancement factors is tackled with two recently developed techniques, namely: bi-analyte SERS (BiASERS) and temperature-dependent SERS vibrational pumping. We demonstrate that SERS EFs as low as 107, as opposed to the figure of 1014 often claimed in the literature, are sufficient for the observation of single molecule SERS signals, with maximum single molecule EFs typically on the order of ∼1010.

Surface-enhanced Raman scattering (SERS) is a powerful technique for trace chemical analysis and single molecule detection in the application of biochemical monitoring and food safety due to its ability to enhance the Raman scattering of molecules near the metallic surface or nanostructures. Here, we present a comprehensive study of the SERS enhancement by the periodically nanostructured surface, where the thin film of silver is deposited onto the surface, except the sidewall of posts, of 1-D lamellar gratings with varying pitch to forming metal-dielectric composite nanostructures. By enhancing the localized and surface-propagating mode in the vicinity of the concaves, the SERS signal can be improved by amplifying the intensity of electric field and increasing the optical path length of the incident light. Experimental investigations show that the enhancement factor can be manipulated by varying the polarization of incident light and the pitch size of gratings. To demonstrate the SERS effects of the proposed structures, thin layers of benzoic acid, which is commonly used as a food preservative, are deposited on the SERS substrates by spin-coating a solution of benzoic acid and dried at room temperature. A Confocal Raman microscope with a 532 nm laser source is used to illuminate light and measure the Raman spectrum of benzoic acid. We demonstrate the Raman signal of benzoic acid can be enhanced on the order of 10 2 on the SERS substrates.

One of the two fundamental mechanisms underlying Surface-Enhanced Raman Scattering (SERS) is the existence of large electromagnetic (EM) fields in the vicinity of the rough metal substrates that are used as substrates. Surface roughness below the micron scale plays a relevant role in this process, due to the roughness-induced excitation of surface-plasmon polaritons. Since in many scattering configurations dipolar and/or electrostatic approximations cannot be employed, we study this EM mechanism from the rigorous standpoint of classical Maxwell equations. By means of numerical simulation calculations based on the Green’s theorem integral equation formulation, the linearly polarized electro- magnetic field scattered from one-dimensional, randomly rough metal surfaces is obtained. In particular, Ag, Au, and Cu surfaces are considered possessing fractal properties analogous to those observed in colloidal aggregates or coldly deposited films commonly used in SERS experiments. We analyze the influence of the roughness parameters on the near field intensity. The enhancement factor of the SERS signal is assumed to be proportional to the square of that of the near field intensity at the pump frequency; in light of the random nature of the roughness, the analysis is performed on the probability density function of the enhancement factor. The optimum pump frequency is obtained from the spectral dependence of both the average field enhancement and the absorption. In addition to the near field intensity calculations, the far field scattered from such random self-affine fractals is studied, revealing interesting features in the angular distribution, such as incoherent peaks at the specular direction, and weak backscattering peaks for the rougher surfaces that also yield large near field enhancements.

Surface-enhanced Raman scattering (SERS) is a powerful spectroscopic technique that has created exciting opportunities in the field of bioanalytical chemistry, where it combines ultrasensitive detection of biologically relevant molecules with vibrational spectroscopy. Due to the difficulties in preparing reproducible SERS active substrates, SERS has been mainly used as a qualitative tool. In order for SERS to be utilized as a viable tool for quantitative analysis, simple, facile SERS substrates which generate clean, highly reproducible signals must be developed. This dissertation deals with the evaluation of three different methods of SERS that led to the development of a novel substrate for the analysis of biological molecules. In this dissertation, I demonstrated the generation of a SERS signal from pyridine adsorbed to the surface of a roughened silver electrode. The Raman signal was detected utilizing a remote fiber optic probe dispersive Raman instrument. The necessary parameters for generating the optimal SERS signal were established. The SERS spectrum of pyridine was successfully characterized. The synthesis of silver colloid solutions for use as a SERS active substrate was demonstrated. Two different synthetic methods were utilized and the conditions for optimal SERS signal strength were established. SERS signals from solutions of tryptophan were successfully generated and detected using an FT-Raman spectrometer. In this dissertation. I report the successful chemical deposition of a thin silver film on the surface of a quartz/glass fiber filter for use as a SERS active substrate. The successful detection of the SERS signal of solutions of phenylalanine and tryptophan utilizing the silver-coated filters and a FT-Raman instrument was demonstrated. Significant progress was made to develop a quantifiable SERS method utilizing the silver-coated filters. The silver-coated filter SERS method presented here is a novel and promising method for biochemical analysis. The demonstrated ability to detect amino acids and mixtures of amino acids can be further extended to the detection of similar biological molecules such as proteins, tumor markers, and nucleic acids. The use of a FT-Raman instrument combined with this new SERS method allows for a method with both high sensitivity and selectivity.

Investigations of the optical response of subwavelength-structure arrays milled into thin metal films have revealed surprising phenomena, including reports of unexpectedly high transmission of light. Many studies have interpreted the optical coupling to the surface in terms of the resonant excitation of surface plasmon polaritons (SPPs), but other approaches involving composite diffraction of surface evanescent waves (CDEW) have also been proposed. Here we present a series of measurements on very simple one-dimensional subwavelength structures to test the key properties of the surface waves, and compare them to the CDEW and SPP models. We find that the optical response of the silver metal surface proceeds in two steps: a diffractive perturbation in the immediate vicinity (2–3µ m) of the structure, followed by excitation of a persistent surface wave that propagates over tens of micrometres. The measured wavelength and phase of this persistent wave are significantly shifted from those expected for resonance excitation of a conventional SPP on a pure silver surface.

Water pollution has created a critical threat to the environment.‎‎ A lot of research has been done ‎recently to use surface-enhanced Raman spectroscopy (SERS) to detect multiple pollutants in water. This study aims to use Ag colloid nanoflowers as liquid SERS enhancer. Tri sodium phosphate (Na3PO4) was investigated as a pollutant using liquid SERS ‎based on colloidal Ag ‎nanoflowers. The chemical method was used to synthesize nanoflowers from silver ‎ions. Atomic Force Microscope (AFM), Scanning Electron Microscope (SEM), and X-ray diffractometer (XRD) were employed to characterize the silver nanoflowers. This ‎nanoflowers SERS action in detecting Na3PO4 was reported and analyzed concerning both shape and size using a 532 ‎nm laser. We observed that the nanoflower's structure produced strong SERS signals. The increase in the SERS signal is related to ‎the deposition of Na3PO4 molecules in the aggregated silver nanostructure ‎in the solution. The concentration of Na3PO4 plays a main role in detection since the Raman ‎signal becomes stronger as the concentration increases. The highest phosphate analytical enhancement factor obtained for SERS in colloidal nanoflowers was 1.7×103 at 0.7×10-6 M which was the lowest concentration.

Nowadays, plasmonic nanoparticles have attracted attention due to the significant improvement in the detection of Raman scattering signals from molecules adsorbed on rough metal surfaces. In the field of surface-enhanced Raman scattering (SERS), it is possible to record spectral signatures of even individual molecules. This phenomenon is partly explained by the significant enhancement of the electromagnetic field at the surface of metal nanoparticles, which actively interacts with the energy levels of adsorbed molecules. The shape, geometry of nanostructures, as well as the size and state of aggregation of the nanomaterial play a key role in the creation of the regions of local enhancement of the electromagnetic field. The localized surface plasmon resonance contributes to a significant enhancement of the electromagnetic field. Due to this, the intensity of the Raman signal of molecules located in these zones increases significantly. There are various approaches to improving the Raman signal of the studied substances. In this work, the extinction cross-sectional spectra of silver nanoparticles under surface plasmon resonance conditions have been calculated using the quasi-dipole approximation, taking into account the influence of the substrate on which the nanoparticles are deposited. The calculations consider the nanoparticle size and the distance between the nanoparticles and the substrate surface. Numerical experiments have demonstrated that the substrate material's permittivity, on which silver nanoparticles are deposited, plays a key role in the design and fabrication of optical structures based on them. It was revealed that a substantial shift of the extinction cross-section peak and an increase in its magnitude at the operating wavelength are primarily driven by surfaces with a high dielectric constant. Based on theoretical research findings, SERS substrates were produced using galvanic replacing and wet chemical methods. Silicon and ITO served as the base materials. Experimental data confirmed that the fabricated SERS substrates are comparable in performance to commercial counterparts. The work was carried out within the SERS project (state registration number 0124U000823). 1. Wang, X., Huang, S. C., Hu, S., Yan, S., & Ren, B. (2020). Fundamental understanding and applications of plasmon-enhanced Raman spectroscopy. Nature Reviews Physics, 2(5), 253-271 2. Schmidt, M. M., Brolo, A. G., & Lindquist, N. C. (2024). Single-molecule surface-enhanced raman spectroscopy: challenges, opportunities, and future directions. ACS nano, 18(38), 25930-25938. 3. William, R. V., Das, G. M., Dantham, V. R., & Laha, R. (2019). Enhancement of single molecule Raman scattering using sprouted potato shaped bimetallic nanoparticles. Scientific Reports, 9(1), 1077.

Mass transport through a sub-10 nm single gold nanopore: SERS and ionic current measurement