Digital surface enhanced Raman spectroscopy for quantifiable single molecule detection in flow†

Surface-enhanced Raman scattering of glyphosate on dispersed silver nanoparticles: A reinterpretation based on model molecules

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

The research reported in this thesis focuses on the fundamental science and engineering required for the fabrication and optimization of nanoparticles functionalized paper as Surface Enhanced Raman Scattering (SERS) bio-diagnostics platform. It addresses the current Sensitivity, Selectivity, Simplicity and Strength (4S) limitations of bioactive paper. The research vision is to develop a nanoparticles treated paper as a generic diagnostic platform to identify and quantify low concentrations of a specific (bio)analyte in aqueous solutions. The product concept is a “dipstick sensor” based on functionalized gold nanoparticle treated paper (AuNP paper) for SERS detection. Such AuNP paper test would allow the direct, rapid and easy detection of (bio)analyte molecules at very low concentrations (below the nanomolar range), making it suitable for biomedical analysis, such as in cancer detection. It also eliminates the tedious preparation of multiple reactants and washing steps involved in current technology such as the Enzyme-Linked Immunosorbent Assay (ELISA) based bioactive paper. Three parallel research length scales are investigated: nano, micro and macro. This involves quantifying the distribution and adsorption state of nanoparticles on paper (nanoscale). The effects of gold nanoparticles (AuNPs) concentration and 3-dimensional (3D) distribution profile on their adsorption and aggregation states on paper were explored. The surface coverage of AuNPs on paper scaled linearly with their concentration profile in solutions. The SERS performances of the AuNPs-treated papers were evaluated with a model Raman molecule, 4-aminothiophenol (4-ATP), and their SERS intensities increased linearly with the density of AuNPs on paper. The role of z-distribution of AuNPs within the bulk of paper was highlighted; their three-dimensional (3D) architecture was able to quench the background noise through interlayer plasmon coupling, thus amplifying the SERS signal. The retention and aggregation state of nanoparticles on paper was controlled to drastically optimize the SERS performance (microscale). Paper substrates were pre-treated with a series of cationic polyacrylamide (CPAM) solutions to control the AuNPs adsorption and aggregation state on paper. The CPAM pre-treated paper produced a more uniform distribution of AuNPs compared to untreated paper. CPAM chains which adsorbed on paper in a high loops and tails conformation promotes efficient bridging of AuNPs to achieve higher surface coverage and aggregation of AuNPs on paper. This configuration is favored by CPAM solutions of higher concentration, charge density and molecular weight. A simple approach to form and visualize polyelectrolyte-nanoparticles chaplet like structures on paper was developed by using negatively-charged gold nanoparticles (AuNPs) to decorate polymer chain of CPAM. AuNP chaplets were adsorbed in an opposite direction to the cellulose fibers and along the length of the CPAM molecules which were draped over the fibers. The effects of CPAM polymer concentration, charge density and molecular weight on the dimension of AuNP chaplets were quantified. Assembling nanometer-scale components (AuNPs) into micrometer-scale arrays (chaplets) on a porous paper substrate can be a promising strategy for low-cost nanoelectronics applications The SERS properties of nanoparticles functionalized paper were quantified and optimized (macroscale). The effect of CPAM concentration, charge density and molecular weight on the aggregation and surface coverage of AuNPs on paper and ultimately the SERS signal of 4-ATP was quantified. The optimized AuNPs-CPAM paper showed a higher sensitivity and Raman enhancement factor (EF), which was almost an order of magnitude higher than the untreated AuNPs paper. The effect of CPAM dissolution kinetic on the SERS reproducibility was also quantified. SERS reproducibility of AuNPs on CPAM pre-treated paper both increase with dissolution time of CPAM, as the heterogeneity of AuNP distribution and aggregate size decreases; increasing CPAM charge density accentuated this tendency. After the SERS efficiency towards the detection of model Raman molecule (4-ATP) was proven, AuNPs paper was upgraded by functionalizing the AuNPs with biotin/streptavidin assemblies for the detection of antibody-antigen binding. The modification of antibody local structure due to the interaction with antigen was detected. Evidence of antigen binding was elucidated from the SERS spectra, confirming the presence of antigen. Reproducible spectra features were observed for the functionalized AuNP papers which were exposed to different concentration of antigen; their intensity increased as a function of antigen concentration. By conducting these studies and merging these three length scales of research, a novel strategy to engineer AuNPs functionalized paper as a low-cost and generic SERS platform for bio-diagnostic applications was developed.

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

The objective of this study was to determine the most stable conformation of L-tryptophan (L-Tryp) on gold and silver nanoparticles. In addition, this work explored how these parameters were affected by analyte concentration, nanoparticle size, and pH. The purpose was to establish whether L-Tryp molecules interact with the nanoparticles through the carboxylate end, the amino group end, or both. This research has brought diverse applications in biophysics and medical diagnostics, potentially opening new avenues in these fields. Moreover, it may enrich the disciplines of chemistry and nanotechnology by offering innovative approaches for future research. These findings represent a significant advancement in understanding the interactions between L-Tryp and nanoparticles, making a meaningful contribution to biophysics and medical diagnostics. Surface-Enhanced Raman Scattering (SERS) spectra of L-Tryp in the 200–3500 cm⁻¹ spectral range were obtained using a 785 nm laser for excitation. Gold and silver nanoparticles were synthesized using the citrate reduction method. The experimental procedure involved the use of electrolytes (such as NaCl) for colloid activation, which resulted in very high SERS signals. Modification of nanoparticle surface charge was achieved by adjusting the pH of Au and Ag colloidal suspensions between 2 and 11. The SERS spectra indicate that small-sized nanoparticles require high concentrations of L-Tryp to achieve high sensitivity, whereas larger nanoparticles perform effectively at lower concentrations. The pronounced enhancement of stretching vibrations in the COO⁻ group in the SERS spectra strongly suggests that the carboxylate group attaches to silver nanoparticles (AgNPs). Conversely, for gold nanoparticle (AuNP), a new band at approximately 2136 cm⁻¹ was observed, indicating that the amino group of L-Tryp interacts with Au in its neutral form. These analyses were complemented with theoretical modeling, employing the Density Functional Theory (DFT) running under Gaussian™ to study molecular models in which L-Tryp interacted with the AgNPs and AuNPs substrates in neutral, cationic, and anionic forms.

Surface-enhanced Raman scattering (SERS) is a process with origins, electromagnetic and chemical. The electromagnetic enhancement consists of the excitation of surface plasmons in the metallic support of the thin film. With only the electromagnetic enhancement mechanism, the surface spectra should not differ from volume Raman spectra. However, between SERS and volume Raman spectra, there are differences resulting from the chemical reactions taking place at the polymer/metal interface, intermediated by solvent molecules, that finally depend on the types of polymers and metallic supports. Polyaniline (PAN) is an excellent material to emphasize the chemical component of SERS. This is due to its particular structure with a repeating unit that contains two entities at different weights—a reduced state and an oxidized state–that, in turn, react differently with a metallic substrate. SERS spectra depend on the oxidizing properties of the metal surface, which involves an intermediate compound of the types Ag2O and Au2O3 when N-methyl-2-pyrrolidinone is used as the solvent. This article presents new results concerning the surface chemical effects that produce variations of the PAN SERS spectra. The SERS spectra of the PAN emeraldine base (PAN-EB) layered on Au support are characterized by a semiquinoid structure that we believe is induced on the intermediate compound Au2O3. In the presence of H2SO4, the SERS spectra change gradually as the degree of acid protonation doping increases. The SERS spectra of the fully protonated PAN-EB are identical to those obtained on PAN emeraldine salt (PAN-ES) synthesized by cyclic voltammetry in an acid medium and are invariable with the type of metallic support. The SERS spectra show that the emeraldine salt can be partially or totally deprotonated with water or NH4OH. The deprotonation is complete for the Ag support and partial for the Au support. The SERS spectra of the fully protonated PAN-EB are characterized by a double band with maxima at about 1330 and 1370 cm−1. Although the generation process of positive charge on the macromolecular chain of PAN-EB doped in the presence of (C4H9)4NBF4 is similar to that due to protonic acid doping, involving cation addition (C4H or H+ ions, respectively) in SERS spectra, the complex band situated at about 1330–1370 cm−1 no longer appears. The doping of PAN-EB with FeCl3 produces two polymer forms: a salt type characterized by a protonated structure similar to that found for PAN-ES and a base type similar to the leucoemeraldine form. © 2000 John Wiley & Sons, Inc. J Polym Sci B: Polym Phys 38: 2599–2609, 2000

The objective of this study was to determine the most stable conformation of L-tryptophan (L-Tryp) on gold and silver nanoparticles. Additionally, this work investigated how these parameters were influenced by analyte concentration, nanoparticle size, and pH. The purpose of this study was to establish whether L-Tryp molecules interact with the nanoparticles through the carboxylate end, the amino group end, or both. This research has diverse applications in biophysics and medical diagnostics, potentially opening up new avenues in these fields. Moreover, it may enrich the disciplines of chemistry and nanotechnology by offering innovative approaches for future research. These findings represent a significant advancement in understanding the interactions between L-Tryp and nanoparticles, making a meaningful contribution to biophysics and medical diagnostics. Surface-Enhanced Raman Scattering (SERS) spectra of L-Tryp in the 100–4000 cm−1 spectral range were obtained using a 785 nm laser for excitation. Gold (Au) and silver (Ag) nanoparticles (NPs) were synthesized using the citrate reduction method. The experimental procedure involved the use of electrolytes (such as NaCl) for colloid activation, which resulted in very high SERS signals. Modification of nanoparticle surface charge was achieved by adjusting the pH of Au and Ag colloidal suspensions between 2 and 11. The SERS spectra indicate that small-sized nanoparticles require high concentrations of L-Tryp to achieve high sensitivity, whereas larger nanoparticles perform effectively at lower concentrations. The pronounced enhancement of stretching vibrations in the COO− group in the SERS spectra strongly suggests that the carboxylate group attaches to silver nanoparticles (AgNPs). Conversely, for gold nanoparticles (AuNPs), a new band at approximately 2136 cm−1 was observed, indicating that the amino group of L-Tryp interacts with Au in its neutral form. These analyses were complemented by theoretical modeling, employing Density Functional Theory (DFT) calculations run using the Gaussian program to study molecular models in which L-Tryp interacted with AgNP and AuNP substrates in neutral, cationic, and anionic forms.

The ability to distinguish between specific and nonspecific binding is important for assessing the interactions between protein receptors and ligands. Surface plasmon resonance (SPR) spectroscopy is an advanced tool to measure binding events, yet the ability to distinguish between specific and nonspecific binding remains a limitation. To address this problem, we use SPR spectroscopy correlated with surface enhanced Raman scattering (SERS). The chemical information present in SERS spectra provides insight into the molecular interactions between functionalized nanoparticles and proteins, which are not detectable by SPR alone. Using a custom instrument with the Kretschmann configuration, we successfully demonstrate simultaneous affinity and the chemical characterization of streptavidin-functionalized gold nanoparticles (STV-NPs) binding to biotin immobilized on a gold film in both air and flowing phosphate buffered saline (PBS). The SPR performance is consistent with that of previous reports. The association constant (KA) for streptavidin/biotin and STV-NPs/biotin interactions observed (2 ± 1 × 107 M-1 and 2.4 ± 0.3 × 1010 M-1, respectively) agree with literature values and show a strong avidity effect associated with the STV-NPs. The SERS scattering from STV-NPs is excited by the surface plasmon polariton and collected from an objective lens mounted over the fluidic channel. The SERS spectra are recorded simultaneously with the SPR sensorgram, and the detected Raman bands provide chemical insight into the binding event. Multivariate curve resolution analysis of the spectra can differentiate specific from nonspecific binding. This label-free, real time, and surface sensitive detection method provides chemical information to protein/ligand binding affinity measurements.

An ultrasensitive method based on Surface enhanced Raman scattering (SERS) has been developed to determine content of a pesticide which is tricyclazole in paddy rice using sliver colloid as a substrate and pyridine as an internal standard. The peaks at 424 and 1035 cm(-1) in a SERS spectrum were selected as analytic and internal peaks, respectively, and their intensity ratio I(t)/I(p) was used to calculate the regression concentration of tricyclazole. The correlation between I(t)/I(p) and concentration showed significant linear relationship with a correlation coefficient of R(2)= 0.995 in a concentration range of 0.05 to 0.70 mg/L and the tricyclazole solution can be detected to be low as 0.002 mg/L by SERS. The method was applied to determine tricyclazole contents of 3 real rice samples with a standard addition method in order to eliminate interference of matrix. The errors of SERS measurements for the 3 samples were 0.0008 to 0.0246, 0.0013 to 0.0028, and 0.0129 to 0.0304 mg/kg, respectively, compared with the results obtained by high performance liquid chromatography method. This also showed a good reproducibility with low values of relative standard deviation (n= 3) for the 3 samples ranged from 3.63% to 4.64%.

Electrochemically renewable SERS sensor: A new platform for the detection of metabolites involved in peroxide production

The adulteration of traditional Chinese medicine (TCM) with synthetic drugs severely harms the TCM effect and public health. Thus, quick detection of synthetic drug found in TCM is crucial. Here, we report surface-enhanced Raman scattering (SERS) coupled with density functional theory (DFT) calculations for the direct identification and detection of aminopyrine (AP) in TCM using silver-coated gold nanoparticles (Au@Ag NPs). It has been demonstrated that the Raman enhancement of Au@Ag NPs to AP was stronger than that of Au, Ag and Ag@Au NPs, and there was a strong dependence of the Raman enhancement on the substrate concentration. Moreover, we obtained the DFT calculation result of AP and found that the calculation result was in good agreement with the experimental one. In addition, the characteristic peak at 999 cm−1, which was the strongest peak in the SERS spectra, was used for a quantitative evaluation of the AP level. The limit of detection (LOD) was 2.50 × 10−7 M (equal to 57.8 ng mL−1) in aqueous solution, which was in the same order of magnitude as that obtained by liquid chromatography-mass spectrometry (LC-MS), and the recovery of AP ranged from 93.9% to 101% in the spiked TCM (Shexiang Fengshi capsule) sample. Each test using the SERS sensor only needed a 10 μL sample solution and the whole test could be accomplished within ∼3 min. The SERS technique can be applied to authenticate and detect trace AP in real herbal products.

The application of nanomaterials in ocular drug delivery is mainly concentrated in nano-controlled release systems. Due to the unique properties of nanomaterials, the use of nanomaterials to carry drugs for treating eye diseases has great advantages compared with traditional drug delivery methods. The nano-formulation of the drug has higher bioavailability and lower side effects. Therefore, the nano-controlled release system has a good application prospect in ophthalmology. In this paper, Fe2O3 magnetic nanoparticles were first prepared by co-deposition, and then the nanoparticles were clad with silica to prepare Fe2O3 @SiO2 core-shell nanostructures. Secondly, an ordered array surface-enhanced Raman scattering (SERS) active substrate was formed on the surface of the porous layer of porous anode aluminium oxide (PAA) and the barrier layer using vacuum electron beam evaporation technology using PAA films as templates. Then, the shell thickness of Fe2O3@SiO2 and the number of coated magnetic particles were controlled by controlling the dosage of ethyl orthosilicate, and a layer of SERS active gold nanoshells was grown after further adsorption of gold seeds to prepare a composite SERS active substrate. Enhanced performance magnetic nanomaterial Fe2O3@SiO2@PAA @Au. Finally, the SERS spectrum and fluorescence quenching characteristics of ocular cells adsorbed on the substrate were detected and analysed. The results show that the magnetic nanomaterial Fe2O3@SiO2@PAA @Au with enhanced performance of SERS active substrate has a good enhancement effect on the Raman scattering signal of eye cells, and to a certain extent, it can eliminate the interference of the fluorescent background. Moreover, the ordered array of SERS active substrates not only has a high SERS enhancement ability and ability to quench fluorescence, but also does not have interference peaks of oxalate impurities remaining in the PAA film, which can obtain more detailed information of the Raman scattering spectrum of eye cells to achieve eye care.

Proteins are one of the most versatile groups of molecules with vital functional roles in living systems. Their enormous diversity and structural flexibility make the detection of these molecules a challenging task. A simple and sensitive label-free protein detection method based on assembly of proteins and colloidal silver nanoparticles (AgNPs) on surfaces and surface-enhanced Raman scattering (SERS) is reported. The SERS spectra from the assembled AgNP/protein films show excellent reproducibility and high quality regardless of the proteins' charge status and size. A detection limit down to 0.5 μg/mL for three acidic proteins; BSA, catalase and pepsin, and three basic proteins; cytochrome c, avidin and lysozyme, is easily achieved. The minimum improvement in detection limit is more than 1 order of magnitude compared to the previously reported detection limits using the technique and the approach has the potential for label-free protein detection and identification.

<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>

Surface enhanced Raman scattering (SERS) has been extensively investigated for rough metal surfaces fabricated by chemical etching and particles fabricated by different methods of nanolithography. Surface roughness is assumed to be necessary for SERS. Sharp edges of nanoparticles are desired for near-field enhancements and, consequently, contribute to the enhanced Raman effect. However, high porosity and roughness decreases the optical near-field strength due to losses by radiation. Arrays of nanoparticles fabricated by nanosphere lithography, are able to generate strong near-fields and do not have reduced roughness. Despite its advantages this fabrication method has an undesired effect: contaminant carbonaceous species attached to the nanoparticles generate strong Raman spectra and prevent their use for other molecules. We have investigated the effect of the particle roughness and preparation method on the characteristics of SERS spectra and how the contamination can be avoided. Samples were prepared by nanosphere lithography and using silver and gold nanoparticles in solution. Dye molecules and alkanethiols were used to decorate the nanoparticles in order to obtain enhanced Raman scattering. SERS spectra were acquired using a scanning confocal Raman microscope. Concentrated solution of alkanethiols reduce or remove completely the SERS spectra typical of the contaminants.