Quantitative Measurement of Multiprotein Nanoparticle Interactions Using NMR Spectroscopy.

An effective intensity-based reference is a cornerstone for quantitative nuclear magnetic resonance (NMR) studies, as the molecular concentration is encoded in its signal. In theory, NMR is well suited for the measurement of competitive protein adsorption onto nanoparticle (NP) surfaces, but current referencing systems are not optimized for multidimensional experiments. Presented herein is a simple and novel referencing system using 15N tryptophan (Trp) as an external reference for 1H-15N 2D NMR experiments. The referencing system is validated by the determination of the binding capacity of a single protein onto gold NPs. Then, the Trp reference is applied to protein mixtures, and signals from each protein are accurately quantified. All results are consistent with previous studies, but with substantially higher precision, indicating that the Trp reference can accurately calibrate the residue peak intensities and reduce systematic errors. Finally, the proposed Trp reference is used to kinetically monitor in situ and in real time the competitive adsorption of different proteins. As a challenging test case, we successfully apply our approach to a mixture of protein variants differing by only a single residue. Our results show that the binding of one protein will affect the binding of the other, leading to an altered NP corona composition. This work therefore highlights the importance of studying protein-NP interactions in protein mixtures in situ, and the referencing system developed here enables the quantification of binding kinetics and thermodynamics of multiple proteins using various 1H-15N 2D NMR techniques.

Understanding the Adsorption of Peptides and Proteins onto PEGylated Gold Nanoparticles.

Polyethylene glycol (PEG) surface conjugations are widely employed to render passivating properties to nanoparticles in biological applications. The benefits of surface passivation by PEG are reduced protein adsorption, diminished non-specific interactions, and improvement in pharmacokinetics. However, the limitations of PEG passivation remain an active area of research, and recent examples from the literature demonstrate how PEG passivation can fail. Here, we study the adsorption amount of biomolecules to PEGylated gold nanoparticles (AuNPs), focusing on how different protein properties influence binding. The AuNPs are PEGylated with three different sizes of conjugated PEG chains, and we examine interactions with proteins of different sizes, charges, and surface cysteine content. The experiments are carried out in vitro at physiologically relevant timescales to obtain the adsorption amounts and rates of each biomolecule on AuNP-PEGs of varying compositions. Our findings are relevant in understanding how protein size and the surface cysteine content affect binding, and our work reveals that cysteine residues can dramatically increase adsorption rates on PEGylated AuNPs. Moreover, shorter chain PEG molecules passivate the AuNP surface more effectively against all protein types.

Quantification of the Photon Absorption, Scattering, and On-Resonance Emission Properties of CdSe/CdS Core/Shell Quantum Dots: Effect of Shell Geometry and Volumes.

Reliable quantification of the optical properties of fluorescent quantum dots (QDs) is critical for their photochemical, -physical, and -biological applications. Presented herein is the experimental quantification of photon scattering, absorption, and on-resonance-fluorescence (ORF) activities of CdSe/CdS core/shell fluorescent QDs as a function of the shell sizes and geometries. Four spherical QDs (SQDs) with different diameters and four rod-like QDs (RQDs) with different aspect ratios (ARs) have been analyzed using UV-vis, fluorescence, and the recent polarized resonance synchronous spectroscopic (PRS2) methods. All quantum dots are simultaneous absorbers and scatterers in the UV-vis wavelength region, and they all exhibit strong ORF emission in the wavelength regions where the QDs both absorb and emit. The absorption and scattering cross-sections of the CdS shell are linearly and quadratically, respectively, proportional to the shell volume for both the SQDs and RQDs. However, the effects of CdS shell coating on the core optical properties are different between SQDs and RQDs. For RQDs, increasing the CdS shell volume through the length elongation has no effect on either the peak wavelength or intensity of the CdSe core UV-vis absorption and ORF, but it reduces the QD fluorescence depolarization. In contrast, increasing CdS shell volume in the SQDs induces red-shift in the CdSe core peak UV-vis absorption and ORF wavelengths, and increases their peak cross-sections, but it has no effect on the SQD fluorescence depolarization. The RQD ORF cross-sections and quantum yields are significantly higher than their respective counterparts for the SQDs with similar particle sizes (volumes). While these new insights should be significant for the QD design, characterization, and applications, the methodology presented in this work is directly applicable for quantifying the optical activities of optically complex materials where the common UV-vis spectrometry and fluorescence spectroscopy are inadequate.

A Divide-and-Conquer Strategy for Quantification of Light Absorption, Scattering, and Emission Properties of Fluorescent Nanomaterials in Solutions.

Optical properties of fluorescent materials including their UV-vis absorption, scattering, and on-resonance fluorescence activities are strongly wavelength-dependent. Reported herein is a divide-and-conquer strategy for experimental quantification of fundamental optical constants of fluorescent nanomaterials including their UV-vis absorption, scattering, and on-resonance-fluorescence (ORF) cross-section spectra and ORF fluorescence and light scattering depolarization spectra. The fluorophore UV-vis extinction spectrum is first divided into a blue and a red wavelength region. The UV-vis extinction cross-section spectrum in the blue wavelength region is decomposed into its absorption and scattering extinction spectra straightforwardly using the established polarized resonance synchronous spectroscopic technique. In its red wavelength region, however, the fluorophores can be simultaneous photon absorbers, scatterers, and anti-Stokes-shifted, on-resonance, and Stokes-shifted fluorescence emitters under the resonance excitation and detection conditions. A polarized anti-Stokes'-shifted, on-resonance, and Stokes'-shifted spectroscopic method is developed for quantifying fluorophore absorption, scattering, one-resonance fluorescence (ORF) cross-section spectra, and scattering and ORF fluorescence depolarization spectra in this wavelength region. Example applications of the presented techniques were demonstrated with fluorescent polystyrene nanoparticles, fluorescent quantum dots, and molecular fluorophores Rhodamine 6G and Eosin Y.

Quantification of Material Fluorescence and Light Scattering Cross Sections Using Ratiometric Bandwidth-Varied Polarized Resonance Synchronous Spectroscopy.

Presented herein is the ratiometric bandwidth-varied polarized resonance synchronous spectroscopy (BVPRS2) method for quantification of material optical activity spectra. These include the sample light absorption and scattering cross-section spectrum, the scattering depolarization spectrum, and the fluorescence emission cross-section and depolarization spectrum in the wavelength region where the sample both absorbs and emits. This ratiometric BVPRS2 spectroscopic method is a self-contained technique capable of quantitatively decoupling material fluorescence and light scattering signal contribution to its ratiometric BVPRS2 spectra through the linear curve-fitting of the ratiometric BVPRS2 signal as a function of the wavelength bandwidth used in the PRS2 measurements. Example applications of this new spectroscopic method are demonstrated with materials that can be approximated as pure scatterers, simultaneous photon absorbers/emitters, simultaneous photon absorbers/scatterers, and finally simultaneous photon absorbers/scatterers/emitters. Because the only instruments needed for this ratiometric BVPRS2 technique are the conventional UV-vis spectrophotometer and spectrofluorometer, this work should open doors for routine decomposition of material UV-vis extinction spectrum into its absorption and scattering component spectra. The methodology and insights provided in this work should be of broad significance to all chemical research that involves photon/matter interactions.

Quantification of Gold Nanoparticle Ultraviolet-Visible Extinction, Absorption, and Scattering Cross-Section Spectra and Scattering Depolarization Spectra: The Effects of Nanoparticle Geometry, Solvent Composition, Ligand Functionalization, and Nanoparticle Aggregation.

Using the recent polarized resonance synchronous spectroscopic (PRS2) technique, we reported the quantification of photon extinction, absorption, scattering cross-section spectra, and scattering depolarization spectra for AuNPs of different sizes and shapes. The effects of the solvent composition, ligand functionalization, and nanoparticle aggregation on the AuNP photon absorption and scattering have also been experimentally quantified. The light scattering depolarization is close to 0 for gold nanospheres (AuNSs) crossing the entire UV-vis region but is strongly wavelength-dependent for gold nanorods (AuNRs). Increasing the dielectric constant of the medium surrounding AuNPs either by solvents or ligand adsorption increases photon absorption and scattering but has no significant impact on the AuNP scattering depolarization. Nanoparticle aggregation increases AuNP photon scattering. However, even the extensively aggregated AuNPs remain predominantly photon absorbers with photon scattering-to-extinction ratios all less than 0.03 for the investigated AuNP aggregates at the AuNP peak extinction wavelength. The AuNP scattering depolarization initially increases with the AuNP aggregation but decreases when aggregation further progresses. The insights from this study are important for a wide range of AuNP applications that involve photon/matter interactions, while the provided methodology is directly applicable for experimental quantification of optical properties for nanomaterials that are commonly simultaneously photon absorbers and scatterers.

Synthesis of C-Unsubstituted 1,2-Diazetidines and Their Ring-Opening Reactions via Selective N-N Bond Cleavage.

C-Unsubstituted 1,2-diazetidines, a rarely studied type of four-membered heterocyclic compounds, were synthesized through an operationally simple intermolecular vicinal disubstitution reaction. 1,2-Diazetidine derivatives bearing various N-arylsulfonyl groups were readily accessed and studied by experimental and computed Raman spectra. The ring-opening reaction of the diazetidine was explored and resulted in the identification of a selective N-N bond cleavage with thiols as nucleophiles, which stereoselectively produced a new class of N-sulfenylimine derivatives with C-aminomethyl groups.

Scattering and absorption differ drastically in their inner filter effects on fluorescence, resonance synchronous, and polarized resonance synchronous spectroscopic measurements.

The sample inner filter effect (IFE) induces spectral distortion and affects the linearity between intensity and analyte concentration in fluorescence, Raman, surface enhanced Raman, and Rayleigh light scattering measurements. Existing spectrofluorometric-based measurements treat light scattering and absorption identically in their sample IFEs. Reported herein is the finding that photon scattering and absorption differ drastically in inducing the sample IFE in Stokes-shifted fluorescence (SSF) spectrum, resonance synchronous spectrum (RS2), and the polarized resonance synchronous spectrum (PRS2) measurements. Absorption with an absorption extinction as small as 0.05 imposes significant IFE on SSF, RS2, and PRS2 measurements. However, no significant IFE occurs even when the scattering extinction is as high as 0.9. For samples that both absorb and scatter light, one should decompose their UV-vis extinction spectra into absorption and scattering extinction component spectra before correcting the sample IFE. An iteration PRS2 method was introduced for the experimental decoupling of the photon absorption and scattering contribution. The methodology presented in this work can be easily implemented by researchers with access to one conventional UV-vis spectrophotometer and one spectrofluorometer equipped with a pair of excitation and detection polarizers. This work should be of broad significance in chemical research given the popularity of fluorescence spectroscopy in material characterization applications.

Predicting protein function and orientation on a gold nanoparticle surface using a residue-based affinity scale.

The orientation adopted by proteins on nanoparticle surfaces determines the nanoparticle's bioactivity and its interactions with living systems. Here, we present a residue-based affinity scale for predicting protein orientation on citrate-gold nanoparticles (AuNPs). Competitive binding between protein variants accounts for thermodynamic and kinetic aspects of adsorption in this scale. For hydrophobic residues, the steric considerations dominate, whereas electrostatic interactions are critical for hydrophilic residues. The scale rationalizes the well-defined binding orientation of the small GB3 protein, and it subsequently predicts the orientation and active site accessibility of two enzymes on AuNPs. Additionally, our approach accounts for the AuNP-bound activity of five out of six additional enzymes from the literature. The model developed here enables high-throughput predictions of protein behavior on nanoparticles, and it enhances our understanding of protein orientation in the biomolecular corona, which should greatly enhance the performance and safety of nanomedicines used in vivo.

Solution NMR of Nanoparticles in Serum: Protein Competition Influences Binding Thermodynamics and Kinetics.

The spontaneous formation of a protein corona on a nanoparticle surface influences the physiological success or failure of the synthetic nanoparticle as a drug carrier or imaging agent used in vivo. A quantitative understanding of protein-nanoparticle interactions is therefore critical for the development of nanoparticle-based therapeutics. In this perspective, we briefly discuss the challenges and limitations of current approaches used for studying protein-nanoparticle binding in a realistic biological medium. Subsequently, we demonstrate that solution nuclear magnetic resonance (NMR) spectroscopy is a powerful tool to monitor protein competitive binding in a complex serum medium in situ. Importantly, when many serum proteins are competing for a gold nanoparticle (AuNP) surface, solution NMR is able to detect differences in binding thermodynamics, and kinetics of a tagged protein. Combined with other experimental approaches, solution NMR is an invaluable tool to understand protein behavior in the nanoparticle corona.

Linear Extrapolation of the Analyte-Specific Light Scattering and Fluorescence Depolarization in Turbid Samples.

Anisotropy and depolarization are two interconvertible parameters in fluorescence and light scattering spectroscopy that describe the polarization distribution of emitted and scattered photons generated with linearly polarized excitation light. Whereas anisotropy is more frequently used in fluorescence literature for studying association/dissociation of fluorophore-bearing reagents, depolarization is more popular in the light-scattering literature for investigating the effect of scatterers' geometries and chemical compositions. Presented herein is a combined computational and experimental study of the scattering and fluorescence depolarization enhancement induced by light scattering in turbid samples. The most important finding is that sample light scattering and fluorescence depolarization increases linearly with sample light-scattering extinction. Therefore, one can extrapolate the analyte-specific scattering and fluorescence depolarization through linear curve fitting of the sample light scattering and fluorescence depolarization as a function of the sample concentration or the path length of the sampling cuvettes. An example application of this linear extrapolation method is demonstrated for quantifying the fluorophore-specific fluorescence depolarization and consequently its anisotropy for an aggregation-induced-emission sample. This work should be important for a wide range of macromolecular, supramolecular, and nanoscale fluorescent materials that are often strong light scatterers due to their large sizes.

Surface Plasmon Resonance, Formation Mechanism, and Surface Enhanced Raman Spectroscopy of Ag+-Stained Gold Nanoparticles.

A series of recent works have demonstrated the spontaneous Ag+ adsorption onto gold surfaces. However, a mechanistic understanding of the Ag+ interactions with gold has been controversial. Reported herein is a systematic study of the Ag+ binding to AuNPs using several in-situ and ex-situ measurement techniques. The time-resolved UV-vis measurements of the AuNP surface plasmonic resonance revealed that the silver adsorption proceeds through two parallel pseudo-first order processes with a time constant of 16(±2) and 1,000(±35) s, respectively. About 95% of the Ag+ adsorption proceeds through the fast adsorption process. The in-situ zeta potential data indicated that this fast Ag+ adsorption is driven primarily by the long-range electrostatic forces that lead to AuNP charge neutralization, while the time-dependent pH data shows that the slow Ag+ binding process involves proton-releasing reactions that must be driven by near-range interactions. These experimental data, together with the ex-situ XPS measurement indicates that adsorbed silver remains cationic, but not as a charged-neutral silver atom proposed by the anti-galvanic reaction mechanism. The surface-enhanced Raman activities of the Ag+-stained AuNPs are slightly higher than that for AuNPs, but significantly lower than that for the silver nanoparticles (AgNPs). The SERS feature of the ligands on the Ag+-stained AuNPs can differ from that on both AuNPs and AgNPs. Besides the new insights to formation mechanism, properties, and applications of the Ag+-stained AuNPs, the experimental methodology presented in this work can also be important for studying nanoparticle interfacial interactions.

Optical Properties and Kinetics: New Insights to the Porphyrin Assembly and Disassembly by Polarized Resonance Synchronous Spectroscopy.

With their unique photochemical properties, porphyrins have remained for decades the most interested chemicals as photonic materials for applications ranging from chemistry, biology, medicine, to photovoltaic. Porphyrins can self-assemble into higher order structures. However, information has been scant on the kinetics and structural evolution during porphyrin assembly and disassembly. Furthermore, quantitative understanding of the porphyrin optical activities is complicated by the complex interplay of photon absorption, scattering, and fluorescence emission that can concurrently occur in porphyrin samples. Using meso-tetrakis(4-sulfonatophenyl)porphyrin as the model molecule, reported herein is a combined UV-vis extinction, polarized Stokes-shifted fluorescence, and polarized resonance synchronous spectroscopic (PRS2) study of porphyrin assembly and disassembly in acidic solutions. Although porphyrin assembly and disassembly occur instantaneously upon the sample preparation, both processes last at least a few months before reaching their approximate equilibrium states. The two processes were monitored in situ by quantifying the porphyrin fluorescence and scattering depolarizations as well as its extinction, absorption, scattering, and fluorescence emission cross sections. In addition to a series of new insights to the porphyrin assembly and disassembly, the methodology described in this work opens the door for the in situ study of the structural and optical properties of photonic materials comprising molecular assembly.