Smart Materials
1.3 Smart Materials for Analytical Determinations
Smart materials provide a wide variety of excellent properties that have contributed to the development of new and improved analytical methods. Figure 1.5 shows a summary of the main smart materials employed in the development of current analytical meth- ods, such as stationary phases for chromatography and electrophoresis, sensors and chips, immunoassays, laser desorption ionization, and SERS (surface‐enhanced Raman spectroscopy) signal enhancement. As can be seen, assorted smart materials have been employed for the aforementioned analytical applications, considering material specific factors such as particle size, electronic properties, selectivity, stability, etc.
1.3.1 Stationary Phases
Chromatography separations are strongly dependent on the nature of the stationary phase, the column length, and the internal diameter. Furthermore, film thickness and particle size of the stationary phase also contribute to the efficacy of analytical separa- tions in gas and liquid chromatography, respectively. Parameters like polarity, particle size, chemical and thermal stability, and homogeneity can be adjusted by using sur- face‐modified silica particles, polysiloxanes, and polymeric materials. In this sense, the use of smart materials with a wide range of physico‐chemical properties allows us to modulate the chromatographic separation providing enhanced resolution as compared with conventional stationary phases.
New stationary phases have been developed for high‐performance liquid chromatography (HPLC), achieving higher efficiency and unique selectivity using different chromatography modes like reversed‐phase, normal‐phase, ion‐exchange, or
Chromatography Electrophoresis Sensors Immunoassays Signal-enhanced Raman spect.
SERS signal Laser
Laser desorption ionization
• MIPs
• Carbon-based
• Monolithic
• MOFs
• Antibodies
• MIPs
• Carbon-based
• Monoliths
• Nanoparticles
• Cyclodextrin- based
• Nanoparticles
• Carbon-based
• QDs
• Aptamers
• MIP
• Antibodies
• Enzymes
• QDs
• Aptamers
• Nanoparticles
• Nanoparticles
• Cage-like materials
• MIPs
• Graphene
• Aptamers
• Nanoparticles
• Carbon-based
• Enzymes
• Ionic liquids
• MOFs
Figure 1.5 Main smart materials employed in the design and development of recent analytical approaches.
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hydrophilic interaction liquid chromatography (HILIC). Conventional packed HPLC columns can be categorized as inorganic (bare silica, modified silica, inorganic oxides, and graphite), organic (nonporous or macroporous polymers), and inorganic–organic hybrids that show the merits of both material types increasing simultaneously thermal and chemical stability [52]. Porous spherical silica particles are the most extended and popular substrates employed for HPLC separations, containing pure alkyl chains (C8 or C18), or alkyl chains with reactive groups (such as amino, chloro, epoxy, mercapto, or isocyanate groups). The use of reactive groups allows the easy tethering of multifunc- tional ligands, such as cyclodextrin, calixarene, crown ethers, and ILs, which offer enhanced chromatography retention and selectivity, because of the heterogeneous interactions with the ligands based on hydrogen bonding, π–π, dipole‐induced dipole, and electrostatic interactions [53].
A wide variety of smart materials have been evaluated for their use as HPLC station- ary phase. MIPs have been traditionally employed as HPLC stationary phase, with the column being packed with ground and sieved bulk polymer, or used as monolith, mono- dispersed spherical MIPs, and composite polymer beads [54]. The production of packed MIP columns is a tedious process, while the production of monolithic columns is sim- pler but efforts must be focused to increase their reproducibility and reusability [55].
Additionally, HPLC stationary phases have been modified with different carbonaceous nanomaterials like CNTs, fullerenes, graphene, and nanodiamonds, with a large sur- face‐area, easy derivatization, and average thermal, and mechanical stability [56]. MOFs have been also employed as stationary phases due to their exceptionally large surface area, tunable pore geometry, and versatile structure and chemistry [57]. The use of high specificity antibodies for affinity chromatography has an important role in character- izing immobilized proteins and provide direct measurements with multiple binding sites [58]. Effective enantioselective separations of pharmaceuticals [59] and even atropisomeric [60] compounds have been carried out using chiral columns based on gold nanoparticles, carbonaceous materials, MIPs, MOFs, ordered mesoporous silica, and capillary monoliths.
Monolithic capillary columns offer optimized porous structures in combination with a rich surface chemistry. The use of ILs as functional monomers and porogenic solvents provides improved selectivity and stability. The incorporation of nanoparticles, such as metal oxides, CNT, graphene, or MOFs, tunes monolith morphology and, as a conse- quence, enhances the separation efficiency [61]. Additionally, fiber‐based monoliths allow the packing of HPLC columns by aligned fibers, woven matrices, or contiguous fiber structures to achieve rapid and effective chromatographic separations [62].
In the same way, as indicated for chromatography, smart materials have also been employed for the improvement of capillary electrochromatography methods. Different materials have been covalently anchored to the capillary walls of open‐tubular capillar- ies in order to prevent analyte adsorption and to modify the rate of the electroosmotic flow [63]. MIP‐based applications have shown superior separation performances using multiple formats, such as packed particles, capillary coating, monoliths, and use of nanoparticle‐based pseudo‐stationary phases [54]. Carboxylated CNTs [64] and gra- phene [65] have also been widely employed because of enhanced hydrophobic, ionic, and hydrogen bonding interactions that take place simultaneously with the analyte.
Ionic liquids covalently bound to the capillary surface were applied for this purpose [63]. Acrylamide‐, methacrylate‐, and silica‐based monolithic capillaries have also been
employed for many capillary electrochromatography applications [66]. Enantiomeric separations have also been achieved by capillary electrochromatography using smart materials like MIPs and nanoparticles such as CNT, silica, TiO2, and Al2O3 coated with cyclodextrin to provide the stereoselectivity [59]. Cyclodextrin‐based capillary polymer monoliths have been efficiently employed as they provide enantioselective separations with a large surface area [61, 67].
1.3.2 Sensor Development
Optical and electrochemical sensors are widely employed in several analytical applica- tions due to the rapid response, easy handling, low cost, portability, and miniaturiza- tion, giving also high sensitivity and selectivity. Consequently, the extended deployment of smart materials in this field has really improved their use and enhanced their analyti- cal characteristics. Nanosensors have received a great attention due to the interaction with target analytes at a scale that makes them suitable for ultrasensitive detection. The exceptional photoelectric properties and size of nanoparticles allows their efficient use as electrode materials. Thus, gold nanoparticles, carbon‐based nanoparticles, and quantum dots QDs materials have played an important role in the development of a wide variety of sensors for analytical applications. Among others, gold nanoparticles are the most employed nanomaterial due to high stability and other particular characteris- tics like large surface area, size‐dependent optical properties, strong adsorption, and easy functionalization. Moreover, gold nanoparticles absorb in the visible spectrum region and can be employed for colorimetric detection [68].
Graphene and related materials have been employed for the fabrication of sensitive sensors and biosensors because of their extraordinary properties, such as electrical con- ductivity, large accessible surface area, and high electron transfer rate. Graphene shows low water solubility and a lack of surface functionality; thus, the use of graphene oxide provides more adequate properties for sensor development than graphene, and pre- sents an improved capacity to immobilize biomolecules [69]. Graphene nanosheets, graphene oxide, and reduced graphene oxide have been employed in the development of chemiluminescence resonance energy transfer and luminescence quenching‐based sensors [70]. Graphene based sensors have been employed for clinical, environmental, and food science applications [69]. MIPS‐graphene oxide sensors have been also employed, based on their extremely high selectivity, in the design and development of selective sensors for several target compounds like dopamine, vanillin, epinephrine, benzenediol isomers, or sulfamethoxazole [70].
Biosensors are sensors that are coupled to a biomolecule, such as an antibody, enzyme, or similar, which provides an especially high selectivity for a series of target molecules, due to the high affinity between antibody and antigen, or substrate and enzyme. Potential applications of antibody‐based sensors (immunosensors) are focused on clinical and diagnostic areas for the analysis of several biomarkers at point‐of‐care application [71].
Electrochemical aptasensors are a new type of biosensor, based on the electro- chemical transduction produced when the aptamer specifically recognizes a target analyte. These sensors provide a high selectivity and sensitivity, low cost, and require simple instrumentation. Aptasensors have been employed for the analysis of small molecules, proteins, and nucleic acids by their coupling to optical, electrochemical,
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and mass sensitive detectors [72]. The miniaturization of aptamer‐based sensors through the integration with nanotechnologies allows the production of sensors in array format, which allows the simultaneous multiplex analysis of several com- pounds [73].
Quantum dots (QDs) are luminescent semiconductor nanocrystals with high quan- tum yield, large extinction coefficient, high photostability, broad absorption, and nar- row emission spectra [74]. Additionally, QDs fluorescence can be easily tunable by the selection of their chemical composition (binary and ternary alloys of heavy metals) and particle size (within the low nm diameter). Sensors have been developed measuring the enhancement or quenching of QD fluorescence as consequence of a direct interaction of target analyte with the surface of a QD particle. QD‐based sensors have been devel- oped for the determination of different organic compounds like spirolactone, tiopronin, dopamine, glucose, TNT, anthracene, p‐nitrophenol, 1‐naphthol, methionine, and enoxacin [75]. These interactions are, in some cases, unspecific, and strongly depend- ent on the QD coating, and ratiometric sensors could be developed [76].
Selective sensors have been developed based on the Förster resonance energy transfer (FRET) phenomenon, which involves the transfer of resonant fluorescence energy from an excited donor to a ground‐state acceptor fluorophore [77]. The efficacy of the energy transfer depends on the Förster radius (distance between the fluorophores) and the spectral overlap between them. QDs show excellent properties such as those of a donor emission fluorophore because of its tunable emission wavelength, as well as wide absorption band, high quantum yield, and easy bioconjugation [78].
Carbon dots are carbon‐based fluorescent materials with excellent optical properties like QDs – tunable with the appropriate control of size, shape, and surface modification.
These materials show a lower toxicity and higher biocompatibility than standard QDs, but a lower quantum yield. However, photoluminescence of carbon dots can be greatly enhanced by doping with nitrogen, sulfur, and phosphorus elements. Additional char- acteristics of carbon dots are simple synthesis, high aqueous solubility, low cost, and suitability for bioimaging [79]. Nanodiamonds, graphite, CNTs, or activated carbon have been applied for their high fluorescence in analytical sensors, with superior per- formance seen with the use of graphene carbon dots due to their exceptional electronic properties, high conductivity, and the high number of reactive sites [80].
1.3.3 Immunoassays
Immunoassays provide analytical methods for the analysis of both biomolecules and small‐size analytes, based on the extremely high specificity and selectivity of antibody–
antigen interactions. One of the most extensively used immunoassays in analytical chemistry is the so‐called enzyme‐linked immunosorbent assay (ELISA) format, a plate‐bound detection based on the use of specific antibodies and enzymes. Different ELISA formats can be employed, such as sandwich, competitive, and antigen‐down assays, depending on the target analyte, the available immunoreagents, and the required dynamic range [81]. ELISA assays usually employ a total analysis time of 1 or 2 h.
However, due to its simplicity and the use of 96‐well plates, the sample throughput is one of its main advantages. Typically, a horseradish peroxidase is conjugated to the antibody (or hapten) and, after the immunological reaction, a concentration‐related signal is generated by adding the appropriate enzymatic substrate.
Fluorescent detection has been also employed in immunosensors using organic dyes like fluorescein or rhodamine coupled to the immunoreagent, but a low quantum yield, and poor stability, is often obtained. In this sense, the use of QDs has been proposed as effective luminescent probes for the development of fluorescent immunoassays. Surface functionalized QDs can be easily conjugated to biomolecules like proteins, antibodies, antibody fragments, aptamers, etc. in order to obtain unique nanoparticles with both excellent optical properties and high specificity [77]. QDs are typically excited by a sin- gle wavelength and those with different emission wavelengths can be used as fluores- cent labels for simultaneous multianalyte immunoassays (multiplexed) with negligible spectral interferences [82, 83].
Aptamers, as short single‐chain oligonucleotides that show high binding affinities against a wide range of target analytes with high selectivity, have been proposed to replace antibodies in many immunoassay systems. Aptamers are easier and cheaper to produce than antibodies and do not require the use of cells or experimental animals [84]. Aptamers are usually immobilized over different supports such as gold, silica and carbon nanoparticles, magnetic beads, graphene, Sepharose, and modified cellulose particles to be employed in biosensors [85].
Recombinant antibodies, often so‐called nanobodies, are variable domain of heavy chain antibodies (12–15 kDa approximate molecular weight) that selectively bind to specific antigens. Nanobodies have been employed in the development of immunoas- says, moreover in FRET‐based approaches where the molecular distances between fluorophores must be reduced [86].
Lateral flow immunoassay is a simple and cost‐effective methodology usually employed for rapid point‐of‐care testing, based on the movement of the sample compo- nents through a membrane that enables the formation and separation of complexes after an immunochemical reaction, avoiding sample pre‐treatments or washing steps [87]. Lateral flow test strips have been widely employed in different areas such as clinical diagnostics, drug screening, or food analysis [88].
1.3.4 Signal Enhancement
Surface‐enhanced Raman spectroscopy (SERS) refers to an inelastic light scattering process from analytes in close proximity to a plasmonic substrate that provides rich vibrational spectroscopic information about the adsorbed molecule with three orders of magnitude enhancement of classical Raman signals. It has been considered a prom- ising non‐destructive technique for chemical, biological, and structural analysis due to its simplicity, rapidity, extreme sensitivity, and high selectivity, even reaching single‐ molecule limits of detection. SERS has been applied to quite different areas like electrochemistry, catalysis, biology, medicine, art conservation, and materials science [89].
Standard plasmonic substrates (Au and Ag) usually show a lack of thermal and pressure stabilities, and they have been replaced by metal oxides like Al2O3, TiO2, and SiO2. A remarkable SERS enhancement can be achieved by the use of capture agents, such as cross‐coupling with metal affinitive ligands, self‐assembled monolayers of thiolated molecules, polymer coatings, molecular recognition agents, aptamers, anti- body fragments, and cage‐like molecular recognition materials (such as cucurbiturils, calixarenes, and cyclodextrins) [89]. Polymer containing nanoparticles have been also
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employed as capture layer to concentrate analytes with a moderate selectivity, leading to increasing attention in recent years to the use of MIPs to improve the specificity of SERS interactions [90]. Graphene quantum dots considerably enhance the SERS effect due to the abundant hydrogen atoms terminated on their surface, which promotes an efficient charge transfer [80]. Other smart materials like graphene oxide or Au─Ag core–shell nanorods have been also evaluated for application as SERS substrates [91].
Furthermore, a new platform, so‐called slippery liquid infused porous SERS (SLIPSERS) has been developed to extend the application of SERS to both aqueous and non‐aqueous media, based on the shape of a film of lubricating fluid on a substrate of Ag nanoparticles using an evaporating liquid droplet [92]. The combination of a SLIPSERS platform with a SERS mapping technique allows the ultrasensitive detection of chemical and biological analytes at the low attomolar level in common fluids.
1.3.5 Laser Desorption/Ionization Mass Spectrometry
Matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐MS) is a soft ionization technique attached to a mass spectrometry detector, usually a time‐of‐flight analyzer, widely employed for the analysis of proteomics, biological cells and tissues, polymers, and small molecules. Direct ionization occurs for molecules that have strong absorption of the laser energy, but non‐absorbing analytes require the use of a matrix that absorbs the laser energy and assists the laser desorption/ionization process of the target analyte. Conventional matrix compounds employed for MALDI‐MS are 2,5‐
dihydroxybenzoic acid, cinnamic acid, sinapic acid, caffeic acid, ferulic acid, naphtha- lene, coumarin, or curcumin [93].
Smart materials based on organic matrices are promising sorbents that provide new functions for MALDI‐MS. The use of nanoparticles with a large surface area, variable pore sizes, and surface functionalization provides new functions and offers new applica- tions for MALDI‐MS analyses, and even may improve the degree of selectivity of ionized analytes [93]. Gold nanoparticles have remarkable advantages, such as easy sample preparation, low background, high salt tolerance, and can be used for the analysis of molecules of less than 500 Da [94]. Carbon nanomaterials usually offer high efficiency and sensitivity in the desorption/ionization of analytes, reduce background noise, and can be applied to both large and small molecules [95]. The incorporation of functional groups to carbon nanomaterials allows an improvement of assay selectivity. Enzyme‐
coupled nanoparticles constitute an effective affinity‐based tool for the study of specific interactions between enzymatic targets and small molecular weight analytes in complex mixtures [91]. The use of IL‐based organic matrices provides enhanced efficiency, tun- able laser absorption, high ionizability, low vapor pressure, low flammability, and low toxicity [93]. MOF materials have also been employed as adsorption materials and due to their rich chemistry have a promising future for MALDI‐MS applications [96].