Table 1:
A list of the most common analytical approaches, with their pros and cons, to probe the interactions between nanoparticles and biomolecules.
| Analytical method | Advantages | Disadvantages |
|---|---|---|
| UV/vis | cheap, fast, flexible, and simple; little sample preparation | nature of the solvent, pH of the solution, temperature, high electrolyte concentrations, and presence of interfering substances can influence the absorption spectrum; experimental variations such as the slit width (effective bandwidth) of the spectrophotometer will also alter the spectrum; to apply UV/vis spectroscopy to analysis, these variables must be controlled or accounted for to identify the substances present |
| fluorescence spectroscopy | sensitive | unstable |
| FTIR | quite cheap, versatile, easy to identify functional groups; sensitive to protein conformation; not constrained by substrate size or material | sample characterization is not possible in complex media; cannot get fine structural detail; time-consuming sample preparation; sample preparation destroys the sample |
| Raman spectroscopy | can be used with solids and liquids; no sample preparation needed; no interference from water; nondestructive; highly specific—like a chemical fingerprint of a material; Raman spectra are acquired quickly (within seconds); samples can be analyzed through glass or a polymer packaging; laser light and Raman scattered light can be transmitted by optical fibers over long distances for remote analysis; Raman spectra can be collected from a very small volume (<1 μm in diameter); inorganic materials are normally easily analyzed by Raman compard to infrared spectroscopy | cannot be used for metals or alloys; Raman effect is very weak; detection needs a sensitive and highly optimized instrumentation; fluorescence of impurities or of the sample itself can hide the Raman spectrum; sample heating through the intense laser radiation can destroy the sample or cover the Raman spectrum |
| mass spectrometry | high-resolution method for characterization of NP-bound proteins; unique technique to obtain protein identities | expensive; requires dedicated facility and trained user |
| NMR spectroscopy | can detect very fine structural components; works for organic and inorganic materials; qualitative and quantitative, versatile; it can be applied to a wide variety of samples for direct structural study and molecular dynamics studies, both in solution and in the solid state | expensive, time-consuming; spectra take a long time to interpret |
| DLS | nonperturbative, fast, and accurate, giving a measure of the vesicle hydrodynamic diameter as this dimension changes in solution | hydrodynamic diameters are influenced by the formation of hydration shells, the shape of the particles, and counterion binding; requires a monodisperse population |
| CD | monitoring conformational changes induced by protein-NP interaction | inherent inconsistency problems in absolute secondary structure determination; CD signal reflects an average of the entire molecular population; CD measurements cannot provide information regarding local structural alterations at the level of individual amino acids |
| ITC | can directly and quantitatively measure the binding affinity constant, enthalpy changes, and binding stoichiometry between NP and proteins in solution; no labeling or immobilization is required; not limited by the ligand or protein size; relatively artifact-free and not affected by the optical properties of the samples | requires relatively high concentrations of samples |
| ζ potential | straightforward method to measure surface charge and changes in surface charge; indicator of stability of NP dispersions | requires a minimum ionic strength and that the NPs be monodisperse as calculates a charge/size ratio |
| chromatography | very sensitive and reliable (provided that the method is carried out carefully without any contamination); complex mixtures can be separated accurately using only a few micrograms of sample; separation takes less time as compared to other techniques; the equipment setups are simple and easy | since the method is very sensitive, improper setup or contamination, even in nanograms, will give different results; sample is generally very diluted afterward and requires reconcentration; time consuming |
| electrophoresis | suitable for separation of complicated protein mixtures; suitable for qualitative and quantitative analysis | proteins are easily adsorbed onto the inner surface of the capillary, and the detection sensitivity is not high |
| SPR | sensitive to changes in the refractive index of the medium surrounding the sensor and to the thickness of the sensor layer; as any change in protein conformation will bring a modification in this parameter, SPR has also been extensively used to study the conformation of immobilized proteins in various environments | sensitivity of the system with a detection limit restricted to 1–10 nM of a 20 kDa protein and even higher for smaller molecules, particularly when the receptor displays a weak affinity |
| QCM | simple, cost-effective, high-resolution mass sensing technique; ease of setup and operation and low cost; QCMs are capable of measuring mass changes as small as a fraction of a monolayer or single layer of atoms; allows a label-free detection of molecules | variations in interfacial parameters, such as surface roughness, surface free energy, surface charge, and viscoelasticity, hamper interpretation of QCM results |
Copyright American Chemical Society 2011 [35]. Abbreviations: Fourier transform infrared (FTIR); nuclear magnetic resonance (NMR); dynamic light scattering (DLS); circular dichroism (CD), isothermal titration calorimetry (ITC), surface plasmon resonance (SPR), and quartz crystal microbalance (QCM).