9.1 Introduction

Nanomaterials are especially interesting in comparison to their bulk form due to their tunable physical, chemical, electronic, thermal, mechanical, and optical properties. Carbon‐based nanomaterials fullerene, carbon nanotubes (CNTs), graphene, carbon dots (CDs) and nanodiamond, have been studied extensively due to their intrinsic properties. Among these, CNTs, graphene, and CDs have attracted tremendous amount of attention for further bioanalytical applications due to their ease of functionalization for detection of analytes of interest. CNTs and graphene are similar in structure where CNTs can be viewed as seamless cylinders rolled by graphene sheets. Despite their electrical and electrochemical properties, these carbon nanomaterials typically requires complicated and costly instrumental setup, specific experimental conditions and synthesis methods such as arc discharge, laser ablation, and chemical vapor deposition. These are not favorable for laboratory settings with limited resources. In view of that, CDs that are easily attainable from renewable sources or waste materials via facile and simple synthesis routes are more ideal and cost‐effective option to be molded and extended for bioanalysis.

CDs are basically nanoparticles that are made up of majority carbon element and in some cases, blended with other impurities such as oxygen and nitrogen. It has caught the attention of the research community as it portrays very similar properties to the well‐known quantum dots (QDs), but with rather easier synthesis route and using less toxic starting precursors. It has been well demonstrated that synthesis of CDs can be from simple organic molecules [1], polymers [2, 3], or even waste biomass [4, 5]. Normally, it is not required to use a specific pure chemical as starting precursor, while the carbonization can be performed in dry form via thermal treatment [6] or just in pure water as solvent in the case of hydrothermal treatment [7]. There is also not much on the generation of toxic side products from the synthesis process, eventually making the whole synthesis process cleaner and greener. All these means that CDs are good alternative to replace the QDs in various applications. CDs have all the unique functionalities of the QDs for an intended application, but less harmful to the environments. It is even more promising when the mentioned environment refers to a bioenvironment in an organism that is often susceptible to toxic compounds. In other words, CDs can be applied directly for in vivo applications. Based on this conditional advantage, researchers have been exploring the practical usage of CDs for application in the biological system. One of the focuses is on bioanalysis, an analytical method that deals with the quantitative measurement of a compound or metabolites in biological fluids, primarily blood, plasma, serum, urine, or tissue extracts [8].

While the general acceptance on the potential application of CDs for bioanalysis is converging, the practical development involves various stages and also technology advancement before the final developed CDs can be adopted as a detection probe. In this case, crude CDs are only acting as signal tags to generate a measurable signal, such as in the form of optical or potential signals using a detector. In order to introduce the analytical characteristics, the CDs need to be first designed with functionality that enables its interaction with the analyte of interest. It can be just of the weaker Van der Waals forces, stronger ionic interactions or the formation of complex via covalent bonds. Such interactions eventually should trigger a change in the initial signal that is concentration dependent. Once this has been achieved, the signal change can be correlated with the concentration of the analyte into a calibration curve and used as model to predict unknown concentration of the analyte based on the change of signal recorded. Specifically, the interaction shall be targeting on small molecules found in the biological system for the bioanalysis application. It will be of further advantage for having the probe to be biocompatible and nontoxic, which will enable its direct in vivo application for real‐time monitoring.

This chapter will particularly discuss the advancements and approaches taken to design CDs into bioanalytical probes. The fundamentals of CDs will be first revealed and served as the foundation for the further discussion on the technology that can be applied to engineer the CDs into a sensing probe. The engineering approaches will be limited to only those that can lead to potential development into a biocompatible probe. In this case, it can be generally divided into two; the advancement on the unique features of the CDs into better suiting the bioanalysis applications and the surface modification with biomolecules to introduce specific interaction of the CDs toward a targeted bioanalyte in the body. The chapter will then discuss the sensing mechanisms that can be adopted with developed CDs, revealing the working principle that causes the change in the initial signals. Most of the transduction systems that are reported to date have adopted optical method, since the CDs show unique photoluminescence (PL) property. In addition, optical method seems to be very practical and involves less sophisticated instrumentation. Some successful examples reported in the literature on the applications of CDs as bioanalysis probe targeting on specific bioanalyte are also included in this chapter. The future prospect of CDs is very promising for their practicality, as real bioanalysis probes and continuous improvements of the system are required to address some of the existing limitations of CDs. These include the identification on the origin of the optical features, consistent protocol for homogenous production of CDs, enhancement of quantum yield (QY) and also improvement of analytical sensitivity.

9.2 Fundamentals of CDs

9.2.2 Optical Properties

9.2.2.1 Absorbance and Photoluminescence (PL)

Ultraviolet and visible (UV–Vis) spectroscopy is an essential tool that has been widely applied to study the electronic energy transition and structural properties of CDs. It is typical to observe CDs displaying strong absorption band in the UV region of 260–320 nm, with a tail extending into the visible range [52]. The absorption in the UV range is often suggested to be ascribed to the π − π * transition of C=C bond and n− π * transition of C=O bond [53–55]. UV–Vis absorption peaks at longer wavelength were also reported at 340 and 451 nm [56]. The former spectrum is attributed to the n− π * transition whereas the absence of a well‐defined band edge in the UV–Vis range was suggested for the latter broad absorption spectrum that extended to the visible range.

The unique PL characteristics of CDs include the effect of excitation wavelength (λ_ex) on the emission wavelength and intensity of CDs. The λ_ex‐dependent emission property of CDs is commonly reported [31, 34, 57]. A tentative explanation of such optical property has been proposed as to be due to the surface states created on the CDs [58]. The complex surface defects could be generated by surface oxidation to trap more excitons during the excited stage. This would subsequently result in radiative recombination of the trapped excitons to generate fluorescence emissions corresponding to the excitation energy, hence red‐shifting the emission wavelength. Different emissive states and heterogeneous types of π conjugations in nanodomains are also suggested to be responsible for the λ_ex‐dependent PL [59, 60]. In contrast, λ_ex‐independent fluorescence emission of CDs has also been observed [61], which is often claimed to be attributed to a lower degree of surface oxidation and surface uniformity [58, 62]. Such λ_ex‐independent emission generally agrees with Kasha's rule, where the emission is originated from the ground vibrational level of the excited singlet state [44, 56].

In addition to λ_ex, pH of the aqueous solution is reported to alter the fluorescence emission wavelength and intensity of CDs. One of the earliest reports has shown that maximum emission of CDs produced from ascorbic acid had red‐shifted from 441 to 550 nm when the pH was increased accordingly [63]. Zhang et al. also reported pH‐induced shift in emission wavelength of CDs from the blue (490 nm) to green (523 nm) region [64]. Moreover, pH change in aqueous solutions of CDs would affect the emission intensity due to the presence of surface functional groups such as carboxyl and amino groups [65, 66]. Wang et al. have observed that CDs exhibited smaller diameter at low pH and were readily dissolved in aqueous solution. However, when at high pH, CDs possess comparatively larger diameter and pH‐induced aggregations could occur and eventually lead to quenching in fluorescence [67].

Although quite a few studies have reported bright blue PL of CDs, their application in biological systems is usually restricted due to blue auto‐fluorescence of biological matrix. This reduces the contrast between the area tagged with CDs and the biological matrix. Besides, there is a potential of photodamage to biological tissues upon excitation of the CDs under UV light [68]. Hence, production of CDs with emission at the longer wavelength and of multicolor is greatly desired for applications dealing with in‐vivo biological environment. Precise control of the synthesis conditions such as reaction temperature and time could effectively produce CDs with two distinct fluorescence properties from the same precursor [2]. Multicolored CDs with relatively high QY of 41% (blue), 43% (yellow), and 39% (red) emission have been fabricated via solvothermal synthesis in dimethylformamide as solvent using citric acid and urea as carbon (C) and nitrogen (N) sources [69]. Besides, different precursors can also tune the color emission of the CDs. CDs with blue to near‐infrared (NIR) emissions, as shown in Figure 9.1 within the range of 480–680 nm have been prepared from various functional precursors based on polythiophene derivatives [70]. Besides starting precursors, the emission of CDs can also be adjusted by controlling the charge transfer process, as demonstrated by Hu et al. [71]. The emissive states of the CDs were tuned by grafting the CDs with molecules consisting of a benzene ring and different heteroatom substituents through peptide bonds. The resulting CDs exhibited bright blue, green, and yellow emission under UV excitation. Interestingly, a recent study has reported the solvatochromism effect where CDs emitted different PL colors in the visible region from green to red when dispersed in various solvents [72]. These CDs were claimed to display λ_ex‐independent emission.

9.2.2.2 Quantum Yield (QY)

QY is one of the vital parameters measured to characterize the PL property of CDs. The CDs produced during the early stage of development have typically shown relatively low QY that is less than 10.0% [73–75]. It was later found that the QY of CDs can be enhanced by doping of heteroatoms such as oxygen (O), sulfur (S), and nitrogen (N) into the CDs [76–78]. When compared to the bare CDs obtained from hydrothermal treatment of citric acid (QY = 5.3%), the QY of N‐doped CDs and N,S‐co‐doped CDs were significantly enhanced to 16.9% and 73.0%, both using quinine sulfate as standard [79]. The work by Liu et al. that prepared N‐doped CDs in a confined layered‐double hydroxide (LDH) host claimed that the QY of CDs could be tuned by adjusting the charge density of the host layer and achieved maximum QY of 61.6% [80]. Recent advances in CDs synthesis have successfully prepared CDs with ultra‐high QY of up to 99% via simple one‐step microwave‐assisted synthesis without the need of additional step of surface passivation [81]. In addition to the aforementioned PL properties of CDs, nonblinking nature and excellent photostability are also the merits of CDs that have inspired tremendous research in this field of study compared to QDs.

9.2.2.3 Photoluminescence Origins

Intensive research has been focusing on unveiling the origin of the PL from CDs; as such knowledge is imperative for fine‐tuning the PL properties to suit a specific application. Although the exact origin of PL is still debatable, several general mechanisms explaining the root of the PL event have been suggested. These include quantum confinement effect, surface states, and molecular origins. Quantum confinement effect of π‐conjugated domains of carbon core can be referred to nano‐sized particles with diameter smaller than the exciton radius, causing the charge carriers to become spatially confined, leading to the rise in energy levels [82]. Through a series of spectroscopic analyses and theoretical calculations (Figure 9.2), Li et al. convincingly showed that the size‐dependent‐PL of CDs obtained from electrochemical approach was solely attributed to the quantum‐sized graphitic structure of CDs [83]. In addition to the particles of different sizes, quantum confinement of a distribution of different emissive energy traps on CDs upon surface passivation was also suggested [11, 14].

There are also some suggestions that PL of CDs is due to the synergistic effect of the graphitic structures in the carbon core and the well‐distributed surface states [84, 85]. Regarding the surface states theory, the PL of CDs is claimed to be originated from the functional groups on the surface of the CDs. The presence of functionalities with O and N is responsible for the λ_ex‐independent emission of CDs due to the different induced vibration relaxation modes [86]. Besides, doping with heteroatoms, especially the N atoms, can improve the emission efficiency of the PL with higher QY. A study by Yuan et al. has claimed that amino groups on the surface of CDs was governing the PL property and could enhance the conjugation degree of the CDs system [87]. As a result, it increases the electron transition probability from the ground state to the lowest excited singlet state that leads to higher QY.

Unlike the surface state where the PL origin was formed by hybridization structure of functional groups and carbon core, molecular‐state‐induced PL is solely due to the organic fluorophore attached on the surface or interior of the carbon backbone [88]. For instance, the dehydration of carbohydrate molecules can produce 5‐hydroxymethylfurfural (HMF) derivatives that serve as aggregated fluorescing units within the CDs, causing it to emit orange‐red fluorescence [89]. Others have suggested the generation of H‐aggregate type excitonic states that has caused the PL of CDs [90, 91]. In a recent study, PL mechanism of multi‐states has been reported for N‐doped CDs obtained via microwave‐assisted synthesis [92]. Treatment at 160 °C produced organic molecule clusters that were rich in amide bonds with molecular states being the PL center. Increasing treatment temperature to 200 °C has facilitated the formation of carbon core, and the PL was attributed to the synergy between the carbon core and surface states; whereas prolonged treatment resulted in PL arise from carbon core.

9.2.2.4 Up‐Conversion Photoluminescence (UCPL)

In addition to the usual PL (sometimes also known as down‐conversion fluorescence), up‐conversion photoluminescence (UCPL) of CDs has also been reported. UCPL refers to the phenomenon where the fluorophores could absorb two or more low‐energy photons and emit high‐energy photons at wavelengths shorter than the excitation wavelength [93]. The UCPL of CDs was suggested by Cao et al., who first observed that CDs emitted visible light upon excitation at NIR (800 nm) using a femtosecond pulsed laser [94]. Following that, UCPL is also reported in CDs obtained via alkali‐assisted electrochemical method [83], thermal decomposition [63], microwave‐assisted pyrolysis [95], etc.

The UCPL is utilizing NIR light as the excitation light source and this offers intriguing advantages especially in the field of biological applications. The benefits of UCPL include noninvasiveness, deep‐tissue penetration, reduced damage caused by photon, low photobleaching, and weak auto‐fluorescence background [96]. In a recent study, UCPL of a nontoxic CDs‐embedded hybrid microgel with good‐biocompatibility was demonstrated for glucose sensing at physiological pH [97]. This has shed light on applying CDs with UCPL property for theranostics purposes. UCPL property is commonly believed to be accounted by multiphoton active process [83, 94]. However, Shen et al. later claimed that multiphoton active process is insufficient to fully explain the mechanism of the UCPL of CDs and therefore speculated the theory of anti‐Stroke transition based on their spectral observation [98]. They have proposed that the UCPL of their CDs was due to anti‐Stokes PL, which involved the process of electrons relaxing back to σ orbital from a high‐energy state when the electrons in π orbital were excited by low‐energy photons.

It is worth mentioning that several reports have claimed that the UCPL observed in CDs systems is actually originated from the equipment artifact. The UCPL is the normal fluorescence excited by light radiation from the second‐order diffraction (λ/2) passing from the excitation monochromators of fluorescence spectrophotometer [99]. This type of misinterpreted PL due to the leaking light radiation can be eliminated by inserting a long‐pass filter in the excitation pathway of the spectrophotometer [100]. Additionally, the interference of second‐order diffraction light can also be eliminated by using a pulsed laser as an excitation source instead of a xenon lamp [101]. Therefore, UCPL of CDs observed by employing laser as the excitation source coupled with quadratic dependence relationship between the two‐photon luminescence intensity and the excitation laser power indicated the genuine UCPL [101]. Nevertheless, the UCPL property of CDs must be carefully evaluated, taking into consideration that second‐order diffraction light (λ/2) might lead to misinterpretation of normal fluorescence as UCPL.

9.2.2.5 Phosphorescence

Phosphorescent materials have been widely used in optoelectronic devices, sensors, and security systems over the past few decades. However, research on room temperature phosphorescence (RTP) of CDs is still at its infancy due to the relatively scarce reports in this area. In a pioneering work by Deng et al., the RTP has been observed for the first time from the CDs obtained via pyrolysis of ethylenediaminetetraacetic acid disodium salt (EDTA‐2Na) that were dispersed into a polyvinyl alcohol (PVA) matrix under UV light excitation [102]. The phosphorescence centered at approximately 500 nm and had a lifetime of 380 ms. Subsequently, RTP has also been observed when CDs were dispersed in different matrices such as potash alum [103], polyurethane [104], LDHs [105], and silica gel [106]. The CDs embedded in silica gel matrix displayed bright green after‐glow had a phosphorescence lifetime of about 1.8 seconds, which was claimed to be the highest magnitude reported for CDs in solid‐state matrices thus far [106].

Apart from single‐component matrices, composite matrices have also shown to activate the RTP of CDs. In one example, N‐doped CDs that were integrated into composite matrices consisting of the recrystallized urea and biuret from the heating of urea has exhibited the RTP [107]. This is due to the more rigid microenvironment of the CDs and the enhanced hydrogen bonding between biuret and CDs that promoted the RTP. The composite matrices have protected the triplet state from a long lifetime quenched by suppressing the nonradiative deactivation processes. The two‐component matrices were claimed to be more superior when compared to single‐component matrices due to the combination of the individual ingredient role [107]. RTP is generally observed when CDs are incorporated onto solid support. However, a ground‐breaking study by Yan et al. reported RTP of water‐soluble CDs in pure aqueous solution without any supporting matrices [108]. The RTP of the functionalized‐CDs could be quenched by ferric ions (Fe(III)) and effectively recovered in the presence of phosphate‐containing molecules such as adenosine‐5ʹ‐triphosphate (ATP).

In terms of potential applications, a novel RTP logic gate based on nucleic acid functionalized CDs and graphene oxide (GO) has been demonstrated [109]. Complementary DNA (cDNA) was first conjugated with CDs (cDNA‐CDs) through carbodiimide chemistry prior to adsorption onto a GO surface by π−π stacking interactions, yielding a cDNA‐CDs/GO complex. RTP of the cDNA‐CDs conjugates was “turned off” upon adsorption onto the GO surface due to photo‐induced electron transfer (PET) from CDs to GO. Subsequent addition of mercuric ions (Hg(II)) or transfer DNA (tDNA) that is complementary to the cDNA could turn the RTP of CDs back “on.” Another study by Chen et al., who demonstrated aggregation‐induced RTP in N‐doped CDs powder hydrothermally synthesized from PVA and ethylenediamine, found potential application in temperature sensing [110]. Interestingly, RTP of the CDs could only be observed in aggregation state but not in aqueous solution due to the postulate that the CDs themselves could serve as both the solidified host and the luminescent guest. The RTP intensity decreased in double exponential decay profile as the temperature increased from 30 to 90 °C; hence, phosphorescence intensity variation could be predicted within this temperature range by fitting relevant equations.

Suppression of radiationless relaxation processes is vital for the phosphorescence mechanism [105]. RTP of CDs is generally claimed to be originated from the triplet excited states of aromatic carbonyl (C=O) groups formed at the surface of CDs. Hydrogen bonding between the solid matrices and CDs could rigidify the C=O bonds on the surface of CDs, and this, in turn, minimizes the nonradiative transitions of triplet excitons and hence promotes phosphorescence [102, 104]. On the other hand, Li et al. argued that the C=N bonds on CDs surface were responsible for the RTP of CDs [107]. The C=N bonds were believed to create a new energy level structure and promote the generation of triplet excitons through n−π* transition. In a recent report, covalent bonds fixation of CDs onto colloidal nanosilica solution was evaluated to be a better alternative than hydrogen bonds due to the extension of long afterglow to solution forms. The presence of unsaturated bonds such as C=C bonds on CDs surface could make them self‐protecting agents from quenching effects as a result of energy transfer from excited triplet states of the chromophore to the dissolved oxygen [111].

9.2.3 Physical and Chemical Properties

CDs are zero‐dimensional, quasi‐spherical nanoparticles with a typical diameter of less than 10 nm, and the carbogenic cores can be amorphous or nanocrystalline [52]. In terms of morphology, scanning electron microscopy (SEM), transmission electron microscopy (TEM), and atomic force microscopy (AFM) can be employed to provide such information. CDs are commonly described as homogeneous, monodisperse nanoparticles with narrow size distribution [32, 37]. Besides, HRTEM, X‐ray diffraction (XRD), and Raman spectroscopy are powerful tools to characterize the structural properties of CDs. Several structural models such as diamond‐like, graphite/graphite oxide, and amorphous structures are often adopted to describe the CDs carbogenic cores. For instance, the selected area electron diffraction (SAED) pattern in TEM image of CDs obtained through laser irradiation indicated a diamond‐like structure [112]; the broad XRD diffraction peak at 2 θ = 22.6° was attributed to amorphous carbon structure [53]; signals of Raman spectrum at 1578 and 1346 cm⁻¹ that corresponded to G and D band, respectively, indicated high degree of graphitization [33].

The carbogenic cores of CDs are mainly composed of C, H, O, and N elements. Microanalysis of the elemental composition and speciation of CDs can be achieved by Fourier transform infrared spectroscopy (FTIR), nuclear magnetic resonance (NMR), and X‐ray photoelectron spectroscopy (XPS). FTIR spectroscopy is widely employed to characterize the surface functional groups that are present on CDs. Carboxylic moieties (─COOH) and hydroxyl groups (─OH) are commonly found on the surface of CDs [112, 113]. CDs usually have high oxygen content due to the oxidized carbogenic cores as well as the presence of surface functional groups. For example, the elemental analysis of the oxidized CDs purified from candle soot have been revealed to have the composition of 36.8% C, 5.9% H, 9.6% N, and 44.7% O, which compares to raw candle soot that consists of 91.7% C, 1.8% H, 0.1% N, and only 4.4% O [12].

The chemical properties of CDs can also be identified by NMR measurements. The ¹ H NMR spectrum can indicate the presence of sp³ C─H protons, aromatic or sp² protons as well as protons attached with hydroxyl, ether, carbonyl, and aldehyde groups [23]. Meanwhile, their ¹³ C NMR spectrum indicated the presence of sp³ carbons and carbons attached with hydroxyl groups and ether linkages, aromatic C=C and C=O carbons.

Furthermore, a typical XPS as shown in Figure 9.3 by Miao et al. indicated the four peaks at approximately 284.3, 285.1, 286.1, and 288.5 eV were corresponding to sp² hybridized carbon atoms in C─C/C=C, sp³ hybridized carbon atoms in C─S/C─N/C─O, ─C≡N, and ─COOH groups [33].

9.3 Bioengineering of CDs for Bioanalysis

9.3.1 Functionalization Mechanism and Strategies

9.3.1.1 Chemical Functionalization

Surface modification via chemical route is practical for tailoring the properties of CDs with desired characteristics to suit a specific application. Sometimes, it is also known as surface passivation or functionalization. The approach can be performed via various types of chemical interactions such as covalent bonding, π−π conjugation, and electrostatic interaction. Among these, the most popular one is the covalent linkage formed using the carbodiimide crosslinking chemistry. This technique is targeting on the activation of carboxylic group that is commonly found on the surface of CDs, in which the activated carboxylic group later can react with its conjugate such as amine group to form a covalent bond. The carbodiimide chemistry involves two‐step reactions where the first is activation of carboxyl groups (─COOH) and the second is peptide bond formation as a result of nucleophilic attack by primary amines (─NH₂) [124, 125]. The new bond formation is often proven by the observation of new absorbance shown in the infrared spectrum. For instance, FTIR spectrum by Fu et al. showing the stretching vibration of N─C=O at 1651 cm⁻¹ has confirmed the formation of amide bonds between ─COOH groups in bare CDs and ─NH₂ groups in arginine (Arg) via carbodiimide chemistry [126]. It is noted that no part of the chemical structure of the carbodiimides is being added to the final product, making them an efficient zero‐length carboxyl‐to‐amine cross‐linker. In some cases, N‐hydroxysuccinimide (NHS) will be added to act as a stabilizer of the intermediate formed by 1‐ethyl‐3‐(3‐dimethylaminopropyl)carbodiimide hydrochloride (EDC). Upon quenching the reaction, the conjugated product is often isolated via various purification options such as column chromatography or dialysis to remove the urea byproducts and excess reagents. The carbodiimide crosslinking is typically favored to conjugate antibody or other protein compounds due to the presence of amine and carboxyl groups.

In addition to the aforementioned chemical interactions, weaker interaction forces can also be employed for the surface conjugation. This can be due to the surface property that is likely to induce attractive forces to some ligands or polymeric chains. For instance, some research reports the electrostatic interactions between negatively charged CDs with positively charged polyethylenimine (PEI) and folic acid (FA) in one of the surface modification efforts [127]. Furthermore, the presence of C=C as shown by the sp² hybridization of CDs has given the nanoparticles leverage for π−π conjugation with desired compounds that are also present with conjugated system. For instance, doxorubicin (DOX), an anticancer drug that contains conjugated system within the chemical structure has been successfully conjugated to CDs through both π−π conjugation and electrostatic interactions for controlled drug release [128]. This characteristic is also useful to functionalize CDs with small molecule dyes that are typically rich with π‐conjugated domains such as rhodamine B [129].

9.3.1.2 Doping

Heteroatoms doping is generally considered as an effective approach to tune the band gap of semiconductors to achieve a particular electrical property of interest. This is by introduction of these heteroatom impurities to the semiconductors intentionally to alter the initial band gaps [130]. Heteroatoms are defined as elements other than C and H. In the case of CDs, doping has been also demonstrated to be efficacious to fine‐tune the properties of CDs. For examples, N‐doping could notably enhance the QY of CDs [77, 80]. Doping with boron (B) has significantly improved the PL intensity and nonlinear optical response of CDs [131, 132]. The presence of S atoms in the carbon precursor has played a vital role in promoting the formation of doped CDs from low‐molecular‐weight precursors [133]. Phosphorus (P)‐doped CDs exhibited aggregation‐induced red shift emission (AIRSE) from blue to orange yellow as schematically illustrated in Figure 9.4 [134].

In addition to nonmetal elements, metal dopants such as zinc (Zn) [135], copper (Cu) [136], cobalt (Co) [137], manganese (Mn) [138], germanium (Ge) [139], and terbium (Tb) [140] have also been reported for CDs. Doping with gadolinium (Gd) endowed CDs to be a potential magnetic resonance imaging (MRI) contrast agent for imaging and radiotherapy of tumors [141, 142]. Moreover, co‐doping provides an effective means to further improve the properties of CDs. Some examples include CDs co‐doped with B, N, and S that later can be fabricated for colorimetric and fluorescent dual mode detection of Fe(III) ions [143] and selenium (Se) ion. Besides, N‐co‐doped CDs exhibited bright green emission with high QY (~52%) evidenced high performance as a dye for fluorescein fundus angiography (FFA) [130].

9.3.1.3 Coupling with Gold Nanoparticles

Gold nanoparticles (AuNPs) have been used for centuries in arts due to their vibrant colors produced as a result of their interaction with visible light. These unique optoelectronics properties have then been studied and exploited for various biomedical applications such as sensory probes, therapeutic agents, drug delivery, etc. The optical and electronic properties of AuNPs can be tuned by altering the size, shape, surface chemistry, or aggregation state. More recently, research on these nanoparticles is extended by the integration with other nanoparticles such as with the CDs for biosensing applications [144, 145]. The CDs have showed an enhanced PL in the presence of AuNPs and this is explained as due to the two synergistic mechanisms of electric field and the induced surface plasmon effects [146]. The same study has also reported that the consistent distance in the proximity of less than 10 nm between CDs and AuNPs as due to the linked polymer, N‐(β‐aminoethyl)‐γ‐aminopropyl methyl‐dimethoxy silane (AEAPMS) has exposed the nanoparticles with increased electric fields. Such effect has improved the local electromagnetic field and, in turn, strengthened the PL of CDs. Simultaneously, the partial transfer of energies from excited state of CDs to AuNPs surface plasmons has induced surface plasmon effects and subsequently increased the PL of CDs. This study has shown that the two nanoparticles can support and enhance each other's properties as a result of coupling reaction. The simplest coupling or modification with AuNPs was by mixing the precursors for both CDs and AuNPs in one‐pot synthesis. A study by Wu's group has utilized citric acid and cysteine as a C source for CDs, which these precursors have concurrently served as reducing agent and could easily form the graphene framework through intermolecular dehydration [147]. Another method of coupling with AuNPs would be by assembly via electrostatic interactions between the AuNPs and CDs. In such cases, both nanoparticles were synthesized individually and assembled in subsequent step. For instance, Wang et al. synthesized CDs via hydrothermal treatment of pancreatin, while AuNPs were obtained from its precursor HAuCl₄ followed by modification with aptamer [148]. The electrostatic interactions were formed upon mixing the two nanoparticles in 10 mM phosphate buffer saline (PBS) buffer.

9.3.1.4 Fabrication onto Solid Polymeric Matrices

The blending of CDs into various solid matrices through sol–gel technology has also attracted immense attention due to the materialization of CDs for real bioanalytical device applications. Molecularly imprinted polymers (MIPs) can be prepared by simulating receptor active sites using target molecules as templates, from which the template molecules will later be removed after the formation of the synthetic polymer [149]. CDs were successfully integrated into a silica matrix through sol–gel based molecular imprinting technique for fluorometric determination of nicotinic acid [150]. Besides, CDs have also been successfully incorporated into other types of polymeric matrices such as agarose hydrogel [151], chitosan hydrogel [152, 153], poly(methyl methacrylate) (PMMA) [154, 155], PVA [156], and gel glass [157]. In a recent study, a polymer/silica hybrid film has been successfully fabricated to entrap CDs [158]. The transparent free‐standing hybrid film that possessed great flexibility could protect the embedded CDs even when treated at a high temperature (550 °C), and its potential in photonic application has also been demonstrated.

9.4 Bioanalysis Applications of CDs

CDs that have been carefully designed and engineered with bioanalytical functionality can be then utilized for specific bioanalytical applications. Bioanalysis has become an important part of biomedical diagnosis as it involves quantification measurement of a drug compound or a metabolite in the biological fluids such as in blood, plasma, serum, urine, or tissue extracts. The excellent properties of CDs, such as good biocompatibility, low cytotoxicity, and rapid clearance from body have prompted many to switch focus from organic dyes or QDs to CDs for bioanalytical applications. These days, the scope of bioanalysis has even extended to the cellular studies via bioimaging, monitoring of the drug delivery process via intracellular tracking, and also the therapeutics including the photothermal or photodynamic therapy (PDT), and theranostics that integrated both diagnostics and therapy. All these will be discussed in greater detail in this section.

9.4.2 Uses of CDs in Bioanalysis

Owing to the unique optoelectronic characteristics of CDs, there are a lot of biosensors being studied and reported in the literature for bioanalysis applications. Among those reported, the most common CDs‐based sensing systems are adopted for detection of heavy metals associated with diseases, DNA, small molecules, pharmaceutical drugs, proteins, antibodies, pH, and other biomarkers as part of disease screening. This section will explore and reveal some examples reported on the use of CDs for bioanalysis.

9.4.2.1 Heavy Metals/Elements

While some metals such as iron and zinc are essential elements for human physiological health, other heavy metals might portray a high toxicity effect to the environment and human health. Most metals when ingested in large amount result in immediate direct poisoning or damaging diseases in a longer run [239]. For instance, heavy metals such as Hg(II), cadmium (Cd(II)), lead (Pb(II)), and arsenic (Ar(II)) pose a high toxicity risk to human and other organisms. CDs can be developed to detect these heavy metals, mostly in their ionic state. In fact, some of the earliest demonstrations on the applications of CDs as sensing probes are on the detection of Hg(II) [240–244]. Most of these CDs‐based sensors adopt cysteine as sensing receptor for the detection of Hg(II) since the thiol group can bind well to Hg(II) [245]. It is worth noting that most of the Hg(II) detection systems reported to date are performed in liquid phase directly using the CDs, while only Gonçalves et al. have reported the development of CDs into a solid platform for the sensing application [242, 246]. The CDs were immobilized on an optical fiber via layer‐by‐layer approach, and the resulting optical fiber sensor had achieved an LOD of 0.01 μM for Hg(II).

Since their practicality for Hg(II) sensing, CDs have also been explored for the detection of other metal ions based on similar sensing mechanism. For example, CDs have been developed for the detection of Pb(II) ions with LOD of 5.05 μM [247] and ferric ions (Fe(III)) with LOD of 1.06 μM [3]. Usually, the surface of the CDs is reported to be oxygen rich due to the presence of hydroxyl (OH⁻ ) and carboxyl (COO⁻ ) groups that are introduced during the carbonization process. This interface is of high affinity to attract the positively charged metal cations. Such interactions could result in the disturbances of the initial origin of the fluorescence, leading to quenching of the fluorescence. The degree of quenching can be correlated to the amount of metal ions present to establish a calibration curve, which later can be analyzed to predict the concentration of metal ions in unknown samples. Most of the CDs synthesized via aerobic carbonization or hydrothermal method have shown fluorescence quenching by Fe (III) ions [24, 248].

More recently, CDs sensing systems have also been developed for selective detection of other toxic metals such as beryllium ions [249], copper ions [95, 250–255], zinc ions [256, 257], arsenic [258], palladium [259], and aluminum ions [260]. Interestingly, the known interaction between CDs and metal cations can also be exploited for selective detection of anions such as sulfides, as demonstrated by Barati et al. [261]. While sulfide ions themselves did not pose any changes on fluorescence of CDs, metal‐mediated sensing has been developed via two pathways. First, Hg(II) ions worked as quencher of the fluorescence of CDs and secondly, the presence of sulfide ions could restore the fluorescence. This approach has allowed the detection of sulfides in the range of 2–10 μM. On the other hand, Ag(I) ions that were found to have no effect on the fluorescence of CDs have facilitated the quenching by sulfides due to the formation of Ag₂S particles, enabling detection within linear dynamic range of 1–100 μM.

9.4.2.2 Reactive Oxygen/Nitrogen Species (ROS/RNS)

ROS is an oxygen‐containing chemical species that is highly reactive, which typically includes peroxides, superoxide, hydroxyl radicals, and singlet oxygen. Similarly, reactive nitrogen species (RNS) is a nitrogen‐containing species that is chemically reactive – namely, peroxynitrite and nitric oxide (NO). ROS and RNS could jointly or individually cause damage to cells, resulting in cellular oxidative or nitrosative stress, and are often associated with blood homeostasis, inflammatory responses during cardiovascular events, or cell apoptosis related to cancer killing [262]. Due to their important roles in biological system, it is essential to monitor the amount of ROS/RNS. Under this motivation, CDs have also been explored for detection of these reactive species. For instance, S and N co‐doped CDs has been developed to detect NO, a class of radical that works as physiological messenger and effector in mammalian cells [263]. The sensing system has achieved a linear response range from 1 to 25 μM and an LOD of 0.3 μM. Another group has also reported sensors for NO using CDs, employing a bimodal sensing system based the colorimetric and fluorimetric observation as a result of reaction between NO and aminoguanidine residues in CDs [264]. The reaction between NO and CDs produced azo residues that give rise to the observable yellow‐red color changes. In the aqueous phase, absorbance increment at 490 nm and fluorescence quenching at 430 nm were observed with the increasing concentration of NO. To further explore the applications in cellular detection, NO generation was tested on treated and untreated mouse leukemic monocyte macrophage cell line RAW 264.7. NO was produced by the addition of lipopolysaccharide (LPS). The CDs have shown fluorescence quenching as an indication of NO production, while not observed by the control cells that were not treated with LPS. In another study, fluorescence detection of both peroxynitrite (ONOO⁻ ) and NO was demonstrated using ethylenediamine doped CDs [265]. The sensing of ONOO⁻ was optimized at pH 10 while the sensitivity of NO was highest under acidic condition at pH 4. The same research team has synthesized tryptophan‐doped CDs for ONOO⁻ sensing at pH 7.4 and reported linear dynamic range within 5–25 μM and LOD of 1.5 μM [266].

CDs have been reported to detect superoxide anion (O₂ ^˙− ) with very minimal interferences (<5%) from other coexisting ROS and small molecules such as amino acids and metal cations [267]. This system has combined CDs with hydroethidine (HE) and it was observed to give an emission at 525 nm upon excitation at 488 nm. HE has served as recognition receptor due to the high binding affinity for O₂ ^˙− . Upon the introduction of O₂ ^˙− to the system, a new fluorescent peak at 610 nm was observed while peak at 525 nm has remained constant. This has allowed a ratiometric detection to be adopted for the detection of O₂ ^˙− . The LOD was evaluated to be 0.1 μM. Besides O₂ ^˙− , CDs have also been explored for the detection of hydroxide radicals (OH^˙ ) by CDs‐ascorbic acid (AA) hydrogel [268]. The hydrogel was constructed from AA derivative as the building block and then further used to encapsulate the amphiphilic CDs. When the OH^˙ was added to the hydrogels, the ROS oxidized the AA constituents and disintegrated the hydrogel scaffold, which, in turn, quenched the strong fluorescence of CDs. This hydrogel sensor has successfully demonstrated for screening the apoptotic activity of 5‐fluorouracil, which is a chemotherapeutic drug compound.

9.4.2.3 Oligonucleotides

Oligonucleotides are short nucleic acid chains containing DNA or RNA molecules. DNA and RNA carry genetic information in biological systems and are often applied in genetic testing and gene‐related bioapplications. Mutagenic changes of DNA are often associated with cancers or other genetically affected diseases. For instance, long‐term exposure to UV has been known to cause DNA damage and can lead to skin cancer. In view of this, there is a need for early detection of DNA damages. There are studies reported that design CDs to monitor the DNA damage. This is by monitoring the FRET interaction between the fluorophore and ethidium bromide (EtBr) that has been preloaded to stain the electrophoretic gel [269]. Although EtBr has been in practice for a long time as a fluorescent tag for DNA detection, there have been some pressing concerns of the health risks caused by its carcinogenic nature and its reputation as mutagen that has high binding affinity toward DNA. The strong fluorescence emission by CDs has allowed the replacement for EtBr in gel electrophoresis [270].

Besides detecting DNA damages, CDs have been demonstrated for the identification of the segment of DNA or genes responsible for particular diseases. For instance, ssDNA coded for human immunodeficiency virus (HIV) has been studied for fluorescence detection based on CDs. Most of these biosensors makes use of FRET between CDs and other fluorescent nanoparticles such as GO/AuNPs [271] and Ag nanoclusters [272]. While most of the HIV genes detections are studied using ssDNA, Liang et al. reported a CDs‐based biosensor for sensing of HIV‐1 dsDNA [273]. In the presence of the dsDNA, the fluorescence of cadmium tellurium (CdTe) QDs pretagged with mitoxantrone (MTX), which could intercalate with DNA was quenched while the fluorescence of CDs remained constant. This has served as a good reference point for ratiometric analysis of fluorescence intensities between the two nanoparticles. Based on this system, the signal‐to‐noise ratio recorded was showing a correlation with the targeted dsDNA and achieved excellent recoveries ranged between 98% and 110% when tested on spiked serum samples of healthy volunteers.

miRNAs are a group of small, noncoding, endogenous, regulatory single‐stranded RNAs that are involved in gene expression regulation. As such, they are often selected as biomarkers for cardiovascular diseases, cancers, and viral infections. Detection of a miRNA, miR9‐1 associated with breast cancer has been reported by observation of fluorescence quenching of CDs [165]. The sensing was designed by adding FAM‐labeled ssDNA to CDs, which no or low fluorescence was observed due to FRET mechanism. However, the emission can be recovered when miR9‐1 is bound to the FAM‐labeled ssDNA forming a duplex helix and being released from the surface of CDs. Furthermore, a DNA‐labeled CDs sensing system based on ECL transduction has been demonstrated for miRNA‐21 with linear dynamic range between 10 aM and 10⁴  fM [166].

9.4.2.4 Small Molecules/Pharmaceutical Drugs/Natural Compounds

Small molecules are organic compounds with typical molecular weight below 900 Da. Some examples include primary and secondary metabolites that assist in regulating the normal biological processes such as amino acids, vitamins, nucleotides, etc. Monitoring of these small molecules in the biological system could be useful in understanding more of a particular disease or their metabolic pathways in finding a cure for the disease. On the other hand, monitoring of pharmaceutical drugs is becoming more important especially in cases where the misuse or overuse of drugs. For instance, the misuse of antibiotic has led to more serious problems such as the birth of superbugs, which may threaten the survival of humankind.

Recently, there have been more reports on utilizing CDs for the detection small molecules. One of the latest is by Fong et al., which demonstrated a simple CDs “turn‐on” fluorescence system for the sensing of vitamin C, also known as AA [274]. In designing the system, the fluorescence signal of the isolated CDs was first effectively quenched by using Fe(III) ions. Once this is achieved, the addition of AA into the system will restore back the fluorescence due to the high affinity of AA toward the Fe(III) ions, where the degree of enhancement in terms of the intensity can be correlated to the amount of AA added. Based on the similar concept, Lin's group has demonstrated a “turn‐on” sensing of AA in rat brain following brain ischemia [275]. The CDs synthesized have been premodified with hexagonal cobalt oxyhydroxide (CoOOH) nanoflakes. During the FRET process, the fluorescence of CDs (λ_em – 450 nm) was turned off by CoOOH nanoflakes when being excited at 380 nm and eventually turned back on when the recognizing unit of CoOOH selectively binds to the enediol group of AA. Cerebral AA in brain microdialysate was determined by implanting the microdialysis probe in the brain cortex region through the guide cannula and was perfused with artificial cerebrospinal fluid. Upon equilibration via microinjection pump, every 30 μL of brain dialysate was collected for AA sensing. This method has enabled in vivo AA sensing from the cortex of rat brain under different physiological conditions including calm and ischemia. Another group has also reported AA sensing in with LOD as low as 5 nM and further quantifying AA in human serum samples and rat brain microdialysate samples [276].

Cholesterol checking is critical, as a high level could result in health complications such as cardiovascular diseases, obesity, and hypertension. Cholesterol can be detected using CDs without involving of enzyme cholesterol oxidase that is commonly adopted to identify cholesterol in existing bioassay. The mechanism is based on the functionalization of β‐cyclodextrin on CDs, in which the core of the β‐cyclodextrin can entrap various hydrophobic molecules with different affinities [37]. In this system, p‐nitrophenol was first bound to the CDs sensing probe, causing quenching of the fluorescence. The fluorescence was then recovered by cholesterol, which has higher affinity toward the β‐cyclodextrin. Another enzyme‐free biosensor has been reported on using Au/CDs nanoconjugates for a dual function (colorimetric and fluorimetric) detection scheme [277]. The Au particles in this system took part in the colorimetric responses and fluorescence quenching in the presence of cholesterol within concentration of 0.208–2.08 mM. When cholesterol is present, a visible color change can be observed together with fluorescence quenching of CDs. The system has very minimal interference from other coexisting entities and recorded a LOD of 2.5 μM. Similarly, a hemoglobin (Hb) conjugated‐CDs (CDs/Hb) complex was constructed for cholesterol sensing [278]. The detection of cholesterol was performed as the CDs break free from the CDs/Hb complex when Hb favors the hydrophobic interaction with cholesterol. A LOD of 56 μM with response time of less than five minutes in human plasma samples has been reported.

Nervous system−related diseases such as Parkinson's disease has been a major concern especially the average age of the world population has grown older. In view of this, DA serves as an important biomarker for such diseases. For the past decades, many works have been invested in search of an accurate sensing system for DA and CDs have also been studied for such purpose. Most of the reported systems for DA determination have shown good correlation between the changes of fluorescence of CDs with the concentration of DA. More recently, Fang et al. have reported a carbon‐fiber microelectrode that has been co‐deposited with CDs and GO nanosheets for selective sensing of DA [279]. In this system, CDs serve as the physical barrier to avoid the complete restacking of GO nanosheets while in the meantime increasing the surface area of accessible sheet surface for DA interaction. This approach has been reported to improve the performance of the sensor. Besides, the group claimed that CDs themselves could interact with DA molecules and increase charge storage capability of the electrodes for signal amplification. Besides using CDs as part of sensing transducer, CDs can be used as matrix for matrix‐assisted laser desorption‐ionization mass spectroscopy (MALDI MS) as demonstrated for screening of three compounds: serotonin (Sr), glutamic acid (Glu), and DA that serve as biomarkers for neurological disorder [280]. The strong absorbance in the UV range (220–350 nm) of CDs was exploited to facilitate the energy transfer from Nc laser (337 nm) of MALDI MS to the three targeted analytes. Due to the significantly reduced background noises, the LOD of Sr, Glu, and DA could be achieved at values as low as 3, 5, and 8 nM, respectively.

Most pharmaceutical drugs or natural compounds are categorized under small molecules. Quantification of such organic compounds known with medicinal benefits is essential for clinical analysis. As such, a substantial amount of effort has been invested to develop biosensors for monitoring these compounds, especially in vivo condition. CDs‐based sensing systems have been explored for this and have successfully demonstrated detection of different drugs such as plant‐derived sinapine [281], melamine [217, 282], sophoridine of traditional medicine [156], isotretinoin or vitamin A [283], allopurinol as uric acid reducing drug [284], patulin as an antibiotic (which was then found to be toxic [285]), amoxicillin as a common antibiotic [286], cisplatin as the anticancer drug [287], and neurotoxins such as tetrodotoxin [288] and hydrazine [289].

9.4.2.5 Proteins

Immunosorbent assay is a widely adopted technique in biochemistry to detect biomolecules based on antibody–antigen interactions. Some efforts have been taken to incorporate CDs into the working principle of this assay to obtain a biosensor that can give direct quantification signal. This is such as the development of a CDs‐linked immunosorbent assay for the detection of human AFP [290]. In this sensing probe, the captured anti‐AFP (Ab₁) was first immobilized onto polystyrene well plates in the presence of BSA as the inhibitor for the unsaturated binding sites. Following that, a CDs‐labeled anti‐AFP pair (Ab₂) was added to the well to form sandwich immunocomplexes with the bound AFP. The concentration of AFP antigen could then be determined based on the direct correlation with the fluorescence intensity measured. Other examples include CD‐labeled antibodies (IgG‐CDs) for in situ visualization of glyphosate distribution in plant tissues as well as detection of glyphosate in river water, tea, and soil samples [291].

There have been innovative explorations with CDs as biocatalysts in immunosorbent assay for the replacement of enzymes in the existing approach. Studies have shown that CDs possess superior peroxidase activity as a result of Fe doping, which mimics the nature of ferriporphyrin [292]. As such, the Fe‐doped CDs (above 70%) were further proven to exhibit excellent catalytic activity of above 70% when bound to antibody, in comparison with the natural horseradish peroxidase (HRP) at below 10%. The catalytic activity of these modified CDs was observed by the color change upon oxidation of 3,3,5,5‐tetramethylbenzidine (TMB) in the presence of H₂O₂. Besides the high catalytic performance, these CDs displayed superiority against the HRP as they are able to catalyze under wider range of pH and temperature conditions. In this classical enzyme‐linked immunosorbent assay (ELISA) kit, CDs labeled with antibody have been employed as biocatalysts to replace HRP for efficient and specific quantification of CEA. In another report, a sandwiched ECL immunosensor for CEA has also been reported with LOD of 1.67 pg ml⁻¹ with dynamic linear range of 5– pgml⁻¹ to 500 ng ml⁻¹ [293]. For this biosensor, primary antibody, Ab₁ was conjugated to the polydopamine and AuNPs nanocomposites while the secondary antibody, Ab₂ was linked to CDs that has been premodified with poly(ethylenimine) functionalized GO. This immunosensor has been applied for real samples analysis in human serum with relative errors below 10% when validated with ELISA kit.

Thrombin is a trypsin‐like serine protease that is involved in converting soluble fibrinogen into insoluble strands of fibrin and catalyzing a variety of other coagulation‐related activities [294]. Due to their pro‐inflammatory character, they are believed to be involved in the onset and progression of atherosclerosis and therefore always being chosen as a diagnosis tool for cardiovascular diseases. Most detections make use of thrombin aptamer to improve the targeting efficiency of the biosensor. A number of thrombin sensors have been demonstrated based on PL phenomena such as color change [295], fluorescence [170], phosphorescence energy transfer [296], and FRET [168]. During a coagulation reaction, the concentration of thrombin in the biological system can range from below 1 to 500 nM [297]. These CDs‐based biosensors for thrombin have mostly reported LOD in the nanomolar concentrations and matched with the practical range for the coagulation process in blood. Hb in red blood cells is involved in transporting oxygen to maintain aerobic physiological activities in human body. Low concentration of Hb will lead to anemia and can be fatal. Therefore, it is important to monitor the concentrations of Hb for clinical diagnosis, especially detecting hemolysis. Huang et al. developed a simple mix and detect fluorescence sensing of Hb by observing the fluorescence changes in the blue region [298]. When added with Hb sample, the initial CDs emission at 430 nm was quenched upon excitation at 340 nm. The quenching was based on static quenching mechanism where ground‐state complex between CDs and Hb was formed. This was further proven by the structural changes in Hb upon interaction with CDs as the α‐helix content of Hb decreased and the secondary structure of Hb rearranged. With low LOD of 0.12 nM, this sensing probe was tested for human urine and blood samples and showed high recovery rates of 95–100%. Besides the direct interaction between Hb and CDs, the presence of a strong oxidizing agent such as H₂O₂ can facilitate the detection of Hb [299]. ROS such as OH^˙ is generated during the reaction between Hb and H₂O₂, which then will quench the fluorescence of CDs. The quenching by OH^˙ has a direct correlation with the quantity of Hb in the linear range of 1–100 nM and LOD of 0.4 nM.

9.4.2.6 Enzyme Activities and Inhibitor Screening

Enzyme activities or kinetics and enzyme inhibition have been important processes employed in clinical diagnosis to derive the cause of a disease. The information harvested will provide insight on the role of the enzyme in metabolism. A drug can later be designed to control or inhibit this enzyme activity to drive to whole metabolism process toward an intended outcome. Many enzymes have a close relationship with certain diseases, and eventually most of them can serve as biomarkers for diseases such as breast and prostate cancer, diabetes, and bone diseases [300]. CDs have been exploited for their applications in enzyme assays. One of the most commonly tested enzymes in the clinical practices is the alkaline phosphatase (ALP), which has also been detected using CDs as the bioanalysis probe. The main aim is to be able to detect ALP in the concentration range of 40–190 U l⁻¹ in human serum [301]. Feng et al. have reported a recyclable biosensor for ALP based on aggregation and disaggregation of CDs via competitive approach [302]. The sensing mechanism follows two steps outlined as followed: (i) CDs tend to aggregate as triggered by cerium ions that can strongly coordinate with the carboxyl groups on the surface of CD and lead to fluorescence quenching; (ii) ALP catalyzes the hydrolysis of ATP to phosphate ions, which has higher affinity to cerium ions than the carboxyl groups on CDs. This leads to the disaggregation of the initial complex and thus will recover the fluorescence of the CDs. The study showed that there is a linear correlation between the degrees of recovery of fluorescence with the ALP concentrations within the range of 4.6–383 U l⁻¹ . Similarly, Cu(II) ions could turn off the fluorescence of CDs, which was then restored by phosphate ions when ALP catalyzed the transformation reaction from pyrophosphate ions to phosphate ions [303].

The fluorescence “off−on” sensing mechanism making use of the catalytic properties of the enzyme has also been demonstrated for detection of other enzymatic activities such as ALP [304–306], acid phosphatase [307], and α‐glucosidase [308]. The enzyme inhibitor screening can be carried as a continuation of the aforementioned two‐step system. This has been applied for acetylcholinesterase (AChE) and its inhibitor screening [309]. Cu(II) ions were first added to aggregate and quench fluorescence of CDs, followed by AChE catalyzing the hydrolysis of acetylthiocholine into thiocholine, inducing the fluorescence recovery. When the inhibitor tacrine is present, AChE loses its catalytic ability to hydrolyze acetylthiocholine and therefore fluorescence of CDs remained quenched instead of being restored due to the absence of thiocholine. This system has demonstrated detection of AChE activity as low as 4.25 U l⁻¹ within linear range of 14.2–121.8 U l⁻¹ . The FRET between CDs and hexagonal cobalt oxyhydroxide nanoflakes has successfully established ratiometric fluorescence analysis for α‐glucosidase inhibitor screening [310]. Within close proximity, the CDs transfer energy to the nanoflakes, which results in fluorescence turn “off.” Subsequently, L‐ascorbic acid‐2‐O‐α‐D‐glucopyranosyl (AAG), which has no reducing power, would be hydrolyzed by the enzyme to release AA that could rapidly reduce the nanoflakes to Co(II) ions. As a result, the FRET was halted and fluorescence of CDs was restored. Acarbose, which was one of the inhibitors for the enzyme, was studied as model and a series of natural compounds have been screened for their inhibitory effects on the enzyme activity as part of anti‐diabetic drug discovery. The tumor‐invasive biomarker, β‐glucuronidase (GLU), was also detected in a similar manner but via the inner‐filter effect of GLU's catalytic product, p‐nitrophenol, which absorbed the fluorescence of CDs due to the complementary spectral overlap between p‐nitrophenol and CDs [311]. This CDs‐based enzyme assay has been applied for tumor screening and the inhibitors screening from natural products that could be potential anti‐tumor drug. Tyrosinase (TYR) activity was monitored using DA functionalized CDs (DA‐CDs), and arbutin was selected as an inhibitor of TYR for screening [312]. It can sensitively monitor the intracellular TYR level in melanoma cells and intracellular pH changes in living cells. Due to the spectral overlap between CDs and neutral red (NR), these two fluorophores were incorporated in a FRET‐sensing system for hyaluronidase (HAase) activity [313]. By a ratiometric analysis of fluorescence emissions of CDs at 525 nm and NR at 630 nm, this system has exhibited LOD as low as 0.05 U ml⁻¹ in aqueous form. Another FRET system of CDs and naphthalimide has been developed for detection of thioredoxin reductase (TrxR), which is commonly observed in most cancers [314]. From the linear correlation between the ratiometric intensities (I₄₅₀/I₅₂₅) and TrxR concentrations, LOD of 0.72 nM was obtained.

9.4.2.7 pH

pH plays a huge role in biological systems especially when the protonation or deprotonation of a biomolecule would lead to a trigger or halt a biological process. pH changes can be due to the presence of foreign species in the body such as drugs during the course of treatment or infection of diseases. Therefore, monitoring of intracellular pH plays an important role besides detecting biomarkers as part of disease screening. This fact holds true especially for the cases of cancerous diseases where a change in the pH is an indication of the presence of tumors [315]. Intracellular pH can be monitored using CDs as a strong emitting fluorophore. The change in pH can lead to variations in the fluorescence emission of CDs, whether by direct monitoring of fluorescence intensity changes or by performing ratiometric measurement of the fluorescence emissions. The surface chemistry of CDs has made them useful for pH sensing due to the presence of reactive oxygenated species such as hydroxyl and carboxyl groups that are sensitive to the uptake and release of hydrogen ions. For instance, the reversible fluorescence changes in response to pH changes exhibited by CDs has been demonstrated and suggested to be due to the presence of acidic/basic sites on the surface [316, 317]. Ratiometric measurement of fluorescence for pH sensing has been a popular practice since the fluorescence changes can be maximally correlated to the pH variations. This sensing technique is applicable to the fluorophores displaying dual fluorescence emissions peaks, where the emissions can each respond specifically to the change of pH. For instance, a two‐photon fluorescent nanoprobe based on CDs has been reported for physiological pH sensing in the range of 6.0–8.5 in living cells and tissues [318]. As shown in Figure 9.5, the nanoprobe consisted of CDs that have been surface bound by a 4ʹ‐(aminomethylphenyl)‐2,2ʹ:6ʹ,2ʺ‐terpyridine (AE‐TPY) molecule, which acted as a selective receptor to detect the change in the concentration of H⁺ . In a surge of H⁺ , a strong luminescence was observed upon two‐photon NIR excitation at wavelength of 800 nm. This observation could be reversed when there was a rise of pH, resulting in weak emission at 498 nm. A similar ratiometric sensor for pH has also been reported based on dual fluorescence emissions between CDs and other fluorophores such as fluorescein isothiocyanate (FITC) [319, 320].

9.4.2.8 Temperature

Temperature is one of the most important parameters for a living organism, as it governs a wide variety of biological reactions from gene expression to cellular metabolism. From a clinical viewpoint, pathological cells are present at slightly higher temperatures than that of normal healthy cells due to their enhanced metabolic activity [321]. Therefore, monitoring cellular temperatures could be useful for better understanding diseases and for developing novel diagnosis and treatment options. In recent years, the concept of a nano‐thermometer has been introduced to measure cellular temperature. Fluorescent nanoparticles can be adopted to map the temperature in living cells since their optical properties such as emission band shape, peak position, intensity, or lifetimes are strongly affected by temperature [322, 323]. The fluorescence of CDs is responsive to the changes of temperature and can be employed to sense change of temperature in liquid from 15 to 60 °C [67]. More recently, CDs have been studied for application as nanoscale thermometry in living cells. Zhang's group has looked into dual‐emission fluorescent responses of a nanohybrid consisting of CDs and gold nanoclusters with single excitation, which was well facilitated within the physiological temperatures of 25–45 °C [324]. The gold nanoclusters were first functionalized with a targeting moiety, GSH to improve the targeting efficiency. The system was then conjugated with amino‐functionalized CDs to form the nanohybrids. The ratiometric analysis of fluorescence dual‐emission at 430 and 605 nm upon single excitation at 367 nm was shown to be more efficient and accurate compared to that of single fluorescence emission. When tested in human embryonic kidney 293T cells, the blue emission from CDs was observed to remain unchanged and served as a reference point, while the red emission corresponding with the gold nanoclusters decreased when the incubating temperature was elevated to 45 °C. Similarly, ratiometric temperature sensing has been demonstrated in vitro when CDs have been coupled with a temperature‐sensitive dye, rhodamine B [325]. On the other hand, Zhang's team has also developed a single‐emission CDs‐based thermometer by observation of the fluorescence “off‐on” using microscopy imaging [326]. When the MC3T3‐E1 cells were incubated with GSH at room temperature, the red fluorescence (λ_em – 615 nm) of CDs was found to decrease but was slowly restored when the temperature was increased to 30 °C and further intensified when the temperature was raised to 40 °C as a result of dissociation of GSH from CDs. This observation has proven that the CDs‐based sensing system could readily enter the cytoplasm of the cell and detect temperature changes in the subcellular level. Besides, since PL lifetimes of fluorophores are dependent on temperature, it could also be applied as nanothermometers in cells, which was demonstrated by Kalytchuk et al. between 15 and 45 °C in human cervical cancer HeLa cells [327]. The PL lifetimes of the synthesized CDs decreased monotonically with the increasing temperatures, without being affected by the pH, concentration of CDs, and the environmental ionic strengths.

9.4.3 Solid‐State Sensing for Point‐of‐Care Diagnostic Kits

Most medical diagnoses are carried out in pathology lab setting, which means that special laboratory knowledge, facilities, and skills are required for the screening process. Besides, longer processing and waiting times are needed before the medical team and patients can find out the cause of a sickness. Point‐of‐care diagnosis has been proposed as a feasible solution to improve the healthcare system. In addition, microfluidics have been developed as miniaturized portable analytical devices for instant test outcome with high accuracy. Biosensors are being studied and improved as part of the process to translate sensory systems into solid platforms for the miniature devices.

There are reported efforts on improving different problems faced while developing solid‐state sensors. The main issue in transferring the sensing probe from liquid phase onto solid platform is the leaching of the fluorophore that can cause tremendous loss in sensing signals. Fabrication techniques become an important factor with the intention to introduce more fluorophores onto the sensor matrix without hindering the interaction with the sensing analytes. Besides layer‐by‐layer immobilization methods as demonstrated by Gonçalves et al. for Hg(II) optical fiber sensor [246], another group has established a fabrication method using colloidal photonic crystals (CPhCs) as a solid matrix without compromising the sensitivity toward the targeted analytes [328]. CPhCs films can act as a Bragg mirror and enhance the fluorescence or incident light with the wavelength located into their photonic band gap, thus improving the sensitivity of the sensor [329]. They can also concurrently modulate electromagnetic waves and exhibit a photonic band gap that is similar to the effect of a semiconductor band gap. Li et al. adopted CPhC comprising three layers of monolithic CPhCs for their solid‐state sensor for Hg(II) [328]. The double hetero‐structure of CPhC was formed by successive deposition of CPhCs in a specific sequence from bottom to top. The Hg(II) targeting CDs were immobilized on the surface of polystyrene spheres as the middle layer in between two monolithic CPhC layers with a periodicity overlapping the excitation wavelength. The excitation efficiency was enhanced as the layers were able to reflect any excitation light that propagate in the CDs layer. Due to the fluorescence enhancement effect from the CPhC structure, the LOD of the solid‐state sensor was obtained as low as 91 pM for Hg(II).

Paper‐based microdevices have attracted a lot of attention as low‐cost sensing platforms since the discovery of microfluidic paper‐based analytical devices (μPADs) [330, 331]. The advancement in the screen‐printing technology has aided the research focus on μPADs in the area of fabrication of designed sensing systems. Wax screen printing is an inexpensive method that has been adopted for fabrication of μPADs, and it involves two fundamental steps: (i) printing patterns of wax on the surface of the paper and (ii) melting the wax onto the paper with heat to form the hydrophobic barrier [332]. A few studies have reported on screen‐printed recyclable biosensors based on ECL transduction for in situ screening for IgG antigen [162], MCF‐7 cancer cells [177], and K562 leukemic cells [333]. Wu et al. coupled RCA method with oligonucleotide functionalized CDs with sensitive ECl signal, within a μPAD for detection of larger target analytes such as proteins [162]. In this system, a three‐dimensional (3D) macroporous Au paper electrode has been employed as the working electrode to capture antibodies. Meanwhile, the RCA is an isothermal DNA amplification strategy that produces tandem‐repeat sequences to serve as template for periodic assembly of CDs, in which each protein recognition event is presented to a few CDs tags for ECL signal readout. The sensing system incorporating the CDs and antibody conjugated AuNPs electrodes were printed onto the paper origami device as depicted in Figure 9.6. The incorporation of RCA has significantly enhanced the sensitivity of the device for monitoring the concentration of IgG as the ECL intensity of CDs increases alongside the cascade DNA nanotags. This device has demonstrated linear dynamic response to IgG in the range of 1.0 fM to 25 pM with LOD of 0.15 fM. Another paper device based on ECL responses of CDs has been reported for observation of MCF‐7 cancer cells and the electrode was then further applied as in situ screening for anti‐cancer drugs [177]. This extension of application was achieved by monitoring the number of apoptotic cancer cells after in situ 3D culturing of the captured cancer cells on the electrode to a drug containing medium. Anticancer drugs such as cisplatin, paclitaxel, fluorouracil have been selected as study model. The relative respiratory activity of captured MCF‐7 cells was investigated to test the chemosensitivity of the μPAD. The result obtained was found in agreement with the validation of drugs induced apoptosis via fluorescence imaging technique. Besides ECL being the commonly applied transduction for paper‐based biosensors, CL‐based paper devices have also been reported for DNA sensing [334]. Similar to other CDs‐based CL sensing mechanisms applied in solution form, this μPAD employed CDs‐dotted nanoporous Au as a signal‐amplification label. N,N‐disuccinimidyl carbonate (DSC) was in charge of capturing DNA by covalently immobilizing on the paper device. In the presence of an oxidizing agent, the CL reaction was induced and produced CL signals corresponding to the targeted DNA and successfully performed at an LOD as low as 8.56 × 10⁻¹⁹  M.

9.4.4 Bioimaging/Real‐Time Monitoring

Bioimaging is referred to as noninvasive method to visualize biological processes in real time. It serves as an important part of clinical diagnosis. Most CDs‐based sensing systems described earlier are based on ex vivo testing, meaning the detection is applied outside of a body using biological samples such as urine and blood serum. However, very few studies have applied in vivo sensing or tracking of biomolecules or other potential analytes directly in body. Therefore, other techniques such as bioimaging are required to work alongside with bioanalysis to enable real‐time monitoring of intracellular processes. Many studies have reported the use of CDs as fluorescent labels in bioimaging due to their excellent optical stability and biocompatibility. In recent years, a few groups have studied CDs sensing in living cells via cellular imaging methods. Most of these studies focus on qualitative detection of heavy metals in living cells, observing the quenching of CDs in the presence of metal ions such as Hg(II) [335] and Fe(III) [336]. A fluorescence turn “off−on” intracellular sensing of Hg(II) and GSH has been demonstrated in HeLa cells based on observation of CDs fluorescence [337]. Most CDs are blue emitting, which could limit their practical in vivo applications due to the universal blue auto‐fluorescence of biological matrices. In addition, UV utilized for excitation can cause photodamage to the biological tissues [338]. Alternatively, CDs emitting at longer wavelength would be preferable, as they can penetrate into deeper tissues. Motivated by these concerns, there has been research into the viability of red‐emitting CDs (λ_em – 600 nm) synthesized via hydrothermal using a mixture of two chemical precursors: 2,5‐diaminobenzenesulfonic acid and 4‐aminophenylboronic acid hydrochloride [143]. They have performed cellular sensing of Fe(III) ions by incubating cervical cancer HeLa cells with CDs and Fe(III) ions. As shown in Figure 9.7, the red fluorescence of CDs was quenched by Fe(III) ions, which served as a qualitative indication of the presence of Fe(III) ions. Fluorescence ratio imaging is an effective approach to quantitative detection of biological molecules in living cells. This is made possible for fluorophores that emit at two different wavelengths upon single excitation. Although excitation‐dependent CDs can provide a series of different fluorescence emissions upon varied excitation wavelengths, it is still a challenge to harness single excitation/dual peak emissions from CDs itself. One possible way is via nanocomposite formation where CDs are coupled with other fluorescing nanoparticles. Zhang et al. grew gold nanoclusters on CDs that served as the skeleton by mixing molecular precursors for both individual nanoparticles in one synthesis [339]. With single excitation at 390 nm, two emissions at 470 (blue) and 565 nm (red) were observed. The fluorescence ratiometric imaging was performed by adding 30 μM of Fe(III) to the normal rat osteoblast MC3T3‐E1 cells. The results showed that no detectable change of blue fluorescence was observed but there was a significant decrease in the red emission. The fluorescence ratio at the two wavelengths has depicted a significant increment after addition of Fe(III).

Other than metal ions in cells, the monitoring on the activation of anticancer prodrug such as cisplatin in real time for living cells using CDs is also possible using ratiometric method. It is based on observing the changes in the multi‐fluorescence peaks of CDs during the activation. Zhao et al. established such a sensing system based on the FRET pairing between CDs and Pt(IV) [287]. In this system, CDs also acted as nanocarriers for the drug and FRET donor while for cisplatin (IV), Pt(IV) was selected as the model drug and the linker to load Dabsyl quencher unit on the surface of CDs. The CDs were capable of emitting at blue (460 nm), green (535 nm), and red (610 nm) fluorescence upon excitation at 360, 480, and 545 nm, respectively. The CDs‐based fluorescent biosensor was tested on the human ovarian carcinoma cell line, A2780 cells under reductive conditions. When the sensor was constructed by the sequence of CDs‐Pt(IV)‐Dabsyl, the blue fluorescence of CDs was effectively quenched by the Dabsyl unit as the FRET acceptor while red and green fluorescence remained unaffected. In the presence of the reducing agent AA, the prodrug Pt(IV) was successfully reduced to Pt(II) species, which resulted in the prodrug activation and at the same time breaking of the bond from the sensor. Consequently, the FRET process became ineffective as the Dabsyl unit was no longer in close proximity with CDs. Therefore, the blue fluorescence of CDs was restored over time, but the red and green fluorescence remained constant throughout and can be used as effective internal reference. The fluorescence ratios, I₄₆₀/I₆₁₀ and I₄₆₀/I₅₃₅ increased gradually and was indicative of the real‐time reduction of Pt(IV) to cytotoxic Pt(II) species due to the activation of the prodrug.

9.5 Future Perspectives

9.6 Conclusions

In summary, CDs have achieved a lot in terms of bioanalytical applications over the past decade since its serendipitous discovery in 2004. As outlined in this chapter, the contributions of knowledge on CDs have been tremendous and vast; however, there are still various areas to be improved and explored. The unique fluorescence properties, excellent biocompatibility, and stability and ease of production from a variety of carbon‐rich sources have been the main driving force for the advancement of CDs. This new type of carbon nanomaterial has a lot of potential as a great alternative to QDs in many areas, especially the biological analysis field. In view of that, more in‐depth research on development of large‐scale synthesis of good‐quality CDs with high QY suitable for bioanalytical applications should be explored for commercialization purposes. On top of that, research and development of CDs‐based biosensors and diagnostics should be further explored and implemented to benefit the medical field.

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