Demonstrated herein is a UV–vis Ratiometric Resonance Synchronous Spectroscopic (R2S2, pronounced as “R-two-S-two” for simplicity) technique where the R2S2 spectrum is obtained by dividing the resonance synchronous spectrum of a NP-containing solution by the solvent resonance synchronous spectrum. Combined with conventional UV–vis measurements, this R2S2 method enables experimental quantification of the absolute optical cross sections for a wide range of molecular and nanoparticle (NP) materials that range optically from pure photon absorbers or scatterers to simultaneous photon absorbers and scatterers, simultaneous photon absorbers and emitters, and all the way to simultaneous photon absorbers, scatterers, and emitters in the UV–vis wavelength region. Example applications of this R2S2 method were demonstrated for quantifying the Rayleigh scattering cross sections of solvents including water and toluene, absorption and resonance light scattering cross sections for plasmonic gold nanoparticles, and absorption, scattering, and on-resonance fluorescence cross sections for semiconductor quantum dots (Qdots). On-resonance fluorescence quantum yields were quantified for the model molecular fluorophore Eosin Y and fluorescent Qdots CdSe and CdSe/ZnS. The insights and methodology presented in this work should be of broad significance in physical and biological science research that involves photon/matter interactions.

Thiols, including organothiol and thiol-containing biomolecules, are among the most important classes of chemicals that are used broadly in organic synthesis, biological chemistry, and nanosciences. Thiol pKa values are key indicators of thiol reactivity and functionality. Reported herein is an internally referenced Raman-based pH titration method that enables reliable quantification of thiol pKa values for both mono- and dithiols in water. The degree of thiol ionization is monitored directly using the peak intensity of the S–H stretching feature in the 2600 cm–1 region relative to an internal reference peak as a function of the titration solution’s pH. The thiol pKa values and Raman activity relative to its internal reference were then determined by curve fitting the experimental data with equations derived on the basis of the Henderson–Hasselbalch equation. Using this Raman titration method, we determined for the first time the first and second thiol pKa values for 1,2-benzenedithiol in water. This Raman-based method is convenient to implement, and its underlying theory is easy to follow. It should therefore have broad application for thiol pKa determinations and verification.

Electrolyte interactions with nanoparticles (NPs) at the solid/liquid interfaces are highly complicated as the charged species can be directly adsorbed onto the NP surfaces, confined in the diffusion layer immediately surrounding the NPs, and dispersed in bulk solutions. Existing studies on electrolyte interactions with NPs are based primarily on the electrical double layer theory that focuses mainly on electrolyte interactions with NPs with fixed pre-existing charges. Demonstrated herein is a comprehensive study of counterion effects during the electrolyte bindings to gold nanoparticles (AuNPs), including halide-induced AuNP aggregation and fusion, quantitative cation and anion coadsorption, selective cation and anion displacement on AuNPs, and surface-enhanced Raman spectroscopic features of the ionic species adsorbed onto AuNP surfaces. In contradiction to previous reports that electrolyte effects are anion-specific, we demonstrated that cations can play a dominant role in the halide-induced AuNP aggregation and fusion and the ion-exchange processes on AuNP surfaces. Mechanistically, these counterion effects are due to the cooperative and competitive cation and anion binding to AuNPs and AuNP-facilitated cation and anion interactions. The insights provided in this work should be of broad importance for NP research and applications in which electrolyte/NP interactions are ubiquitously implicated.

Organothiol binding to gold nanoparticles (AuNPs) in water proceeds through a deprotonation pathway in which the sulfur-bound hydrogen (RS-H) atoms are released to solution as protons and the organothiol attach to AuNPs as negatively charged thiolate. The missing puzzle pieces in this mechanism are (i) the significance of electrostatic repulsion among the likely charged thiolates packed on AuNP surfaces, and (ii) the pathways for the ligand binding system to cope with such electrostatic repulsion. Presented herein are a series of experimental and theoretical evidence that ion pairing, the coadsorption of negatively charged thiolate and positively charged cations, is a main mechanism for the system to reduce the electrostatic repulsion among the thiolate self-assembled onto AuNP surfaces. This work represents a significant step forward in the comprehensive understanding of organothiol binding to AuNPs.

Fluorescence and Raman inner filter effects (IFE) cause spectral distortion and nonlinearity between spectral signal intensity with increasing analyte concentration. Convenient and effective correction of fluorescence IFE has been an active research goal for decades. Presented herein is the finding that fluorescence and Raman IFE can be reliably corrected using the equation Icorr/Iobsd = 10dxAx + dmAm when the effective excitation and emission path lengths, dx and dm, of a fluorophotometer are determined by simple linear curve-fitting of Raman intensities of a series of water Raman reference samples that have known degrees of Raman IFEs. The path lengths derived with one set of Raman measurements at one specific excitation wavelength are effective for correcting fluorescence and Raman IFEs induced by any chromophore or fluorophore, regardless of the excitation and emission wavelengths. The IFE-corrected fluorescence intensities are linearly correlated to fluorophore concentration over 5 orders of magnitude (from 5.9 nM to 0.59 mM) for 2-aminopurine in a 1 cm × 0.17 cm fluorescence cuvette. This water Raman-based method is easy to implement. It does not involve complicated instrument geometry determination or difficult data manipulation. This work should be of broad significance to physical and biological sciences given the popularity of fluorescence techniques in analytical applications.

Ion-pairing, the association of oppositely charged ionic species in solution and at liquid/solid interfaces has been proposed as a key factor for a wide range of physicochemical phenomena. However, experimental observations of ion pairing at the ligand/solid interfaces are challenging due to difficulties in differentiating ion species in the electrical double layer from that adsorbed on the solid surfaces. Using surface enhanced Raman spectroscopy in combination with electrolyte washing, we presented herein the first direct experimental evidence of ion pairing, the coadsorption of oppositely charged ionic species onto gold nanoparticles (AuNPs). Ion pairing reduces the electrolyte concentration threshold in inducing AuNP aggregation and enhances the competitiveness of electrolyte over neutral molecules for binding to AuNP surfaces. The methodology and insights provided in this work should be important for understanding electrolyte interfacial interactions with nanoparticles.

Nanoparticles (NP) can modify fluorophore fluorescence in solution through multiple pathways that include fluorescence inner filter effect (IFE), dynamic and static quenching, surface enhancement, and fluorophore quantum yield variation associated with structural and conformational modifications induced by NP binding. The combined contribution of the latter three effects is termed the collective near-field effect because (1) they affect only fluorophore fluorescence in molecules close to the NPs, and (2) it is impossible to differentiate these effects with steady-state fluorescence measurements. A generalized model (F0corr/FNPcorr = (1 + K[NP])/(1 + K[NP]S) was developed for the determination of the NP collective near-field effect S on the fluorophore fluorescence in the surface-adsorbed molecules. The popular Stern–Volmer equation (F0corr/FNPcorr = (1 + K[NP]) used in current fluorescence studies of NP interfacial interactions is a special case of this generalized model, valid only under situations in which the surface-bound molecules are completely fluorescence inactive (S = 0). In addition, we excluded the possibility of NPs inducing significant dynamic fluorescence quenching under realistic experimental conditions on the basis of a simple back-of-the-envelope calculation. Furthermore, using an external reference fluorescence IFE correction method developed in this work, we demonstrated that gold nanoparticles (AuNPs) only slightly attenuate, but do not completely quench the fluorescence signal of the protein, bovine serum albumin (BSA), on AuNP. This result undermines the reliability of the BSA/AuNP binding constants calculated using the Stern–Volmer equation in earlier studies of BSA/AuNP interfacial interactions. The methodology and insights provided in this work should be of general importance for fluorescence study of nanoparticle interfacial interactions.

The wavelength-dependent correlations between UV–vis intensities and surface enhanced Raman spectroscopic (SERS) enhancement factors (EFs) of aggregated gold and silver nanoparticles (AgNPs and AuNPs) were investigated using two experimental approaches. The first is to study the time-resolved SERS EFs under three fixed excitation wavelengths (532, 632, and 785 nm), each as a function of nanoparticle (NP) aggregation states. The second is to compare SERS EFs at these three excitation wavelengths for a series of protein-stabilized AuNP or AgNP aggregates. The SERS EFs were determined using a solvent internal reference method. The NP UV–vis intensity is an excellent indicator for identifying the optimal aggregation state for the AgNP-based SERS acquisitions under each of the three excitation wavelengths and for the AuNP-based SERS under 632 nm excitation. However, the NP UV–vis intensity is an unreliable predictor of the optimal excitation wavelength for either AuNPs or AgNPs. Computational simulations reveal that the NP SERS enhancement is much more sensitive than NP UV–vis intensity to small changes in the NP aggregation states. In addition to enhancing the understanding of the correlation among NP aggregation, UV–vis intensity, and SERS activity, the techniques and insights derived from this work should be important for developing sensitive and reproducible colloidal-NP-based SERS applications.

The mechanism of sodium borohydride removal of organothiols from gold nanoparticles (AuNPs) was studied using an experimental investigation and computational modeling. Organothiols and other AuNP surface adsorbates such as thiophene, adenine, rhodamine, small anions (Br– and I–), and a polymer (PVP, poly(N-vinylpyrrolidone)) can all be rapidly and completely removed from the AuNP surfaces. A computational study showed that hydride derived from sodium borohydride has a higher binding affinity to AuNPs than organothiols. Thus, it can displace organothiols and all the other adsorbates tested from AuNPs. Sodium borohydride may be used as a hazard-free, general-purpose detergent that should find utility in a variety of AuNP applications including catalysis, biosensing, surface enhanced Raman spectroscopy, and AuNP recycle and reuse.

The resonance Raman (RR) enhancement factors of Rhodamine 6G (R6G) in water and on gold and silver nanoparticles (AuNPs and AgNPs) were determined using a double ratiometric method where adenine is used as the internal reference. The RR enhancement factor for R6G on AgNPs upon laser excitation at 532 nm is 537.6 ± 214.8. This is ∼5 times lower than the experimental (2.7 ± 0.3) × 103 RR enhancement factor for R6G in water. These experimental RR enhancement factors for R6G in water and on AgNPs are 104 smaller than the 107 RR enhancement proposed in literature for R6G in water and on SERS substrates. In addition, a simple back-of-the-envelope calculation showed that even with this damped RR for R6G on AgNPs in comparison to R6G in water, a SERS enhancement factor of 106 is sufficient to explain the single-molecule resonance SERS activities reported for R6G located in nanoparticle junctions. This conclusion is deduced from fact that normal Raman spectrum could be readily obtained with 24 fmol of adenine at laser focal volume of ∼150 fL at 532 nm excitation. This work provides the first direct experimental evidence for the recent theoretical predication that plasmonic nanoparticles quench the resonance Raman signal. In addition, the double ratiometric method reported in this work represents a significant technique development in Raman and SERS, which should pave the way for quantitative investigations of the RR for dye molecules dissolved in solution or adsorbed on plasmonic nanoparticles.

Recent research has demonstrated that the nanoparticle (NP) surface enhanced Raman spectroscopy (SERS) substrate modifies an analyte’s Raman signal through two competitive mechanisms, SERS enhancement and NP inner filter effect, instead of SERS enhancement alone as commonly believed. Using a combination of time-resolved Raman spectroscopy and a solvent internal reference method, reported herein is a quantitative determination of the SERS enhancement factors (EFs) of mercaptobenzimidazole (MBI), a model organothiol, adsorbed onto gold and silver nanoparticles (AuNPs and AgNPs). The peak MBI SERS EF depends only on the type and size of NPs, but not analyte and NP concentrations, or the type (KF, KCl, KBr, and K2SO4) and concentrations of the electrolytic aggregation agents. The experimental SERS EFs of MBI on both AuNPs and AgNPs can be fully explained by the electromagnetic mechanism alone. This result, combined with our recent findings that a series of structurally diverse organothiols have similar SERS EFs, argues quite strongly against the possibility of large chemical enhancement (e.g., >10 times) for organothiols adsorbed onto colloidal AuNPs and AgNPs.

Proteins and organothiols (OTs) are known to have high affinity for gold nanoparticles (AuNPs). Systematic investigation of protein and OT coadsorption onto AuNPs is, however, mostly an unexplored area. Presented here is a comparison of simultaneous and sequential protein and OT interactions with AuNPs in which a protein and an OT are either simultaneously or sequentially added to colloidal AuNPs. Using bovine serum albumin (BSA) as the model protein and eight model organothiols, both the protein and the OT were coadsorbed onto AuNPs in samples formed by sequential or simultaneous addition. AuNP stability against OT-adsorption-induced AuNP aggregation differed significantly among the AuNP/OT and AuNP/BSA/OT mixtures. The stability of AuNPs in the AuNP/BSA/OT mixtures with the same compositions increased from (AuNP/OT)/BSA to AuNP/(BSA/OT) and finally (AuNP/BSA)/OT (where the two components inside the parentheses are mixed first followed by the addition of the third component). Aging the (AuNP/BSA) mixtures before OT addition also increased the AuNP stability in (AuNP/BSA)/OT samples. This sequence and aging dependence of AuNP stability indicates that protein and OT coadsorption onto AuNPs is kinetically controlled. It also offers a plausible explanation to the large discrepancy in the binding constants reported for the BSA interaction with AuNPs (from 105 to 1011 M–1). The work is important for AuNP biological/biomedical applications because AuNPs encounter a mixture of proteins and OTs in addition to other molecular species in biofluids.

The protein and gold nanoparticle (AuNP) interfacial interaction has broad implications for biological and biomedical applications of AuNPs. In situ characterization of the morphology and structural evolution of protein on AuNPs is difficult. We have found that the protein coating layer formed by bovine serum albumin (BSA) on AuNP is highly permeable to further organothiol adsorption. Using mercaptobenzimidazole (MBI) as a molecular probe, it is found that BSA interaction with AuNP is an exceedingly lengthy process. Structural modification of BSA coating layer on AuNP continues even after 2 days’ aging of the (AuNP/BSA) mixture. While BSA is in a near full monolayer packing on the AuNPs, it passivates only up to 30% of the AuNP surfaces against MBI adsorption. Aging reduces the kinetics of the MBI adsorption. However, even in the most aged BSA-coated AuNP (3 days), 80% of the MBI adsorption occurs within the first 5 min of the MBI addition to the (AuNP/BSA) mixture. The possibility of MBI displacing the adsorbed BSA was excluded with quantitative BSA adsorption studies. Besides MBI, other organothiols including endogenous amino acid thiols (cysteine, homocysteine, and glutathione) were also shown to penetrate through the protein coating layer and be adsorbed onto AuNPs. In addition to providing critical new understanding of the morphology and structural evolution of protein on AuNPs, this work also provides a new venue for preparation of multicomponent composite nanoparticle with applications in drug delivery, cancer imaging and therapy, and material sciences.

Studying the correlation between the molecular structures of SERS-active analytes and their SERS enhancement factors is important to our fundamental understanding of SERS chemical enhancement. Using a common internal reference method, we quantitatively compared the Raman activities, SERS activities, and SERS enhancement factors for a series of organothiols that differ significantly in their structural characteristics and reported chemical enhancements. We find that while the tested molecules vary tremendously in their normal Raman and SERS activities (by more than 4 orders of magnitude), their SERS enhancement factors are very similar (the largest difference is less than 1 order of magnitude). This result strongly suggests that SERS chemical enhancement factors are not as diverse as initially believed. In addition to shedding critical insight on the SERS phenomena, the common internal reference method developed in this work provides a simple and reliable way for systematic investigation of the correlation between molecular structures and their normal Raman and SERS activities.Keywords: enhancement factor; gold nanoparticle; Raman; SERS; silver nanoparticle;

Dye conjugation is a common strategy improving the surface enhanced Raman detection sensitivity of biomolecules. Reported is a proof-of-concept study of a novel surface enhanced Raman spectroscopic tagging strategy termed as acid-cleavable SERS tag (ACST) method. Using Rhodamine B as the starting material, we prepared the first ACST prototype that consisted of, from the distal end, a SERS tag moiety (STM), an acid-cleavable linker, and a protein reactive moiety. Complete acid cleavage of the ACST tags was achieved at a very mild condition that is 1.5% trifluoroacetic acid (TFA) aqueous solution at room temperature. SERS detection of this ACST tagged protein was demonstrated using bovine serum albumin (BSA) as the model protein. While the SERS spectrum of intact ACST-BSA was entirely dominated by the fluorescent signal of STM, quality SERS spectra can be readily obtained with the acid cleaved ACST-BSA conjugates. Separation of the acid cleaved STM from protein further enhances the SERS sensitivity. Current SERS detection sensitivity, achieved with the acid cleaved ACST-BSA conjugate is ∼5 nM in terms of the BSA concentration and ∼1.5 nM in ACST content. The dynamic range of the cleaved ACST-BSA conjugate spans four orders of magnitudes from ∼10 nM to ∼100 μM in protein concentrations. Further improvement in the SERS sensitivity can be achieved with resonance Raman acquisition. This cleavable tagging strategy may also be used for elimination of protein interference in fluorescence based biomolecule detection.

Determination of the ligand conformation on gold nanoparticle (AuNP) is of fundamental importance in nanoparticle research and applications. Using a combination of surface-enhanced Raman spectroscopy (SERS), density function calculation, and normal Raman spectroscopy, the pH dependence of mercaptobenzimadazole (MBI) adsorption onto AuNP was systematically studied. Structures and conformations of MBI adsorbates on AuNP were determined together with their binding constants, and saturation packing densities were determined at three different pHs (1.4, 7.9, and 12.5). While MBI thione is the predominant tautomer in solution with a pH value lower than 10.3, MBI thiolate is the main adsorbate on AuNP surface in solution with pH > 2. MBI thiones dominate the AuNP surface only in solutions with pH < 2. While MBI thione has a higher saturation packing density (∼632 pmol/cm2) than MBI thiolate (∼540 pmol/cm2), its binding constant (2.14 × 106 M−1) is about five times smaller than that for MBI thiolate (10.12 × 106 M−1). Using the MBI footprint deduced from its saturation packing density on AuNP, the conformation of MBI was determined. While the MBI thione binds monodentately to the AuNP with a perfectly upright orientation, MBI thiolate binds bidentately to AuNP with a tilt angle that allows interaction of AuNP with both the sulfur and the nitrogen atoms in MBI thiolate. In addition to the new insights provided on MBI binding onto gold nanoparticle, the methodology employed in this study can be particularly useful for studying AuNP interactions with other imidazole−thiol compounds, a class of heterocylic compounds that can exist in different tautomeric forms.

Glycomic analysis is an increasingly important field in biological and biomedical research as glycosylation is one of the most important protein post-translational modifications. We have developed a new technique to detect carbohydrates using surface enhanced Raman spectroscopy (SERS) by designing and applying a Rhodamine B derivative as the SERS tag. Using a reductive amination reaction, the Rhodamine-based tag (RT) was successfully conjugated to three model carbohydrates (glucose, lactose, and glucuronic acid). SERS detection limits obtained with a 633 nm HeNe laser were ∼1 nM in concentration for all the RT−carbohydrate conjugates and ∼10 fmol in total sample consumption. The dynamic range of the SERS method is about 4 orders of magnitude, spanning from 1 nM to 5 μM. Ratiometric SERS quantification using isotope-substituted SERS internal references allows comparative quantifications of carbohydrates labeled with RT and deuterium/hydrogen substituted RT tags, respectively. In addition to enhancing the SERS detection of the tagged carbohydrates, the Rhodamine tagging facilitates fluorescence and mass spectrometric detection of carbohydrates. Current fluorescence sensitivity of RT−carbohydrates is ∼3 nM in concentration while the mass spectrometry (MS) sensitivity is about 1 fmol, achieved with a linear ion trap electrospray ionization (ESI)-MS instrument. Potential applications that take advantage of the high SERS, fluorescence, and MS sensitivity of this SERS tagging strategy are discussed for practical glycomic analysis where carbohydrates may be quantified with a fluorescence and SERS technique and then identified with ESI-MS techniques.

A novel ratiometric Raman spectroscopic (RMRS) method has been developed for quantitative determination of protein carbonyl levels. Oxidized bovine serum albumin (BSA) and oxidized lysozyme were used as model proteins to demonstrate this method. The technique involves conjugation of protein carbonyls with dinitrophenyl hydrazine (DNPH), followed by drop coating deposition Raman spectral acquisition (DCDR). The RMRS method is easy to implement because it requires only one conjugation reaction, uses a single spectral acquisition, and does not require sample calibration. Characteristic peaks from both protein and DNPH moieties are obtained in a single spectral acquisition, allowing the protein carbonyl level to be calculated from the peak intensity ratio. Detection sensitivity for the RMRS method is approximately 0.33 pmol carbonyl per measurement. Fluorescence and/or immunoassay-based techniques only detect a signal from the labeling molecule and, thus, yield no structural or quantitative information for the modified protein, whereas the RMRS technique allows protein identification and protein carbonyl quantification in a single experiment.