Fluorescence “Giant” Red Edge Effect
5.2 Methodology
5.2.1 Hydrothermal carbonization
Hydrothermal carbonization, a propitious technique among all surface passivation technologies, is a chemical process for the conversion of organic compounds to structured carbons. This process can be classified into two basic constituents based on the application of temperature. The high-temperature method occurs from 300C to 800C, allowing multiwalled CNTs, graphic carbon materials, fullerenes, acti- vated carbon materials, and carbon spheres with different nanostructures to be syn- thesized. The low-temperature hydrothermal carbonization process is performed
below 300C to synthesize functional carbonaceous materials where carbon spheres have been obtained as main products via dehydration reaction followed by polymer- ization reaction[2,3].
5.2.1.1 Amino-functionalized fluorescent carbon quantum dots Highly amino-functionalized fluorescent CQDs can be synthesized by low- temperature hydrothermal carbonization of chitosan. Here the formation of CQDs and their surface functionalization take place simultaneously during hydrothermal carbonization, which occurs through the dehydration of chitosan (Fig. 5.2). The functional groups make them water-soluble and reduce their potential bio-toxicity.
The advantage of this one-step synthetic method is that no acid solvent or surface passivation reagent is needed and this occurs in an aqueous solution. The process is very cheap, absolutely green[4].
The transparent yellowish CQDs solution is clear under sunlight, whereas it shows strong blue luminescence under UV light. Some defect sites on the surface of CQDs can be created due to the surface modification. These defects trap the radi- ative recombination of the excitons resulting in high fluorescence emission. The CQDs show low cytotoxicity and do not pose any significant toxic effects. This result concludes that CQDs can be used in a high concentration for bioimaging or other medical applications.
5.2.1.2 Branched polyethylenimine functionalized carbon quantum dots
The branched polyethylenimine (BPEI) CQDs can be synthesized by low- temperature (,200C) carbonization of citric acid (CA) in the presence of BPEI.
This is basically an easy one-step bottom-up synthesis method. The temperature should not be higher than 200C, to avoid the degeneration of BPEI. Here CA is used as the carbon precursor because of its facile low carbonization temperature (,200C). Since BPEI plays a role in surface passivation of CQDs with some amines, it helps to detect different analytes with the other free amines due to its polyamine structure. It can be used as capping and functionalizing reagent. The principle of the synthesis of BPEI functionalized CQDs is shown inFig. 5.3.
Figure 5.2 A schematic illustration of the preparation procedure of CQDs by hydrothermal carbonization of chitosan[4].
The polyamine functionalized CQDs can both selectively recognize trace amount of certain metal ions, such as Cu21 ion with high FL quantum yield. The amino groups at the surface of the CQDs can bind the Cu21to form cupric amine, result- ing in a selective and strong quenching of the CQDs’ FL via a so-called inner filter effect, and give a very sensitive signal response, providing their promising applica- tion in analytical chemistry[5,6].
5.2.1.3 Amino-functionalized carbon quantum dots
Amino-functionalized CQDs can also be prepared from anhydrous CA and diethyle- netriamine by a low-temperature (200C) hydrothermal approach. Then the synthe- sized CQDs can be attached covalently to 4-carboxyphenylboronic acid (CPBA) to form CPBA-CQD (Fig. 5.4).
The CPBA functional groups are reactive toward vicinal diols. So they can cova- lently bridge the highly toxic catechol with the vicinal diol structures and as a result, the fluorescence gets quenched significantly via the static quenching mecha- nism[7].
5.2.1.4 Spiropyran-functionalized carbon quantum dots
Fluorescent CQDs can be synthesized by the low-temperature hydrothermal carbonization of ethylenediaminetetraacetic acid disodium salt (EDTA.2Na). Then the synthesized CQDs are functionalized with spiropyran by linking them covalently. Spiropyran, a photoisomeric dye, is known for its photochromic properties that provide this molecule with the ability of functionalizing some low dimensional materials like CNTs, noble metal nanoclusters, and semiconductor quantum dot. These spiropyran-functionalized materials have extensive use in medical and technology areas. To synthesize spiropyran- functionalized CQDs, the functionalization of the CQDs with ethylenediamine (EDA) is needed at first. So to synthesize EDA-CQD, excess amount of EDA is added to the CQD solution, activated by N-Hydroxysuccinimide (NHS) for 1 hour. Then, carboxyl containing spiropyran is treated with EDA-CQD to functionalize them with spiropyran.
Spiropyran-functionalized CQDs show reversible fluorescence modulation, excellent photoreversibility, high stability, and relatively fast photoresponsivity. This suggests that Figure 5.3 Synthesis route of BPEI functionalized CQDs (BPEI-CD)[5].
the photoresponsive spiropyran-functionalized CQDs can be potentially applied in biolog- ical imaging and labeling, reversible data storage/erasing, as well as individual light- dependent nanoscale devices[8].
5.2.2 Microwave-assisted pyrolysis
The thermal decomposition of matter at elevated temperatures in an inert atmo- sphere is known as pyrolysis. Microwave-assisted pyrolysis is a special type of pyrolysis that comprises microwave dielectric heating and temperatures as high as 800C. The traditional surface engineering process of nanoparticles usually embraces multiple steps and makes the processing time-consuming. But the microwave-assisted pyrolysis process is very fast[9].
5.2.2.1 Hyperbranched polyethylenimine and isobutyric amide functionalized C-dots
Strong photoluminescent polyethylenimine (PEI) functionalized C-dots (CD-PEI) have been synthesized directly via microwave pyrolysis of glycerol in the presence of hyperbranched PEI under 700 W microwaves for 10 min. The role of PEI as the sur- face passivation agent will aid in the generation of C-dots, which will help in gene delivery and intensify fluorescent properties. In this synthetic route, the formation of
+ 85ºC
CPBA
200ºC, 5h Hydrothermal
+
CA DETA CDs
CPBA-CDs Figure 5.4 Synthesis route of CPBA functionalized CQDs (CPBA-CD)[7].
carbon nanoparticles and the surface passivation with PEI occur simultaneously in one pot. Isobutyric amide (IBAm) groups can be coupled with CD-PEI through the amidation reaction of isobutyric anhydride and HPEI moiety to produce multistimuli- responsive CD-PEI-IBAm (Fig. 5.5). This type of photoluminescent CQDs is able to host organic molecules as nanocarriers. Thus they are applicable in the area of drug delivery systems and biotechnology as “smart” materials[10].
5.2.2.2 Organosilane functionalized carbon quantum dots
For real device application, it is required to synthesize nontoxic and ecofriendly CQDs by embedding them in solid-state architectures or in a suitable solid matrix.
Silica gel and organically functionalized silicates are significantly used as solid matrices because of their optical properties and inherent stability. But aggregation, low doping concentration, or phase separation during the preparation of polymer hybrid materials or silica gel make their use limited. So nanomaterials can be modi- fied with organosilanes instead of silica gel after their formation. The highly lumi- nescent amorphous CDs can be synthesized by the decomposition and pyrolysis of anhydrous CA, which produces gas and condensable vapors with the surface passiv- ation reaction of the amine groups of N-(β-aminoethyl)-γ-aminopropylmethyldi- methoxysilane (AEAPMS) and the carboxyl groups derived from the pyrolyzed Figure 5.5 Synthesis of hyperbranched PEI and IBAm functionalized CD[10].
species in 1 minute. Here AEAPMS acts as a coordinating solvent. The AEAPMS- CDs can be forged into pure CD monoliths or fluorescent films simply by charring them at 80C for 24 h. Furthermore, the water-insoluble AEAPMS-CDs can be con- verted into water-soluble CDs/silica particles by treating them with tetraethylortho- silicate (TEOS) (Fig. 5.6). This silica precursor TEOS helps to hydrolyze and condense the terminal methoxysilane groups to form a silica overlayer[11,12].
Those biocompatible and nontoxic CDs/silica particles have promising imple- mentations in different fields like medical diagnostics to catalysis and photovoltaics due to their cost-effectiveness, magnificent chemical stability, ready scalability, and special properties.
5.2.2.3 Organic dye-functionalized carbon quantum dot
Extremely fluorescent crystalline CQDs can be synthesized from sucrose upon 100 W microwave irradiation for 3 min 40 s in presence of phosphoric acid. Here, sucrose and phosphoric acid play the role of carbon precursor oxidizing agents.
This type of CQDs exhibits bright green fluorescence upon excitation of UV light.
To further modify the fluorescence property as well as to reduce the cytotoxicity of the synthesized carbon nanoparticles of the resulting CQDs, they are functionalized by different dyes, for instance rhodamine B, fluorescein, andα-naphthylamine. To functionalize those dyes, the CQDs should, at first, be activated by1-Ethyl-3-(3- dimethylaminopropyl)carbodiimide (EDC). Then those activated CQDs are treated with an ethanol solution of corresponding dyes to prepare CQD-Naph, FCQD, and CQD-Rh (Fig. 5.7).
The particles functionalized with fluorescein exhibit maximum fluorescence intensity. All of the above compounds, as well as CQDs, have been successfully implemented with minimal cytotoxicity into the erythrocyte-enriched proportion of
+
CA AEAPMS
24h 80ºC 100% CDS
film/monolith
240ºC 1 min
AEAPMS-CD
TEOS
CDs/silica particles Figure 5.6 Synthesis route of organosilane functionalized CD[11].
healthy human blood cells. In the future, CQD-Naph could also be used as a unique material for bioimaging and drug delivery due to the following benefits:
G It manifests a very negligible amount of cytotoxicity among all the compounds.
G The synthesis procedure is way simpler than that of the FCQD.
G After entering living cells, it emits the brightest green fluorescence[13].
5.2.3 Sol gel reaction
A novel ecofriendly molecularly imprinted polymer (MIP) can be produced by an effective one-pot sol gel polymerization at room temperature. There are different types of methods to prepare MIP such as multi-step swelling polymerization, sus- pension polymerization, and precipitation polymerization. Sol gel polymerization shows superiority over others as it manifests three advantages:
1. Thin films and/or bulk gels enable simple fabrication.
2. The ecosustainable reaction solvent (ethanol or water) differs from the general solvent (chloroform, acetonitrile, or toluene) used in the polymerization reactions mentioned above.
3. The reagents can be readily introduced within the porous and extensively cross-linked host structure without the problems of chemical or thermal degradation due to the mild proper polymerization conditions.
The surface of AEAPMS functionalized CDs can be modified with MIP matrix (CDs@MIP) by one-pot sol gel molecular imprinting method at room temperature.
Here dopamine (DA) is used as a template. Silica molecular imprinted nanospheres can be synthesized by hydrolysis and condensation reaction between CDs template molecules (DA), functional monomer (3-aminopropyl-triethoxysilane, APTES), and cross-linker (TEOS) in the presence of aqueous ammonia solution that acts as catalysis (Fig. 5.8). In the presence of light and air, as DA molecules are oxidized easily, the Figure 5.7 Synthesis of organic dyes (fluorescein, rhodamine B, andα-naphthylamine) functionalized CD[13].
reaction should perform in the dark inert (nitrogen) environment. These CDs@MIP are used to recognize DA in aqueous solutions and in human urine samples. Optical recognition of DA is significant in diagnoses, prohibition, and treatments of some neu- rological diseases; for example, Parkinson’s disease, Schizophrenia, and Hunting-ton’s disease[14].
5.2.4 Condensation reaction
The polyene polyamine (PEPA) functionalized CDs can be prepared by the hydro- thermal carbonization of PEPA and CA at 170C for 0.5 2 hours. This PEPA-CD is modified with the anticancer drug oxaplatin derivative oxa(IV)-COOH. The amino groups on the surface of the fluorescent CDs and the carbonyl groups of the oxa(IV)-COOH, undergoing the condensation reaction, result in the functionaliza- tion (Fig. 5.9). This functionalization induces the optical properties of CDs, so this
CDs:
1 min
+
CA AEAPMS
240ºC
AEAPMS-CD
Rebind Extract
CDs@MIP
DA:
Figure 5.8 Synthesis of CDs@MIP[14].
CD-oxa(IV) EDC, sulfo-NHS
CD COOH
oxa(IV)-COOH
Figure 5.9 Synthesis of CD-oxa(IV)[15].
CD-oxa(IV) is useful for fluorescent tracking. Besides that, due to the anticancer function of oxaplatin CD-oxa(IV), it has been an intense research focus in the medi- cal field[15].
5.2.4.1 Europium-adjusted carbon dots
The CDs treated for the functionalization by europium (Eu31) are synthesized by the condensation reaction of CA and 11-aminoundecanoic acid. The availability of car- boxylate groups on the surface of CDs makes them water-soluble. Europium is the most reactive element in the lanthanide series. Eu31 is one of the rarest earth ele- ments on earth and generally it occurs in the 13 oxidation state. Like other lantha- nides, Eu31 exhibits high coordination number i.e., C.N 6. It shows preference for the O-donor atoms and acts as bridging element between the neighboring carboxylate groups. So, Eu31 can easily coordinate with the carboxylate groups attached on the surface of the CDs, resulting in the aggregation of the CDs. Thus, the presence of Eu31 ion makes the fluorescence emission of CDs quenched through charge or energy-transfer process (Fig. 5.10). The Eu31 functionalized CDs (CDs-Eu31) flaunt high selectivity towards phosphate (Pi) by showing specific fluorescence “turn on”
response towards Pi[16].
5.2.5 Oxidation polymerization reaction
The synthesis of a novel carbon dot polyaniline (CD PANI) complex can be done in the presence of hydrochloric acid and CD through an efficient one-pot chemical- oxidative polymerization reaction. At first, the CDs are synthesized by the dehydra- tion reaction between D-(1)-glucose and concentrated sulfuric acid. Aniline monomer is treated under acidic conditions for the protonation of aniline molecule, i.e., for the emergence of anilinium ion. In the aqueous solution, the anilinium ions combine with COO- groups present on the surface of the CDs via charge charge interactions and Figure 5.10 Schematic representation of Pi detection based on the off-fluorescence probe of carbon dots adjusted by Eu31[16].
form an anilinium ion CD composite. The aniline monomers undergo polymeriza- tion reaction with the help of ammonium persulfate, which acts as the oxidant for 16 hours at room temperature, and CD PANI is generated[17](Fig. 5.11).