Carbon Dots as Optical Nanoprobes for Biosensors

12 Carbon Dots as Optical Nanoprobes for Biosensors Sing Muk Ng*,† *Fa cult y of E ngi nee rin g, C omputi ng, an d Scie nce, Swi nburne Uni versi ty of Te ch nol ogy Sara wak Cam pus, Kuchi ng, Mal ays ia † Res ear ch Cen tre fo r Sustain able Techn olog ies, Swi nburn e Uni versi ty of Te ch nol ogy Sa rawa k C ampu s, Kuchi ng, Mala ysi a

12.1 Introduction Optical sensors are a class of sensors that have attracted a great deal of attention for analytical applications, such as for monitoring the environment, or human health conditions. This is chiefly due to their straightforward working principle and simple hardware configuration. A typical optical sensor consists of a light source, a sensing platform, light waveguides, a light detector, and a data processor (Naseer et al., 2013). Most of these components are readily available from the market with high performance and comparatively low prices due to mass production in order to supply the telecommunications and electronics industries. The sensing mechanism works by detecting the change in how the light interacts with the sensing receptors before and after exposing to them to the analyte of interest. The basic change can be of light intensity alteration, or the shift in the wavelength of the light, due to the presence of the analyte of interest in the sample. Advancement efforts in the field of optical sensors often concentrate on making the device smaller, consume less power, use fewer components, offer more sensitivity and selective receptors, and be deployed in sophisticated and harsh sensing conditions. One of the major breakthroughs is the merging in technology of optical sensors with biotechnology that has led to the development of optical biosensors. Cutting edge technology has enabled the hybridization of biological species, such as enzymes, antibodies, and even living cells as part of the optical sensing receptors; and the bio-interaction with the respective biomolecules can be captured from the change in the optical signal (Borisov and Wolfbeis, 2008). Later, this is transformed into analytical information via modeling and data analysis of the raw recorded signals. It allows the performance of a more efficient medical diagnosis process in the health care sector, where important biomarkers associated with specific diseases can be detected; and subsequently, personalized treatment can be provided more accurately. Recently, the development of nanotechnology has led to a revolution in the design of biosensors into the nanometer scale dimension, which are more well-known as

Nanobiosensors for Biomolecular Targeting. https://doi.org/10.1016/B978-0-12-813900-4.00012-9 © 2019 Elsevier Inc. All rights reserved.

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nanoprobes. This means the sensor is made more powerful and effective because of its ability to enter a very small environment, and to perform diagnosis at the molecular and cellular levels. The convergence of all these technologies has enabled the detection of biological process to be done in vivo, in real time, within a single cell. This is because the monitoring of cellular function by detecting metabolites produced over a period of time, and predicting abnormalities of the biological system use the chemical profiles obtained from the nanoprobe. There is a report that demonstrates the possibility of using a nanoprobe for the monitoring of peroxynitrite in living cells (Wu et al., 2017). The principle is based on using nanomaterials that are surface modified with biological species as the sensing platform to interact with the biomolecules of interest. The nanomaterials will need to have specific property that is recordable, such as electrical signals, surface plasmonic waves, or an optical signal that will show the response when interactions are taking place between the biological species and the targeted biomolecules. One of the most popular choices is the nanomaterials that have a luminescence property due to the ease of detection in using a light detector. Correlation can be made between the degree of enhancement or quenching of the luminescence with the concentration of the analyte that is present within the microenvironment, such as in a particular cell under study. In view of the optical nanoprobes for biosensing applications, the recent accidental discovery of carbon dots (CDs) during the purification of carbon nanotubes have portrayed various characteristics that meet the criteria required to develop an optical nanoprobe (Xu et al., 2004). CDs are nanoparticles made of mainly carbon in the hybrid form of sp2 and sp3, which the structure varies from amorphous to crystalline, depending on the synthesis process. In some cases, the CDs also contain a small portion of heteroatoms such as oxygen, nitrogen, sulfur, or phosphorus. Uniquely, CDs have similar physical and optical properties to the well-known quantum dots (QDs), but portray a less toxic effect, while offering better biocompatibility with living cells. This is mainly due to the nontoxic nature of the carbon building blocks. As such, it becomes a major advantage of CDs as compared with the QDs, especially as optical nanoprobes in biological systems. It can eliminate the main concern of heavy metal ion leaching that is present in the majority of the QDs. Besides being safer for the biological environment, there are many options for producing CDs using greener approaches, and the selection of starting precursors can be from vast choices, ranging from pure compounds to complex matrices. In addition, research has slowly revealed that there are options to fine-tune the luminescence property, modifying the CDs with specific functionality and designing the luminescence to generate signals that are of analytical significance. By understanding the common properties and behaviors of CDs, planning can be done accordingly to engineer the CDs into a nanoprobe that can best suit the intended biosensing application. The consideration of the plan should include the best method for the synthesis of CDs, choice of starting precursor, modification of the surface of the CDs, integration into a working sensing system, establishment of the sensing mechanism, and the practical utilization aspect in real applications (Fig. 12.1).

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Synthesis method for CDs Choice of starting precursor for CDs

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FIG. 12.1 Some planning aspects to be considered when developing CDs as nanoprobes for biosensing applications.

12.2 CDs as Biosensor Receptors There are several merits and advantages to utilizing CDs as biosensor receptors in a nanoprobe. The consideration should be made based on the overall development processes; starting from synthesis process of the raw CDs up to the practical use in various applications. In this view, the major aspects include the practicality of each process, quality assurance, sustainability, economic implication, and suitability as a nanoprobe for real applications. There is a need to strike a balance between all these aspects to meet the final requirements of an intended application. This section will provide some reviews on the fundamental features of CDs and some of the advancements in technology that have enabled the fine tuning of some of these features in suiting the final intended application.

12.2.1 Fluorescence Properties and Characteristics One of the features that has attracted the most attention from researchers in the field of sensor development is the fact that CDs show fluorescence properties. This means there is a readily available property to be recorded as a potential sensing signal. The next effort is really to design a sensing mechanism that will affect the fluorescence property in an analytically significant manner. Mathematical models are used to establish the relationship between the recorded changes in the fluorescence property with the presence of the analytes of interest. This can be the relationship in terms of the type of analyte present to generate qualitative information, or the relationship with the concentration of the analyte in giving quantitation information. Although this is the main motivation, the actual origin of the fluorescence for CDs is still under debate, and continuous studies have strongly

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suggested that the fluorescence might be generated from different fluorescent pathways, instead of just one specific fluorescence transition. Despite the uncertainty, the general spectra of CDs are quite consistent with mostly symmetrical shape on the wavelength scale. The strokes’shift is often larger compared with organic dyes with considerably more broad emissions within the blue to green region. There is still very limited reporting available on the CDs emission at the longer wavelength region. In many cases, the emission peak is independent from the excitation wavelength at the short wavelength region; but it becomes excitation-dependent in the longer wavelength region. The dependency effect shifted the emission peak, but showed different emission efficiencies, that is, showing a remarkable decrease in the intensity as the excitation wavelength moved away from the optimum peak (Fig. 12.2). The cause of this is suspected to be due to the nonhomogenous origin that portrays a continuous distribution. This can be the size factor of the CDs, or the surface state of the CDs. There are several postulates that seem to explain the origin of fluorescence for CDs. Taking the analogy to the QDs counterpart, the quantum confinement effect shown by the QDs has been adopted to be one of the possible reasons to explain the origin of the fluorescence. The energy band gap becomes discrete once the size of a crystal hits the smallest dimension limit. This can be explained using the Bohr exciton radius theory, where the shrinking of a crystal dimension can lead to the quantum confinement effect. It causes the increase of the excitonic transition energy, causing the blue shift in the absorption and luminescence band gap energies. This quantum confinement effect can be observed clearly from a fractionalized sample of CDs into different size groups, where larger sizes CDs have shown a clear red shift in the emission to a longer wavelength

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Wavelength (nm) FIG. 12.2 The excitation-dependent emission of carbon dots. Reproduced with permission from Chin, S.-F., et al., 2017. Nitrogen doped carbon nanodots as fluorescent probes for selective detection and quantification of ferric(III) ions. Opt. Mater. 73 (Supplement C), 77–82.

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(Zhao et al., 2008). In another study, further oxidation processes on the CDs of different sizes has showed no obvious change in the wavelength of the emission peaks. This indicates that size is the major factor that has caused the shift in the wavelength of the peaks, and thus the origin should be from the quantum confinement effect (Dong et al., 2010). The correlation between sizes and the emission peaks can be theoretically established based on the evaluation of the HOMO-LUMO gap of a given size of CDs (Zhang et al., 1998). Fig. 12.3 shows a clear trend on the change of the emission color due to the different sizes of CDs and the change in band gap energy that is dependent on the size of the CDs. Although the quantum confinement effect is a possible explanation for the fluorescence origin of CDs, some researchers have proven that surface states could be the origin instead. Early studies have shown that surface passivation is crucial to enhance the

FIG. 12.3 The change in the color of the fluorescence emission of the CDs and its correlation to the size of the respective CDs. The photograph at (A) shows the CDs under normal room light and under the UV light where (B) shows the different emission for the CDs and (C) shows the correlation between energy band-gap with the particles size. The (D) shows the HOMO–LUMO gap dependence on the size of the CDs. Reproduced with permission from Li, H., et al., 2010. Water-soluble fluorescent carbon quantum dots and photocatalyst design. Angew. Chem. Int. Ed. 49 (26), 4430–4434.

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fluorescence property of CDs. Raw carbon nanoparticles such as those generated from laser ablation of a pure carbon source will only portray the fluorescence property after the reflux with mineral acid (Sun et al., 2006). The reason is mainly due to the oxidation process that has occurred on the surface of CDs. The surface is decorated with functional groups such as carboxylic and hydroxyl terminals that can serve as emission sites for the fluorescence to happen. A study has shown a clear trend of a red shift in the emission of CDs from batches of CDs with similar sizes, but of increasing degrees in surface oxidation (Fig. 12.4) (Ding et al., 2016). In a separate study, the oxidation state of the similar sized CDs has been tuned electrochemically, and the result revealed a red shift in the emission that coincided with the increased degree of oxidation on the surface of the CDs (Bao et al., 2011). Many studies have shown that the surface modification can shift the emission accordingly, and be used as a tuning parameter for the synthesis of the CDs (Sun et al., 2006; Tang et al., 2012). There is also another postulate that combines the two possibilities of the quantum confinement effect with the surface states as the origin of the fluorescence, where a broad band of the CDs is from the overlapping of two spectrals. The shorter wavelength at the blue region is associated with the core of the CDs, while the longer wavelength above that is most likely to be caused by the surface state. The ultrafast time-resolved fluorescence and carrier dynamics have been used to reveal that there are two overlapped bands in the broad band, which can be ascribed as the intrinsic and extrinsic fluorescence (Wen et al., 2013). The result has suggested that the intrinsic band at the higher energy is due to the sp2 nano domains of the core, while the extrinsic band at the lower energy end is originated from the surface states. A systematic investigation of the formation mechanism of CDs via the pyrolysis of organic precursors at different temperatures has showed clear evidence on the dual band emission property of CDs (Krysmann et al., 2012). A shift in the emission band is clearly observed during the formation of CDs, monitored starting from

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FIG. 12.4 The result showing that the band-gap of the surface traps is getting smaller with the increase in the degree of surface oxidation of the CDs. Reproduced with permission from Ding H., et al., 2016. Full-color light-emitting carbon dots with a surface-state-controlled luminescence mechanism. ACS Nano. 10 (1), 484–491.

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the precursor rich stage until the formation of the core of the nanoparticles as due to the rise in the temperature during the synthesis. The observation has been further supported by a carefully controlled temperature-dependent experiment (Yu et al., 2012). The fluorescence of CDs is measured at different temperatures, and the change in profile has suggested the dual band profile that attributed to the emission from the core and the surface states. In addition to the three possible fluorescence mechanisms discussed, there are also some other suggestions to explain the fluorescence origin of the CDs. Regardless, this fluorescence emission is a merit to the CDs that enabled its practical utilization in biomedical applications, including nanoprobes for medical diagnostics. The effort focuses on harvesting fluorescence signals for useful analytical information. Suitable mathematical models are used to fit the observed trend against the change in the analyte information, such as the type or the concentration. This can be as simple as the linear relationship between the change and the concentration of the analyte. In a more complex scenario, the commonly adopted Stern-Volmer relationship can be employed, which states that the normalized change of the fluorescence will have a linear relationship with the amount of the analyte present. The degree of quenching, governed by the Stern-Volmer constant, will explain the effectiveness of the interaction between the CDs with the analyte, which eventually causes the quenching. Usually this can serve as the information to identify the type of analyte that is present in the sample, as different analytes will interact differently with the CDs and cause different degrees of quenching. In some cases, the Stern-Volmer model might not be able to fit the trend in a linear manner, and this is when the modification of the model is required.

12.2.2 Preparation of CDs In the consideration of utilizing a novel material for any application, the physical and chemical characteristics must suit the requirement of the intended application. For example, CDs have the potential to be used as sensing receptors in nanoprobes, because they are physically small and emit fluorescence that can be used as a sensing signal. However, one should not neglect practicality in producing the novel material, and the cost associated with the synthesis process. This might not have direct connection with the usability in the application, but definitely will have an impact on the practical utilization and the potential to be commercialized in the market. In the case of CDs, there are various synthesis options to produce CDs, and this is a great advantage over other nanomaterials, such as QDs. In fact, the first reported CDs is also ironically isolated from the side product during the purification of single-walled carbon nanotubes (Xu et al., 2004), an indication of a simple synthesis process. Since then, a number of reports have appeared in the literature with different approaches to obtaining CDs, but with similar general properties. Some common examples include laser ablation of the carbon source, electrochemical oxidation of carbon electrodes, and carbonization of organic matters. It is also very interesting to see that different carbon rich/organic precursors, such as agriculture biomass

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(Li et al., 2016) and food leftovers (Park et al., 2014), have been used to produce the CDs. Many of these are unique in the sense that they are waste that is being converted into CDs, supporting the concept of sustainability and the principles of greener chemistry. They involve the use of less organic solvent, no toxic chemicals, and the side products are mostly nontoxic., Additionally, the cost associated with the whole process is reduced significantly. Despite the diverse synthesis routes and starting precursors that can be used to produce CDs, the production of CDs can be generally classified under just two main categories; the top-down and bottom-up approaches, likewise to the synthesis of other nanomaterials (Fig. 12.5). This means the CDs are either produced from the bulk format into nanoparticles using a top-down approach, or formed from smaller precursors into nanoparticles via the bottom-up approach. Usually, the starting materials for the top-down approach are from bulk carbon sources, such as graphene, that will be crushed into small carbon nanoparticles via harsh processes. Due to this, the instrumentation and setup for this approach are more advance and costly. Setup can include using high power lasers or electrochemical oxidation. Most of the isolated carbon nanoparticles, after the harsh process, will need to undergo surface passivation processes such as refluxing in acid in order to introduce the fluorescence to the CDs. Although the whole process seems to be more expensive and lengthy, the CDs produced have more control of the purity due to the known grade of the starting materials, and the fact that formation involves a controllable physical process, rather than a chemical reaction. The probability of having CDs with varied chemical content and structures will be low. This is an advantage to suit some later

FIG. 12.5 The common synthesis process of nanoparticles. Reproduced with permission from Ng, S.M., Koneswaran, M., Narayanaswamy, R., 2016. A review on fluorescent inorganic nanoparticles for optical sensing applications. RSC Adv. 6 (26), 21624–21661.

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applications, such as the use of nanoprobes for biosensing purposes, which requires high consistency in the standard of the deployed CDs. Furthermore, the engineering aspect of the CDs, such as surface modification or grafting of biological species on the CDs, will be easier as well because the chemical property of the CDs is highly homogenous. In contrast, the formation of CDs via a bottom-up approach involves a chemical reaction that combines small molecular precursors into the carbon rich nanoparticle. In view of this, the selection of starting precursor is very diverse, and it appears that any organic materials with high content in carbon can be used. When activated via thermal or strong acid, small molecules can undergo hydrolysis, and the high active intermediate species can combine to form larger clusters that are rich in carbon. For example, sugar molecules will be hydrolyzed using strong mineral acid, and eventually the intermediates can combine into CDs that show strong yellow emission. Larger structure compounds, such as polymers, can also be converted into CDs via the bottom-up approach. The mechanism involves an additional step of the depolymerization process first before proceeding with the general formation route. However, this usually requires higher activation energy that can be achieved by using stronger acids or higher carbonization temperatures. Because it involves a chemical reaction, the chemical composition can vary according to the synthesis conditions. Thus, optimization of the carbonization process is crucial, as the products formed will be condition-dependent. For example, insufficient energy provided will not convert the starting precursor into CDs; while under harsh conditions it can completely decompose the starting precursors into basic products, such as water and carbon dioxide (Tan et al., 2014). In most cases, an additional surface passivation step is not required to introduce the fluorescence property, as the oxidation process can occur simultaneously during the formation of the CDs. As a whole, this approach is comparatively cheaper and involves only simple instrumentation, which makes it attractive to produce CDs for nanoprobes’ application. The homogeneity of the CDs can be improved by using a pure compound as the starting precursor, as compared with complex matrices such as biomass wastes, while the carbonization process needs to be optimized and controlled carefully to ensure the reaction will proceed in a similar fashion to produce the CDs. In general, some of the production methods offer the possibility of producing CDs in larger scale. This is quite important, especially in meeting the commercialization requirement, where the amount produced should eventually meet the demands required. Also, large-scale production offers the advantage of reducing the overall cost associated in getting the CDs. There have been reports in the literature that mentioned the production of CDs in the range of grams, which can be considered a large amount because the CDs are nanoparticles. In the application as ink, it is possible to produce in the amount of 10 g aqueous solution of CDs to be used as injection ink for printing purposes (Wang et al., 2012). More recently, this has been demonstrated on the production of about 120 g of CDs from 100 kg of food waste using an ultrasound irradiation method at room temperature (Park et al., 2014). The CDs showed no significant difference in the properties of the CDs compared with what has been reported in the literature. Once the production can be scaled up, there will also be concerns about the reproducibility of the CDs, as reliability of

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the nanoprobes developed will rely on the quality of the CDs. A detailed study has been performed on this aspect by using commercial bee pollens as a starting precursor, and investigating the impact of different scales for synthesis (Zhang et al., 2015a). The result has shown to have no significant change in the quality and properties of the CDs produced from the different scaled-up batches. In addition, repeated synthesis using similar ingredients and conditions also produced a similar yield of CDs, which clearly demonstrates the possibility of reproducing CDs of similar quality in selected applications.

12.2.3 Isolation and Fragmentation Isolation of CDs from a crude mother sample, and later their separation into smaller sample fractions are also important steps to producing clean, homogeneous, and high purity CDs that can be used as nanoprobes for biosensing applications. This is to remove most of the impurities and non-CD products that formed, and were involved in the synthesis process. In most cases, the isolation is made directly via centrifugation to remove the larger particles, and dialysis to exclude lower molecular weighted species. CDs can often be suspended in the aqueous media to form colloidal solutions due to their small sizes, while larger particles will be precipitated out. In some cases, density gradient centrifugation can be performed to separate the colloidal CDs into different regions of the centrifuge tube under a mild spinning rate. The density gradient within the tube is introduced by filling it with a solution such as sugar, which creates a decreasing density from the bottom to the top of the tube. By doing this, CDs with the intended property can be extracted out easily by just collecting the solvent within the region (Fig. 12.6) (Oza et al., 2015). Similarly, two

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FIG. 12.6 The separation of CDs in a centrifuge tube that have different concentration gradients of sucrose solution. Reproduced with permission from Oza, G., et al., 2015. A green route towards highly photoluminescent and cytocompatible carbon dot synthesis and its separation using sucrose density gradient centrifugation. J. Fluoresc. 25 (1), 9–14.

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major species of CDs have been successfully separated using this technique from the crude sample obtained from the direct carbonization of citrus juice under ultrasonic conditions (Pandey et al., 2013). The separation can also be achieved via direct filtration using millipore membranes with pore sizes in the nanometer range. There is no issue, because the solution used is aqueous, and not organic solvents that could possibly dissolve the membrane. In terms of highly soluble impurities such a salt, the solvent extraction method can be used for the isolation. A study has shown that acetone can be used to isolate CDs from the aqueous solution that contains a high concentration of salt (Chang et al., 2017). Two obvious layers have formed where the CDs will partition on the acetone layer, leaving the salt crystalline in the water layer (Fig. 12.7). Acetone can then be removed easily from the CDs via evaporation. Salts are often present in the CDs sample, especially for those CDs that were formed via carbonization using strong mineral acids and subsequently neutralized using a base solution, which will produce large amounts of salt as a side product. In promoting the homogeneity of the CDs, one can opt to perform fragmentation of the crude sample via separation techniques into smaller potions. This separation can group the CDs with similar properties into smaller portions, and thus often show different emission properties for each of the isolated portions. This matches with the suggestion that the origin of the fluorescence is from one of these physical properties, that is, size or surface states. As such, the efficiency for the end sensing application will be enhanced, as CDs with specific emissions can be selected with less interference from the other CDs that emit under similar excitation wavelengths. In addition, the contrast from the biological environment that often shows auto fluorescence can be optimized by selecting CDs that emit

FIG. 12.7 Formation of 2 layers due to the addition of acetone into a CD’s solution containing a high salt concentration. The CDs will partition on the acetone layer, while the salt will remain in the aqueous media. Reproduced with permission from Chang, M.M.F., Ginjom, I.R., Ng, S.M., 2017. Single-shot ‘turn-off’ optical probe for rapid detection of paraoxon-ethyl pesticide on vegetable utilising fluorescence carbon dots. Sensors Actuators B Chem. 242, 1050–1056.

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wavelengths out of the auto-fluorescing range. For example, CDs with longer emission wavelengths, such as in the green to red regions, are preferable because the biological environment often strongly reflects in the blue region. Also, better contrast enhances sensitivity, and better homogeneity after fragmentation will also ease the surface modification process of the CDs, which is a crucial step to introducing selectivity into the sensing nanoprobe. Organic synthesis can be performed more effectively with less interference and deactivation as compared with the use of crude samples that contain various side products. Few separation techniques are available for the fragmentation of a crude CD sample. A chromatographic technique using column separation is one of the common methods used. This is done by pouring the crude sample into a column filled with a stationary phase, such as porous silica particles. Partitioning will occur, and different fragments can be collected after different eluent times. Those that interacted strongly with the stationary phase will stay in the column longer, and elute later compared with those that have less interaction. A study using high performance liquid chromatography (HPLC) has demonstrated success in separating crude CDs into smaller fragments with more homogenous properties using a C18 column (Lu et al., 2014). The surface chemistry of the CDs has found to be a crucial element in the separation phase in this case, and the use of a suitable gradient mode of different solvents has increased the separation efficiency. Additionally, the size factor will also influence the time required to travel down the column. Smaller CDs will tend to take a longer time because they have a longer pathway to travel while moving through all the micro-channels available from the stationary phase, while this is not the case for the larger nanoparticles. For example, low-pressure gel filtration chromatography using a column packed with Sephadex LH-20 can be used to separate the size of the CDs, where the larger-sized CDs clearly show the atomic force microscopy (AFM) is eluted first (Arcudi et al., 2016). In addition to column separation, gel electrophoresis separation is another method to separate the crude CDs. The working principle is based on the movement of CDs across a porous gel medium, due to the push of an electrical field through the gel. The CDs travel through the pores in the gel with a rate that is proportional to the charge and size factors. Smaller CDs with a higher charge will travel faster, and cover a greater distance, as compared with those with larger sizes and with a lower charge. Because the electrical field is involved to move the CDs, it is crucial to have the surface modified with functional groups that carry charges to interact with the field. In fact, the first reported isolation of CDs was purified using 1% agarose gel slab, where the crude suspension separated into three bands with different properties (Xu et al., 2004). Later, the CDs prepared from candle soot also successfully separated using polyacrylamide gel electrophoresis (PAGE) into different fragments that emitted multicolour fluorescence (Liu et al., 2007).The candle soot was the first to be refluxed to introduce various chemical functional groups to enable separation. Similarly, a sharp, well-separated blue fluorescent band is observed from the separation work performed on the crude sample of CDs prepared from the caramelization of polyethylene glycol (PEG) ( Jaiswal et al., 2012).

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In cases where the sample amount is small, capillary electrophoresis is usually preferable for the separation. The working principle is similar to the gel electrophoresis, while only different in the setup. Instead of using a gel slab, a fine silica capillary is used where both ends will be immersed in two different vials that have been applied with high potential. CDs can be separated based on their size and charge on the surface across the capillary. Other than just separation, the technique can also be employed to perform comparison in terms of the effectiveness of a surface modification step. For example, the success in tagging of antibodies can be proved using this technique as compared with bare CDs (Wu and Remcho, 2016). The CDs tagged with antibodies eventually show higher mobility due to the enhanced charge intensity as carried by the antibodies. Thus, it can directly separate out those CDs that have been successfully tagged from those bare CDs.

12.2.4 Physiochemical Properties and Their Effect Within the Environment CDs are often in quasispherical shape, and most commonly have diameter sizes of 1.0–10.0 nm. Some CDs are larger, but still exhibit similar general properties (Chin et al., 2012). The morphology of the CD is often reported to be quite smooth and chemically inert. The nanometer size range is very suitable for penetration into a single cell wall, and can act as a nanoprobe when engineered with sensing receptors on the surface. Nanoparticles with an overall controlled size below 5.5 nm are usually able to be excreted by renal clearance (Choi et al., 2009). Unlike organic dyes, CDs are reported to be very stable, and will not degrade easily within the biological environment. The tendency of being metabolized by the cell is also low, due to the physical strength compared with the molecular strength of an organic dye. The removal from the body after the sensing process is highly possible, as there is a study showing that CDs with sizes even larger than the recommended dimension are able to be efficiently excreted via urine (Chen et al., 2014). In this case, they will not accumulate within the body after the sensing application. Besides being physically stable, CDs are also reported to be stable toward photobleaching, and can remain the same in water for more than half a year without a fluorescence decrease (Mao et al., 2010). This is important, as the signal will reflect on the presence of the analyte and its analytical information. Any fluctuation in the intensity should be caused by the presence of the analyte, and not be a result of the photobleaching activity. The luminescence property of CDs, similar to fluorophores, is subjected to change under different microenvironments. This could include a change in salinity or the pH of the environment. For example, most CDs change in intensity when introduced to different pH conditions (Wang et al., 2016), while some also shift the emission peak to different wavelengths (Kong et al., 2014). The possibility of degradation due to pH change is low, because most of the cases show repeatable trends under changing pH conditions. One of the possible causes of the change could be the protonation and deprotonation of the chemical terminals at the surface of the CDs. Once this occurs, the surface charges will be modified, possibly leading to aggregation. In an aggregated form, the CDs can

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self-quench the fluorescence intensity. Such an effect is being observed in CDs produced from the carbonization of starch nanoparticles where lower pH has caused the reduction in the fluorescence intensity that has been postulated due to the formation of aggregates by the enhanced hydrogen bond at lower pH (Mohd Yazid et al., 2013). Other reasons could be due to the disturbance of the origin of the fluorescence that is connected with the terminals that undergo protonation and deprotonation. The surface of CDs that undergo the oxidation process is often decorated with carboxylic and hydroxyl groups, and these groups contain a proton that can equilibrate with the surrounding condition. Once this happens, it can directly influence the fluorescence property if some of these moieties are directly connected to the origin of the fluorescence. A detailed study of CDs produced from the hydrothermal carbonization of lemon juice revealed that the equilibrium of phenol and the phenolate group is actually responsible for the changes in the fluorescence property under different pH conditions (Dutta Choudhury et al., 2017). Other groups, such as the carboxylic group, showed deprotonation as well, but does not play a direct role in affecting the fluorescence profile. The reason is simply that the phenolic group is directly coupled to the emissive moiety, and can influence the nature of the energy levels and the electronic transitions in the CDs. This is a specific example, and other batches of CDs might have other terminals that are linked to the origin of the fluorescence. Although the environment can influence the fluorescence property of the CDs that are taken as the sensing signal, it is not necessarily interference that will reduce the reliability of the nanoprobes constructed from CDs. Instead, an understanding of the mechanism that triggered the change could be used as another function of the nanoprobe in providing information related to the microenvironment. An accurate modeling on the pH effect can add another sensing dimension to the nanoprobes. This pHdependent property by itself is a good nanoprobe for in vivo pH sensing for cell, as there is no such pH electrode that can reach such a small scale. For example, CDs can be utilized for intracellular pH sensing by monitoring the fluorescence emission change of the peak that is known to have been caused by the pH factor (Chandra and Singh, 2017). This signal can also normalize against other emissions from the same CDs that are not influenced by the change of pH. This is known as the ratiometric method, and is powerful in eliminating other interference, such as change in temperature or salinity concentration within the cellular microenvironment. Additionally, the effect of pH can be used to tune better sensitivity of the nanoprobes toward an intended analyte. This is to precondition the binding site with higher affinity to the targeted analytes. In cases where the nanoprobe is targeting metal ions, the increase in pH can create deprotonation of the functional groups at the surface of the CDs to form a negatively charged layer. This eventually can increase the attraction with the positively charged metal ions. This phenomenon is observed in one of the studies that deal with the use of CDs as potential nanoprobes for mercury (II) ion sensing, where no effective quenching is observed at low pH, but concentration dependent quenching is achieved at higher pH conditions (Dutta Choudhury et al., 2017).

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12.2.5 Biocompatibility and Toxicology Regarding nanoprobes intended for biosensing applications, the biocompatibility and toxicity aspects are of great concern. It is not acceptable to utilize a nanoprobe in a biology host if it is harmful and causes damage to the biological system, even if the nanoprobe is showing superior analytical performance. By nature, CDs are generally considered to be safe, because the building block mainly consists of carbon elements. In addition, the starting precursors, reagents, and carbonization method can be selected from options that are green and nontoxic. This helps increase reliability, especially if the CDs are extracted from foods such as those chosen from direct isolation, such as instant coffee ( Jiang et al., 2014) or the carbonization of edible starch (Chin et al., 2012; Basu et al., 2015; Yan et al., 2015). Although the general perspective sounds safe, detailed studies on the toxicity and biocompatibility of CDs for biomedical applications are still required. The MTTassay is one of those commonly used methods for checking the cell viability in the presence of the CDs. The MTT assay is a sensitive and reliable indicator of the cellular metabolic activity as reflected by the reduction of MTT, a yellow water-soluble tetrazolium dye injected into purple formazan crystals by the mitochondrial dehydrogenases from the cells. The formation of formazan crystal can be quantitatively monitored using UV-vis absorption spectroscopy. In a study using HeLa cells, the addition of CDs up to the concentration of 300 μg mL 1 has shown no significant effect on the cells’ viability after an incubation period of 24 h (Wang et al., 2017a). Other studies have also demonstrated no significant effect of CDs on the cell viability for 93T human kidney cells (Zhao et al., 2008), HepG2 human liver hepatocellular cells (Ray et al., 2009), and human colorectal adenocarcinoma HT-29 cells (Yang et al., 2009a). All of these, collectively, are good indications on the low toxicity nature of CDs. Besides MTT, a trypan blue exclusion test can also be adopted to study cell viability for CDs (Bhaisare et al., 2015). It is based on the principle that the membrane of a live cell is intact and could exclude trypan dye from entering the cell, but not for dead cells. This can be visually observed under the optical microscope where blue stained cells are taken as dead cells, and unstained will be taken as live cells, as shown in Fig. 12.8. Usually, the CDs will be incubated with the cells over a fixed period of time, and with varying concentrations before the cells are harvested, mixed with trypan dye, and viewed under the microscope. Cell viability is not the ultimate indicator, but rather a quick test on the toxicity of the CDs. For real biomedical application, the in vivo study using a live host is also crucial, as the CDs might not be directly affecting the metabolic activity in cells, but could still pose concern over the risk of disturbing the biological system at the functional level. Animal studies can be a good alternative for more in-depth exploration of the toxicity effect of CDs on a host. A mortality study is one of the more straightforward studies, where a host can be introduced with different concentrations of CDs, and the survival rate of the host over a period is evaluated. Such a study has been performed on the embryo of a zebrafish using different concentrations of CDs produced from human hair and pigskin as precursors. The different studies performed, such as direct soaking, injection, and hypotoxicity

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FIG. 12.8 Examples of photos taken under the microscope view for the trypan assay where (A), (B), and (C) show the effect of increasing the concentration of CDs from 1, 3, and 5 mg mL 1. The blue stained cells are taken as dead cells, while the clear ones are taken as live cells. Reproduced with permission from Bhaisare, M.L., et al., 2015. Synthesis of fluorescent carbon dots via microwave carbonization of citric acid in presence of tetraoctylammonium ion, and their application to cellular bioimaging. Microchim. Acta 182 (13), 2173–2181.

tests, showed no significant impact from the CDs toward the embryo, as compared with the control hosts that do not include the addition of CDs (Zhang et al., 2016). The use of mice is another model reported for the testing of CDs for the toxicity effect. Various assays such as acute toxicity, subacute toxicity, and genotoxicity experiments can be performed on the mice for systematic evaluation of the CD’s toxicity (Wang et al., 2013). In a particular study, the intake of CDs by mice has shown to have no significant impact or clinical symptoms toward the activity and behavior over a period of 4 weeks (Yang et al., 2009b). Further biochemistry studies are being carried out on the mice, and have found no significant effects on heptic injuries and kidney functions. The indicators of alanine amino transferase, aspartate amino transferase, uric acid, blood urea nitrogen, and creatinine are reported to be at similar levels for the mice exposed to different dosages of CDs and the control group. The success of the nanoprobe application also depends strongly on the understanding of the bio-distribution, retention, and clearance. In the case of CDs, studies show that it can be efficiently and rapidly excreted from the body after the introduction to the host (Huang et al., 2013). This will lower the risk of bioaccumulation that later might trigger

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other biological malfunctions. Other studies show that most of the CDs can be cleared by 6 days postinjection without accumulation in vital organs and tissues (Parvin and Mandal, 2016).

12.3 Design and Modification of CDs for Biosensors In a nanoprobe system, the CDs function as a platform that interacts with the analyte of interest, and subsequently shows the change in the fluorescence profile that can reflect on the presence of a specific species of analyte and/or the concentration of the analyte. In this case, it is crucial that the CDs’ platform has the functional terminals for the interaction to occur. More importantly, the interaction needs to be at least selective, if not too specific, by allowing only the intended analyte group to approach the terminal. Otherwise, all the matrices within the sample will contribute to the signal change via random collision, and this can cause a serious interference effect. The near proximity in distance of the specific analyte with CDs through the functional terminals will have a high probability of inducing a disturbance into the initial fluorescence of the CDs. Thus, the engineering of the CDs with the functional terminals is an important aspect that will ensure the success of the CDs as nanoprobes. The functional terminals can be of simple molecules, longer polymers, or complex biological agents, such as enzymes and antibodies. There are several approaches to introduce these functional terminals that will be briefly discussed in this section.

12.3.1 Simple Passivation (Thermal/Acid) Surface passivation of CDs via simple acid oxidation is one of the earliest approaches to introducing chemical functionality to the surface of the CDs. The initial motivation is to introduce fluorescence to the CDs by forming the energy gaps via surface states. The normal practice is by refluxing the carbon nanoparticles with nitric acid over a long period. The characterization found that these CDs are rich in chemical groups, especially those with oxygen, such as the carboxylic and hydroxyl functional groups (Li et al., 2016). Although this is not intentionally performed to introduce the chemical functionality of the surface, the presence of these groups has promoted the binding affinity toward some other species, which is potentially of interest to be monitored. This means the surface passivated CDs turned into potential nanoprobe-sensing applications. There are reports on the use of surface passivated CDs for the sensitive detection of tin(II) ions, which showed good quenching when the CDs were added with the ions (Mohd Yazid et al., 2013). The reflux with nitric acid introduced functional groups rich in oxygen that have high affinity toward positively charged metal ions. The formation of a complex between the CDs and the metal ions is clearly reflected in the UV-vis spectrometry study, which has been suggested to have caused a disturbance to the initial fluorescence. In addition to performing simple surface passivation by refluxing with acid, direct thermal and hydrothermal carbonization methods used to produce the CDs are also reported

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to introduce chemical functionality to the surface of CDs. For example, CDs produced from the thermal carbonization of sago wastes in a furnace have shown to complement chemical groups such as the carbonyl groups (Tan et al., 2014). There is no need to perform additional step to introduce the chemical groups on the surface; instead, the synthesis process in the presence of oxygen will undergo oxidation at the same time during the carbonization process. Similarly, these chemical functional groups have indirectly introduced the CDs with binding affinity to some potential analytes of metal ions (Tan et al., 2014). The CDs produced by a hydrothermal carbonization process have shown to be able to detect various metal ions without any further chemical modification on the surface of the CDs (Wang et al., 2017b). The chemical analysis from the Fourier Transform Infrared Spectroscopy (FTIR) is often used to confirm the presence of these chemical functional groups that are introduced indirectly during the synthesis process. The CDs produced from pork meat using hydrothermal methods have found to contain COOH, OH, and NH2 groups on the surface of CDs, where they will be under negative charge conditions under a weakly alkaline solution (Zhao et al., 2018). Under these conditions, it can interact with uric acid via an electrostatic interaction on the secondary amine group in the molecule that is positive charged; causing quenching that can be evaluated for analytical information.

12.3.2 Specific Surface Modification (Molecules/Biomolecule) Instead of random formation of different chemical groups on the CDs, advanced effort has been concentrated on grafting specific receptors on the CDs. This has really been the key revolution for CDs to be used as nanoprobes in biosensing applications. Various high specific probes can be developed by applying biotechnology with nanotechnology. Specific molecules of different sizes, structures, and functionality are tagged on the CD’s surface to create binding affinity. CDs decorated with tyrosinase can be a nanoprobe that is efficient and sensitive in detecting levodopa (Li et al., 2015). Simple boric acid can also be functionalized onto the CDs, and show great sensitivity in terms of quenching toward the presence of glucose (Shen and Xia, 2014). The mechanism is via the formation of glucose-boronic acid complexes that will aggregate the CDs and eventually destroy the fluorescence due to a self-quenching event. More inert layers, such as the organosilane layer, have also reported to be successfully capped on the CDs, and have shown great stability and low toxicity to the cell lines tested (Wang et al., 2011). Larger biomolecules of specific structures and shapes can also be tagged on the surface of the CDs. The advantage is that these molecules have a cavity that fits only specific analytes, following the lock and key theory. CDs that are tagged with aptamers, and singlestranded RNA or DNA oligonucleotides have shown to promote binding affinity and specificity. For example, the tagging of AS1411, a nucleolin aptamer on the surface of CDs, can be used as a nanoprobe for the detection of several types of cancer cells (Motaghi et al., 2017). The aptamer will be released from the CDs, specifically, only when cancer cells overexpressing nucleolin are present, leading to the increase of fluorescence of the CDs

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that is initially quenched by the aptamer. A similar nanoprobe can be developed for the detection of dopamine, simply by tagging the surface of the CDs with dopamine aptamer (Zhu et al., 2016). On the less specific side, the use of macromolecules as a pocket on the surface can also create a unique cavity on the CDs in which to fit small hydrophobic molecules. One example is the use of cyclodextrin molecules as a capping agent, which has been demonstrated to be able to detect small biomolecules, such as hemin (Baruah et al., 2013). The antibody is another candidate that is being used to tag on CDs for the development of nanoprobes. Studies have demonstrated that the CDs tagged with goat antihuman IgG antibodies can detect the presence of human IgG (Zhu et al., 2014). CDs that are labeled with antihuman α-fetoprotein have also successfully demonstrated detection of human α-fetoprotein within the range of 0–350 ng mL 1 (Wu et al., 2015). Deoxyribonucleic acid (DNA) can also be used as the receptor that is tagged on the surface of the CDs. Interaction of the tagged DNA with the conjugate can cause a change in the fluorescence signal of the CDs, and be used as an analytical signal (Takalkar et al., 2017).

12.3.3 Doping of Heteroatoms In the application of biosensing, the nanoprobes developed from the CDs are ideally to have excitation and emission at the longer wavelength regions. The reasons are simply that UV excitation is harmful to the biological system, while emission in the far visible sector will give better contrast with the biological system. There have been different attempts to red shift the fluorescence profile. One of the common ones is by doping of the heteroatom, such as through nitrogen, oxygen, phosphorus, and sulfur to the CDs. Nitrogen is one of the more famous candidates because it has a comparable atomic size and five valence electrons to form bonding with carbon atoms. The fluorescence peak has demonstrated that it will shift to a longer wavelength under the same excitation when the nitrogen content increases in the CDs (Zhang et al., 2012). The rational suggested on the observation is on the formation of new fluorescence origins by the nitrogen doped. In some studies, nitrogen is also observed to increase the emission efficiency instead of shifting the emission. The mechanism is suggested to be the introduction of an upward shift of the Fermi level and electrons in the conduction band (Xu et al., 2013). In addition, nitrogen as an impurity can cause disorders to the carbon hexagonal rings and create emission energy traps that contribute to the fluorescence mechanism. Both peak redshifting and increase of intensity due to the nitrogen dopant are merits that can enhance the performance of the nanoprobe made from these nitrogen-doped CDs toward some specific analyte of interest (Zhang et al., 2014). Sulfur can also be doped into the CDs, and has shown to promote the quantum yield of CDs that can be useful in enhancing the sensitivity of the nanoprobe made from the CDs (Xu et al., 2015). The sulfur, in this case, can act as a catalyst in a redox reaction during the synthesis, and will directly introduce more passivated surface detects onto the CDs that will enhance photoluminescence efficiency. The sulfur element can be introduced via

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many channels, and one of the simple methods is by adding sulfuric acid to the precursor during the synthesis process (Hu et al., 2014). The use of sulphate salt is also possible where a particular study has added sodium sulphate into the starting precursor during the synthesis of the CDs to form sulfur-doped CDs (Xu et al., 2015). These doped CDs showed high quantum yield and can be utilized for the detection of iron (III) ions via a fluorescence quenching method. Oxygen is another famous element often introduced to improve the fluorescence property of the CDs. This matches the suggestion that one of the possible origins for the fluorescence is from the surface defect caused by the functional group that is rich with oxygen. Oxygen atoms can be present in various chemical functional groups, such as the carbonyl and hydroxyl groups. In an electrochemiluminescence study on CDs, a secondary emission at the longer wavelength region has been suggested to be caused by the defect energy level at the periphery introduced by oxygen-containing groups (Qin et al., 2017). The result of the study showed low emission after the reduction of CDs, further affirming the role of oxygen in the generating the fluorescence property of CDs. The presence of a rich oxygen condition during the synthesis has promoted the aromatization of the starting precursors that lead to enriching the sp2 hybrid, which is postulated to be the florescence origin of the CDs. In a separate study, the doping of oxygen is also found to shorten the reaction time for the formation of CDs and enhance the quantum yield (Zhao et al., 2017a). Phosphorus is also a common element doped onto CDs to fine-tune the optical property. Initial observation has shown that the presence of phosphorous can lead to the red shift of the emission profile. This effect can be observed when the carbonization of the starting materials is performed using two different acids, where one contains the phosphorus element, but the other does not. A well-controlled study has demonstrated clearly a significant red shift in the emission of the isolated CDs when the use of sulfuric acid to carbonize the sucrose has been changed to phosphoric acid (Loi et al., 2017). The atomic size of the phosphorus is larger than the carbon, but studies have shown that it can form substitutional defects in diamond sp3 thin films (Sternschulte et al., 1999). Such impurities can lead to the site behaving as an n-type donor, and eventually can modify the electronic and optical properties of the material. Such phenomena is also expected in the phosphorus doped CDs. Although the exact change in the fluorescence mechanism due to the phosphorus doping still remains unclear, there have been some suggestions made to explain the governing phenomena based on the theoretical and experimental methods. For example, it is postulated that the addition of phosphorus into CDs can cause more isolated sp2 carbon clusters, and this could increase the efficiency that leads to the increase of a band gap, and eventually creates a rise in the fluorescence (Zhou et al., 2014). In some cases, co-doping of more dopants is also possible, and has shown very promising results in enhancing the optical property of the CDs. For example, CDs doped with just nitrogen are recorded to have the quantum yield of around 5%, and the efficient can be increased 10 times by co-doping with sulfur (Ding et al., 2014). The nitrogen bound to the carbon is identified to be the main origin causing the fluorescence, and the addition of

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sulfur can have a synergistic effect that promotes efficiency of the emission. This is in terms of exhibiting higher catalytic activity from the combination of two dopants toward the oxygen reduction reaction, compared with those CDs doped with just a single element. A theoretical simulation has suggested the presence of both the dopants can lead to the redistribution of spin and charge densities that has promoted the quantum yield (Liang et al., 2012).

12.3.4 Optical Tuning During Synthesis Besides doping, the control of the rate and condition during carbonization have also shown to affect the final emission wavelength. A bottom-up approach via harsh carbonization methods using a strong acid, such as sulfuric acid, leads to rapid nucleation during the formation of the CDs. This leads to smaller nanoparticle sizes that tend to show a blue shift in the emission wavelength (Loi et al., 2017). However, when milder conditions are applied, using less harsh acids such as phosphoric acid, the condition flavors particle growth compared with nucleation. This tends to red shift the wavelength of the emission peak. Other than controlling the carbonization rate using types of acid, the control of temperature during the carbonization has shown to have a similar observation in the ability to shift the peak of the emission. When carbonization is carried out at a lower temperature using a strong acid, the synthesis rate will be reduced, and can promote the emission into longer a wavelength (Ng et al., 2017). Similarly, performing acid carbonization at a higher temperature will promote the formation of CDs in the shorter wavelength region. Interestingly, when the initial carbonization process is performed at a lower temperature, and then eventually raising the temperature to a higher degree for the same sample, the CDs obtained still show a strong tendency to emit at a longer wavelength. This suggests that the initial stage of the carbonization is the determination factor because at this stage; the precursors start to form the CDs via nucleation and particle growth. A lower temperature at the initial state will not favor nucleation, but will favor particle growth. A later increase of temperature will only increase the rate of the particle growth that is the main reaction occurring at the later stage of the carbonization process.

12.3.5 Matrix Blended (Immobilization/MIPs) CDs also can be coated with a thin layer of matrix to introduce the binding affinity toward some intended analytes of interest. The matrix is often from long polymeric materials that can form a thin coating on the CDs. This often can enhance the fluorescence intensity due to the shielding of potential quenchers from the CDs, reducing the possible nonfluorescence pathways of the CDs. In some cases, the coating of polymer can cut the interaction point on the CDs, making it more inert toward the surroundings (Zhang et al., 2015b). Conversely, the backbone of the polymer may contain specific chemical functional groups that can have interaction terminals. One common, and the earliest, coating for CDs is the use of PEG. PEG has an oxygen-carbon backbone, where the oxygen contains the electron lone pairs that can interact with the surrounding species. For example, CDs passivated

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with PEG have provided the suitable surface chemistry to interact on biological sites, making it suitable to be adopted for bio-labeling applications (Sachdev et al., 2013). Other studies have shown the use of a PEG surface to achieve better interaction between the analytes with the CDs in achieving a better, more sensitivite probe. In a particular work, PEG is used to enhance the interaction of CDs with iron (III) ions via the hydroxyl group, which has managed to improve the detection limit from the bare CDs of 7.4 μM to the PEG capped CDs of 2.56 nM. Other polymers are also used to form the capping layer on CDs with similar motivation, and some reported examples include the use of hyberbranched polymer (Wu et al., 2013; Yin et al., 2013) and polyethyleneimine (Wang et al., 2015a). Besides just performing simple coating techniques using polymer, the use of molecularly imprinted polymers (MIPs) is one of the latest technologies that enable the introduction of specific binding sites on the surface of the CDs. The concept is simple, where the formation of the polymer coating from its respective monomers and reagents is done in the presence of the analyte of interest. Before the polymerization process takes place, the selected monomers and the cross-linkers will interact with the analyte of interest and form complexes via various modes of interaction, such as hydrogen bonding, van der Waals forces, coordination bonding, covalent bonding, and so forth. Once the polymerization is completed, the complexes’ structure and conformation will be frozen within the polymeric matrices. Subsequent removal of the analyte species will leave the polymer with cavities of specific shapes and sizes to the analyte species. These cavities have high binding affinity toward the analyte species because they have matching shapes and sizes. Thus, as the layer coating the CDs, the MIPs will have the role of attracting the analyte toward the core of the CDs, and eventually cause the change in the fluorescence profile. This will enable the selectivity to be tuned specifically for a targeted analyte, or even biomolecules for biosensing applications. A report has demonstrated that the CDs can be utilized to specifically detect sterigmatocystin toxins via the MIPs’ coating (Xu et al., 2016). The MIPs are formed using a dummy template of 8-hydroxyquinoline using a nonhydrolytic sol-gel process, which upon removal, showed good selectivity toward the sterigmatocystin. Such selectivity will not be achieved using just the sol-gel without the imprinting process. Similarly, the use of MIPs coating on the CDs has managed to introduced the sensing selectivity toward various analytes, such as the antiinflammatory drug celecoxib (Amjadi and Jalili, 2018), promethazine (Ensafi et al., 2018), bisphenol A (Liu et al., 2016), tetracycline (Hou et al., 2016), and so forth. In this case, the materials and polymers used can be of a similar type, but the imprinting process is the key factor that introduces the specificity to the nanoprobes.

12.4 Typical Optical Sensing Mechanism When an analyte interacts with the receptors engineered on the surface of the CDs, it can provide analytical information once the initial fluorescence profile of the CDs is being disturbed by the interaction; causing the change in emission intensity or the shift in

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the emission peak. Otherwise, the system will not be functioning as a nanaprobe; even the analyte is interacting effectively with the CDs. In view of this, the development of the nanoprobes should ensure the proper design of a sensing mechanism that can trigger the change in the fluorescence profile of the CDs once there is interaction with the analyte of interest. The change will be taken as a signal and translated into useful analytical information. This section will reveal some common sensing strategies employed for nanoprobes developed using CDs.

12.4.1 “Turn-Off” Strategy Because the CDs are showing fluorescence, the quenching of this emission due to the interaction with the analyte of interest can be used as a sensing signal. It is also known as a “turn-off” strategy, as the intensity of the fluorescence emission will be reduced in the presence of the analyte as an effective quencher. Often, the degree of quenching will have the correlation with the amount of the analyte that is present in the sample. Several € rster resomechanisms have been proposed to cause the quenching. It can undergo Fo nance energy transfer (FRET), where the energy of the emission from the CDs overlaps with the absorbance band of the analyte, leading to a nonradiative pathway for the transfer of the energy of the excited state. FRET becomes more effective when the quencher and fluorophore are of near proximity, and this is where the receptor designed on the CDs becomes important to attract the analyte near the CDs. In a study, CDs that were produced from the hydrothermal carbonzation of aminopolysaccharide chitosan have showed FRET quenching behavior toward nitroaromatics-based compounds (Liang et al., 2016). The CD’s surface is found to be rich in carbonyl, carboxyl, and amino functionality that can interact with the nitroaromatics compounds, and the study revealed that the distance ˚ . In this case, the absorbance of one of the nitroarobetween the two is around 10–100 A matics compounds is shown to have overlapped strongly with the emission of the CDs, allowing the emission energy to transfer from the CDs that eventually turns off the fluorescence of the CDs (Fig. 12.9). In cases where there is no direct overlap of the spectral, a secondary reagent can be used as an alternative strategy to promote the FRET. For example, sodium rhodizonate can promote FRET between CDs with ammonia that initially does not show any quenching effect (Ganiga and Cyriac, 2016). Besides FRET, photoinduced electron transfer (PET) is another common mechanism that can cause quenching of the CDs by the analyte for a nanoprobe. In general, PET is a process where the excited electrons of a fluorophore have matching energy with a quencher electronic state, allowing the transfer of the excited electrons to the quencher. In the case of CDs, the excited electrons from the CDs will be transferred to the electronic orbital of a matching analyte, where the CDs are the donor and the analyte is the acceptor of the electrons. Because the excited electrons did not have the opportunity to relax back within the CDs system due to the transfer, the fluorescence process is being disturbed, and is causing the reduction in the intensity of the emission. For example, the CDs produced by microwave treatment of poly(ethylenimine) have shown a great quenching effect in the

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1.2

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FIG. 12.9 The overlapping spectral between the nitroaromatics compound (TNP) with the CDs (CQDs). Reproduced with permission from Liang, Z., et al., 2016. Probing energy and electron transfer mechanisms in fluorescence quenching of biomass carbon quantum dots. ACS Appl. Mater. Interfaces 8 (27), 17478–17488.

presence of copper (II) ions (Wang et al., 2017c). Upon further investigation, the quenching observed is found due to the transfer of the excited electrons from the lowest unoccupied molecular (LUMO) energy levels of CDs to the d-orbital of the copper (II) ions that formed a complex with the amine group that presents on the surface of the CDs. This would prevent the direct electron-hole recombination of CDs, and eventually stop the initial fluorescence process. This has enabled the system to be employed as a nanoprobe for the monitoring of copper (II) ions in a cellular system. A separate study has reported that CDs that have been surface modified with α-cyclodextrin could form a complex with derivatives of methyl viologen by encapsulation. This leads to the formation of aggregates, and promoting the PET from the CDs to the derivatives of methyl viologen, where strong fluorescence quenching is observed (Mondal and Purkayastha, 2016). This study has demonstrated that PET can also occur between CDs with molecules instead of metal ions that are more commonly observed; providing wider application potential for the CD’s nanoprobe to detect various analytes of interest.

12.4.2 “Turn-Off-On” Strategy It is possible to specifically design the sensing mechanism of CDs by first turning off the fluorescence signal, where later addition of the targeted analyte will turn the signal back on. This means a quencher that will interact with the analyte will be introduced to the CDs, which eventually will quench away the fluorescence signal. Once the analyte is added later in the sequence, they will interact with the quencher and disturb the initial quenching activity by giving rise to the fluorescence signal. This approach is known as the turn-off-on mechanism. One example is the use of CDs produced from the carbonization of alginate, where the fluorescence can be totally quenched by the addition of iron

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(III) ions in a concentration-dependent manner. Once the signal is totally quenched, the addition of ascorbic acid, commonly known as vitamin C, can restore the initial fluorescence signal (Fong et al., 2016). It is postulated that the initial iron(III) ions have stronger binding affinity toward the ascorbic acid, where it will be removed from the initial interaction terminal with the CDs. The previous study has shown that the synthesis process of the CDs has introduced carbonyl-rich groups on the surface, as shown by the FTIR study, which can form binding with the iron (III) ions (Fong et al., 2015). Such a technique has successfully detected the presence of the ascorbic acid with the linear response range of 24–40 μg mL 1 of ascorbic acid. In a separate study, a similar approach has been adopted by using methylene blue as the initial quencher to turn off the fluorescence, and the later introduction of specific deoxyribonucleic acid (DNA) can restore the fluorescence signal (Bai et al., 2011). This is very relevant for the system to be used as a nanoprobe for biosensing, as DNA-based sensing systems are often used for the accurate detection of diseases.

12.5 Practical Utilization of CDs in Biosensors Nanoprobes based on CDs have been practically demonstrated to be utilized for various biosensing applications. This includes the detection of various biological analytes, either in vitro or in vivo. The integration into nanoprobes is well supported by the current technology advancement in terms of the electronic, light source, detector, and waveguide. As a nanoprobe, the CDs will be introduced to the bio-environment, including the possibility of introduction into a single cell due to the small size; and the CDs are expected to interact with the analyte of interest or the surroundings. A low-power excitation light source will be provided to excite the CDs, and the interaction will cause a change to the optical property of the CDs. The use of cheap and efficient light-emitting diodes (LED) can be used to replace the conventional light source that generates from the high power system, such as the helium light source. This change in the property will be then recorded by a light detector and analyzed based on an optimized mathematical model to generate useful analytical information. CDs have been proven to be adopted for metal ion sensing via the interaction of metal ions with the surface chemical functional groups on the CDs (Tan et al., 2014; Ngu et al., 2016). Some metal ions are important elements within the biological system, which will be an advantage if the monitoring can be made toward the molecular level. Some metal ions are crucial for the metabolism processes, while others might be harmful, and able to suppress normal biological processes. Some metal ions might play the role of electrolytes in transmitting the biological information from one part to another. Thus, monitoring using CDs as nanoprobes will be of additional benefit in the field of biomedical diagnosis. Besides metal ions, metabolites produced from biological processes are also an important group of analytes, where they can be used as biomarkers for various diseases. In view of this, CDs have shown very good potential in detecting this group of simple molecules. Cholesterol and xanthine are metabolites that participate in the hydrogen peroxide bio-process, and can be detected using CDs nanoprobes (Ma et al., 2017). Another

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example is the use of CDs for the detection of dipicolinic acid, a biomarker for anthrax that can cause serious infection to humans (Chen et al., 2015). Other groups have reported the detection of cancer, and disease biomarkers using CD-based probes (Pasinszki et al., 2017). The biomedical field often studies molecular biology to understand the expression of different genes and proteins under different conditions, such as under stress or under contamination of pathogens. In this case, the detection is not focused directly on the analyte, but rather, performing an indirect measurement of the expressed products, such as proteins. Proteins are basically macromolecules that of a certain shape and size, while also often portraying biological activities. In this case, even in the range of macromolecular sizes, CDs can be adopted as nanoprobes for the detection of different proteins. In a study, polyethylenimine, ethylenediamine branched-functionalized CDs have been made possible for protein sensing (Freire et al., 2018). The report has shown the success in detecting eight different proteins within the detection range of 5–40 nM. In a more advance setting, biomolecules, such as aptamers, can be grafted onto the CDs, during which the presence of the specific protein can induce a sandwiched aggregate structure that eventually will quench the fluorescence signal. In a specific example, the aptamer has been tagged on CDs and silica nanoparticles to form the nanoprobe system. In the presence of thrombin, both the sets of the nanoparticles will interact and eventually form a cluster and turn off the initial fluorescence intensity of the CDs (Xu et al., 2012). Target analytes of larger molecular sizes can be detected as well using nanoprobes developed from CDs. For example, CDs capped with hydrocarbon chains can interact with bacterial cells after a short incubation time and cause the fluorescence signal to change (Nandi et al., 2015). This proof of concept will be very useful in the future, by enhancing the specificity, especially toward those pathogenic bacteria that are harmful to humans. One example reported is by grafting a specific aptamer onto the surface of the CDs, which later the nanoprobes can use to detect a specific pathogen, such as Salmonella typhimurium (Wang et al., 2015b). DNA-based CDs have also been used to detect bacteria and viruses. This is based on the matching in the DNA sequence using a single-strand DNA that is attached on the surface of the CDs. One example is the success in developing an ultrasensitive homogeneous nanoprobe for the detection of DNA sequences related to HIV using CDs (Qaddare, 2016). The mechanism is based on FRET between the CDs and gold nanoparticles as nanoquenchers. For the large area of detection, such as tissues and tumors, the working principle of the nanoprobes is based on the fluorescence imaging technique. The CDs will be modified specifically to be able to attach or diffuse into the tissue layers, where under the excitation wavelength, the area will glow and show a clear boundary between the different functions. This technique is very popular in testing as part of cancer research to detect tumors or cancerous cells. in vitro study has shown very promising results where the CDs showed excellent cancer cell cytoplasm and nucleus targeting in MCF-7 cells (Aiyer et al., 2016). Similarly, CDs coupled with folic acid have shown clear uptake by cancer cells, but not by normal cells (Zhao et al., 2017b). This has been very useful for diagnostic

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purposes. The in vivo imaging can also be performed using CDs to identify the area infected with cancer cells. CDs having aspartic acid functional groups have shown great targeting properties toward C6 glioma cells, where in vivo fluorescence images have shown high-contrast bio-distribution of the CDs.

12.6 Summary and Outlook In summary, nanoprobes developed from CDs definitely have great potential to be employed in real biosensing applications. This is especially true when examining the practical benefits of CDs that are easy to prepare, economical, sustainable, and adopt greener synthesis approaches; and more importantly, have proven to have the low toxicity effect that makes them suitable for adoption in biomedical applications. All these are far more superior to their QDs counterparts. The quantum yield has also been improved significantly, although those initial isolated CDs showed weaker emission, creating a concern for analytical applications. Although there are still some fundamental functions of the CDs that remain unclear and under debate, the possibility of utilizing CDs as nanoprobes is clear as proven by the huge amount of successful studies that demonstrated the usability of CDs for biosensing, as reported in the literature. In order to step forward, efforts should now shift more toward the commercialization requirements that will be the key enabler for this technology to be transferred into the market for real use for diagnostics. Stakeholders and relevant authorities should set standards and specifications for the CDs to be used for nanoprobe development. This is for quality assurance, and to facilitate acceptance by the public of the reliability of the developed nanoprobes from CDs for biosensing. This might not need to be just from the aspect of safety, but also in terms of sensitivity from the cultural and religious points of view. This aspect is crucial, because the production of CDs can create endless possibilities from its selection of starting materials, carbonization methods, and the options for surface modification. Only those that meet the requirements should be allowed to transfer from the research stage into the real application stage. With this next effort, the real use of CDs as nanoprobes that are commercially available will soon be a reality.

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Further Reading Chin, S.-F., et al., 2017. Nitrogen doped carbon nanodots as fluorescent probes for selective detection and quantification of ferric(III) ions. Opt. Mater. 73 (Supplement C), 77–82. Li, H., et al., 2010. Water-soluble fluorescent carbon quantum dots and photocatalyst design. Angew. Chem. Int. Ed. 49 (26), 4430–4434. Ng, S.M., Koneswaran, M., Narayanaswamy, R., 2016. A review on fluorescent inorganic nanoparticles for optical sensing applications. RSC Adv. 6 (26), 21624–21661.