Carbonaceous Nanoparticle Air Pollution: Toxicity and Detection in Biological Samples

Among the different air pollutants, particulate matter (PM) is of great concern due to its abundant presence in the atmosphere, which results in adverse effects on the environment and human health. The different components of PM can be classified based on their physicochemical properties. Carbonaceous particles (CPs) constitute a major fraction of ultrafine PM and have the most harmful effects. Herein, we present a detailed overview of the main components of CPs, e.g., carbon black (CB), black carbon (BC), and brown carbon (BrC), from natural and anthropogenic sources. The emission sources and the adverse effects of CPs on the environment and human health are discussed. Particularly, we provide a detailed overview of the reported toxic effects of CPs in the human body, such as respiratory effects, cardiovascular effects, neurodegenerative effects, carcinogenic effects, etc. In addition, we also discuss the challenges faced by and limitations of the available analytical techniques for the qualitative and quantitative detection of CPs in atmospheric and biological samples. Considering the heterogeneous nature of CPs and biological samples, a detailed overview of different analytical techniques for the detection of CPs in (real-exposure) biological samples is also provided. This review provides useful insights into the classification, toxicity, and detection of CPs in biological samples.


Introduction
Air pollution has been a major concern for many years due to its harmful effects on the environment and public health [1]. Worldwide, outdoor air pollution is estimated to be the cause of approximately four million premature deaths annually, of which about half a million occur in the European Union (EU). An additional four million premature deaths have been attributed to household air pollution in the World Health Organization's (WHO) report [2][3][4]. Air pollution is a complex mixture of gaseous components and particulates originating mainly from human activities and natural processes [5]. The composition of the pollutants depends on various factors such as their sources, emission rate, and wind conditions [5]. The major gaseous pollutants are nitrogen oxide (NO 2 ), ozone (O 3 ), and carbon monoxide (CO), whereas common particulates in the atmosphere include various particles of natural and anthropogenic origins [6,7]. Particulate matter (PM), or simply particulates, are extremely small particles and liquid droplets suspended in the atmosphere containing a mixture of different salts, organic chemicals, metals, dust particles, and CPs [8,9]. PM pollutants can be classified based on their sizes into coarse PM (PM 10 ; size < 10 µm and >2.5 µm), fine PM (PM 2.5 ; size < 2.5 µm), and ultrafine PM (PM 0.1 ; size < 100 nm) (Figure 1a). The particulates in PM can either be directly emitted into the atmosphere, i.e., primary PM, or can be formed in the atmosphere from gaseous precursors-the secondary PM [9,10]. Different emission sources from anthropogenic activities and natural processes contribute to the release of the primary components of PM Despite the considerable improvements in the atmospheric concentrations of PM recently, the environmental and health effects of PM are still manifold [9,21]. The environmental effects of PM depend on the chemical composition and include global warming, damage to crops and forests, and contribution to acidic rain [22]. The impact of PM on public health is very complex due to their variability in size, morphology, and composition [23]. A few well-known effects include, for example, the worsening of many respiratory diseases such as asthma and chronic obstructive pulmonary disease (COPD) caused by short-term exposure to PM 10 [24], while exposure to PM 2.5 has been associated with premature death through existing pulmonary diseases and reduced lung function in children [25]. In addition, exposure to PM 2.5 results in cardiovascular morbidity and mortality through the deterioration of cardiac function [26]. Epidemiological studies indicate that the carcinogenic effects of PM 2.5 result in increased mortality, for example, due to lung cancer [27,28]. PM 0.1 is present in the atmosphere in large concentrations and generally enters the body through inhalation and can translocate to different organs through the bloodstream [29]. PM 0.1 causes pulmonary inflammation and can be retained in the lungs for a long time. In addition, it is also transported through the olfactory nerve into the brain and hence results in autonomic dysfunction [30,31]. PM 0.1 exposure is also associated with diabetes, cancer, and low birth weight [32,33].
Since PM is a complex mixture of components with different chemistry, variable sizes, and originating from different sources, it is a complicated task to link the adverse health effects of PM to a specific constituent. Specifically, the fine and ultrafine constituents of PM are more toxic compared to others [23,34,35]. CPs constitute a major fraction of PM 2.5 and PM 0.1 , and their abundant presence in the atmosphere makes them among the most harmful on its manufacturing process, CB can be divided into acetylene black, channel black, furnace black, and thermal black [63]. Approximately ten million tons of CB are produced every year globally, making it one of the top 50 industrially manufactured chemicals [42,64]. Almost 90% of commercially produced CB is used in rubber applications, namely, tirerelated automotive uses and other automotive and non-automotive uses of rubber [64,65]. The remaining 10% is used for various applications, e.g., as a black pigment or a conducting agent in plastics, inks, paints, and even in food (E153) [64][65][66][67]. The size of primary CB particles ranges from 15 to 300 nm [42]. There is a huge risk of occupational exposure to CB during different stages of its manufacturing process, production, collection, and handling [42,65]. The risk of occupational exposure to CB is also possible in downstream CB user industries such as the manufacture of rubber, paint, and ink; printing; in the plastics, paper, and ceramics industries; and in carbon electrode production [42,68].

Black Carbon (BC)
BC is a collective term used for different carbonaceous substances ranging from partly charred plant residues to highly graphitized soot particles resulting from incomplete combustion [69]. BC particles are a heterogeneous mixture of different species from various sources consisting of large aromatics and few functional groups [70]. Compared to CB, there is no universally defined chemical definition of BC. However, in a Report to Congress on Black Carbon, the United States (US) Environmental Protection Agency (EPA) defined BC as a "solid form of carbonaceous component of PM that absorbs solar radiation at all wavelengths" [71]. BC mostly originates from the incomplete combustion of biomass and fossil fuels linked to human activities [39,42]. Among these, diesel exhaust engines are a major contributor to the emissions of BC in the environment in the proximity of heavytraffic areas [72]. In addition, the natural sources of BC emissions are volcanic eruptions and wildfires [73]. The level of global BC emissions from both natural and anthropogenic sources is estimated to be approximately nine million tons per year [42]. The size of BC particles ranges from a few nm to a few hundred nm for atmospheric BC usually sourced from diesel exhaust processes [74]. Atmospheric exposure to BC depends on the daily activities of individuals, whereas persons associated with the transportation sector are prone to higher exposure to BC [72].

Brown Carbon (BrC)
In contrast to CB and BC, BrC is a fraction of OC that does not absorb all visible light and is characterized by its (light) brown appearance [75]. Recently, BrC attracted the attention of the scientific community due to its strong light-absorbing abilities in the ultraviolet (UV) and visible (Vis) regions and for playing a role in climate change [76]. BrC is chemically very complex as it originates from the incomplete combustion of various materials but can also stem from non-combustion processes either through primary or secondary sources [75]. The major primary BrC sources include low-temperature biomass and coal burning through human activities, biogenic emissions from plant residue, and humic matter [77,78]. Secondary BrC is produced through atmospheric transformation reactions [79]. Compounds of high molecular weight and a light-absorbing nature can result from atmospheric multiphase transformations in the presence of gas-phase or cloud micro-droplets [80][81][82]. BrC is a dynamic mixture of organic compounds and very little information is available about the relationship between its chemical composition and emission sources. Atmospheric humic-like substances have been considered major components of BrC in addition to polycyclic aromatics and biopolymers such as lignin [39,43,61]. Atmospheric BrC can also be classified based on its soluble and insoluble fractions both in water and methanol [83,84]. The global level of BrC emissions is estimated to be approximately seven million tons [75,85]. Although the role of BrC in climate change is already known, the adverse health effects of BrC are also receiving more attention from scientists [86,87]. The characteristics of CB, BC, and BrC are provided in Table 1. Humic-like substances, PAHs, Biopolymers.
Various sizes, depending on the molecular structure.

Exposure sources
Environmental and Occupational.
Environmental: anthropogenic and natural sources.
Environmental: anthropogenic and natural sources.

Environmental/Health effects
Cytotoxicity, inflammation and oxidative stress, cardiovascular and respiratory diseases.
Global warming, cytotoxicity, inflammation and oxidative stress, cardiovascular and respiratory diseases, carrier for harmful substances.

Optical properties
Absorbs light at all wavelengths from UV-Vis-nIR.
Absorbs light at all wavelengths from UV-Vis-nIR.
Absorbs light strongly in the UV region.

Environmental Impact of CPs
Carbonaceous aerosols have a significant impact on the weather and climate through the absorption and scattering of sunlight [88]. When suspended in the atmosphere, BC contributes to the warming of the atmosphere by absorbing solar radiation at all wavelengths from UV-Vis to nIR and converting it into heat [89]. BC is the second most important chemical contributing to climate change after carbon dioxide [90]. The BC emitted through human activities and natural sources can also be deposited on the ice and snow, and the consequent decreased albedo effect results in an increased temperature and hence the melting the snow [91]. This also results in the enhanced melting of the ice caps in the arctic and other glaciated regions [92]. In addition, BC also has consequences on the health of the global ecosystem by changing rainfall patterns through its increased absorption of sunlight and by changing the number of liquid cloud droplets [93,94]. This can in turn affect both ecosystems and human living conditions, for example, by affecting agricultural productivity [93,95]. BC can also deposit on the leaves of trees, which consequently increases their temperature [96]. In the past few years, BrC has also received the attention of scientists, particularly in atmospheric research, due to its ability to strongly absorb light in the UV-blue region and hence contribute to climate change [97]. In contrast to BC, the light absorption efficiency of BrC is highly variable as it depends on the chemical composition and source [98]. BrC from the burning of biomass can cause strong circum-arctic warming and indirectly affect the ecosystem and the economy [99,100].

Health Effects of CPs
The toxicity of CPs is known to strongly depend on various factors such as their physical characteristics (size, shape, etc.) and chemical composition [101]. Environmental and occupational exposure to ambient CPs is associated with a multitude of diseases as well as a higher mortality rate [102][103][104][105]. CPs from the burning of biomass cause toxicity through the stimulation of oxidative stress, inflammation, and genotoxicity through localized or systemic toxicity [106]. Being a major fraction of ultrafine PM, very fine CPs are highly toxic due to their translocation to different organs via the bloodstream, and a large surface area can result in an enhanced inflammatory response [29]. The toxic effects of CPs on human health are discussed in detail in this section.

Pulmonary/Respiratory Effects
The lungs are the prime organs exposed to atmospheric CPs after inhalation [107]. The deposition of CPs in the lungs depends on the particle size, whereas the composition of the particles determines their toxicity [108]. CB particles consist mainly of EC whereas BC and BrC also contain organic (polyaromatic) components with chemical toxicity towards the lungs [39,42]. Exposure to CPs can cause two pulmonary diseases: COPD and asthma [109][110][111]. The pathophysiology of COPD and asthma includes inflammation of the airways, tissue remodeling and fibrosis, mucociliary dysfunctions, and structural changes [107,112]. Exposure to CPs can also cause alterations in the lungs through the interruption of different lung functions [107]. For example, the inflammation of the airways after the inhalation of CPs causes serious damage to lung function, as diesel exhaust particles (DEPs) alter the production of cytokines [113]. Furthermore, tissue remodeling and fibrosis are observed after inflammatory conditions leading to the accumulation of collagen fibers [107]. Susceptible individuals that already have COPD or asthma are more prone to CP-mediated oxidative damage. In addition, through the generation of reactive oxygen species (ROS), ultrafine CPs cause adverse effects in susceptible individuals with COPD or asthma [114]. The effects of CPs on various biochemical and molecular mediators can cause respiratory dysfunction. In vivo studies have shown the dose-dependent toxicity of synergized CB and Cd resulting in the inflammation of lungs [115]. In an inhalation study, rats were exposed to CB at 7 mg/m 3 and 50 mg/m 3 for 6 h per day and 5 days per week. After 13 weeks, inflammation and oxidative stress were observed in the bronchioalveolar lavage fluid from rats [116,117].

Cardiovascular Effects
Exposure to CPs from the atmosphere also affects cardiovascular functions, and several direct or indirect pathways explain the link between CPs and cardiovascular effects [118][119][120]. Through direct pathways, fine, and specifically ultrafine CPs, can translocate from the lungs into the bloodstream and hence target remote organs leading to potential cumulative toxicity [119]. Exposure to ultrafine particles causes cardiac depression effects leading to the deterioration of cardiac function [121]. Indirect pathways are mediated by pulmonary oxidative stress and the inflammatory response, as well as interaction with the autonomic nervous system through specific lung receptors [122]. After the deposition of particles in the lungs, they trigger an inflammation-related cascade resulting in an increased circulating level of pro-inflammatory cytokines, thereby contributing to the risks of atherosclerosis progression [26,123]. The dose-dependent exposure of rats to CB enhanced their cardiovascular risk by inducing hyperhomocysteinemia and platelet hyperactivity [124]. Furthermore, the ROSdependent mechanism also involves the pro-inflammatory pathway triggered by CPs, which is linked to vascular dysfunction, cardiac arrhythmias, and myocardial infarction [125,126]. In addition, CPs can also stimulate the autonomic nervous system, resulting in impaired autonomic balance and a hyper-activated sympathetic tone that is related to increased cardiovascular risk [127]. Exposure to CPs is also linked with myocardial infarction and accelerated cardiovascular changes [128][129][130].

Reproductive and Developmental Toxicity
Maternal exposure to CPs can result in developmental toxicity and affect three major organ systems in offspring, namely, the central nervous system, the male reproductive system, and the immune system [131]. The dose-dependent maternal exposure to CPs (Printex 90) was linked with changes in the histology of different cell populations in the central nervous system (CNS) and the altered open-field behavior of the offspring in a murine model [132]. Maternal exposure to CPs resulted in the enlargement of lysosomal granules in brain perivascular macrophages (PVMs), as well as increased glial fibrillary acidic protein (GFAP) expression levels in astrocytes, indicating reactive astrogliosis in six-week-old offspring [132][133][134]. The observed changes in the CNS can cause early brain aging, and the offspring have an increased susceptibility to age-related brain disorders [132]. Additionally, the exposure to CPs, particularly CB particles, during gestation has effects on sperm counts and causes structural testicular changes in male offspring [135,136]. The exposure of mice to CB for 10 weeks decreased their daily sperm count and testosterone levels [131]. Maternal exposure to diesel exhaust and tobacco smoke particles can cause allergic immune responses in offspring [137]. Prenatal exposure to CPs can cause immune system impairment, resulting in the frequent onset of allergies during childhood [138]. Maternal exposure to CPs also affects the fetus, resulting in low birth weight [139].

Neurotoxic Effects
Epidemiological research along with in vivo and in vitro studies are further clarifying that exposure to fine particulates damages the nervous system and the brain [31]. Ambient CPs have toxic effects on the brain after potentially translocating through the olfactory tract/olfactory nerve, gastro-intestinal tract/vagus nerve, or blood-brain barrier [140]. The different potential pathways damaging the brain and CNS include direct toxic effects, neuroinflammation, and oxidative stress [141,142]. Ultrafine CPs can have a direct toxic effect through their deposition on the olfactory mucosa of the respiratory tract and subsequent translocation to the brain through the olfactory nerve [31]. Chronic exposure to particles from diesel exhaust can induce oxidative stress, neuroinflammation, and impaired neurogenesis in different brain regions, subsequently leading to brain cell death [143,144]. The dose-dependent exposure of mice to ultrafine soot-iron particles through inhalation was associated with indicators of neural inflammation [145]. Long-term exposure to fine particulates during adulthood accelerates the effects of aging in the brain, thereby increasing the risks of developing dementia or neurodegenerative diseases such as Alzheimer's disease or Parkinson's disease [146,147]. Long-term exposure to traffic exhaust CPs is associated with ischemic stroke, resulting in elevated stroke incidences [148].

Genotoxic and Carcinogenic Effects
The carcinogenic effects of CPs in humans are either caused at the exposure site such as the respiratory tract or at a distal location after the translocation of particles through the bloodstream [107,149]. Occupational exposure to PAHs, accounting for a significant fraction of BrC and BC, is linked to respiratory, urinary tract, and prostate cancers [150,151]. The regular inhalation of DEPs and CB particles results in pulmonary carcinogenic effects [152,153]. Exposure to CPs, particularly soot, can cause DNA mutations, while higher concentrations of PAHs are responsible for genotoxic effects by damaging DNA [154,155]. Exposure to CPs causes DNA modifications due to DNA adduct formation after the reaction of PAHs with DNA molecules [156,157]. CPs-particularly wood smoke particles-cause oxidative stress, which is associated with a failure of the DNA repair mechanism [158]. Exposure to fine and ultrafine particulates causes DNA methylation changes at the molecular level resulting in changing the expression profiles of genes such that they cause cancer [159]. Soot particles from the 1991 oil fires in Kuwait have shown that a dose-dependent increase results in the induction of genetic effects under in vitro conditions [107]. PAHs also cause epigenetic effects through histone modification, whereas CB can potentiate single-and double-stranded DNA breaks and hence cause genotoxic effects [160,161]. The presence of metal ions in CPs brings about chemical toxicity through the enhanced ROS formation capacity of CPs, resulting in genotoxic effects [162,163]. In addition, BrC particles attract persistent organic pollutants (POPs) and act as carriers for carcinogenic materials such as benzo[a]pyrene (Bap) [75,76,164].

Dermal Toxicity
The skin is the second most important route-after the respiratory tract-for the interaction and penetration of nanoparticle pollutants in the body [165]. Therefore, epidermal cells, like cells in other organs, are exposed to carbonaceous pollutants from the atmosphere, resulting in the production of pro-inflammatory cytokines by human dermal keratinocytes [166,167]. Diesel exhaust CPs and those from cigarette smoke are responsible for damaging effects on skin tissues and premature skin aging [167,168]. For example, DEPs are responsible for the development of allergic and non-allergic skin inflammation after the generation of ROS through redox reactions [169]. Organic extracts from CPs caused dose-dependent cytotoxicity in murine epidermal cells in model studies [170]. Although CPs have cytotoxic effects on the skin, no study was found regarding the toxic effects of CPs in other organs after full penetration through the skin. The toxic effects of CPs on human health are shown in the schematic in Figure 3.

Characterization Techniques for Carbonaceous Particles (CPs)
Adequate sampling and qualitative and quantitative characterization are essential to evaluate the adverse effects of CPs on the environment and humans. However, CPs' identification is not a simple task, as they are a heterogeneous group of particulates with a diverse chemical and structural nature and large variability in size [42,47]. In addition, various sampling conditions, such as those presented by polluted air or biological samples, pose many challenges for the qualitative and quantitative identification of CPs [39,171]. For example, elevated background signals from biological settings such as fluids or tissue samples are very intense compared to signals from CPs [171]. Due to the variability in different sampling conditions, it is difficult to develop generally applicable techniques for CPs' identification [171,172]. The generally used techniques for the characterization of CPs in atmospheric and biological samples are discussed in this section together with their advantages and limitations.

Characterization Techniques for Atmospheric CPs
The identification and quantification of CPs in atmospheric samples is not straightforward and the currently available techniques cannot accurately perform the sensitive detection of the different constituents of PM [37,38,173]. The detection of CPs in atmospheric samples is generally performed using light absorption and thermal radiation techniques; however, these two classes of techniques do not necessarily give similar results [40, 94,174,175]. Light absorption techniques correlate the light absorption or attenuation from the samples to the mass (per volume) of the absorbing material using a light absorption coefficient and assuming that the absorbed or attenuated light is proportional to the mass of the CPs [55,176]. Using light absorption techniques, the bulk analysis of CPs is performed using a filter-based approach in which a gas stream (polluted air) is passed through a filter to concentrate the samples. Aethalometers use a filter-based technique to measure the light absorption of CPs and can estimate the contribution from different CPs based on wavelength-dependent light absorption measurements [176,177]. For example, emissions from the burning of fossil fuel absorb light dominantly from the Vis-nIR region, indicating the presence of BC, whereas emissions from the burning of biomass have more BrC, and hence an enhanced absorption in the UV and blue wavelengths [178,179]. In addition, based on the attenuation coefficient, the concentration of the CPs can be estimated [180]. Although this technique can identify and quantify CPs in environmental samples, there are several limitations to this technique such as: (i) the shadowing effect, (ii) the scattering of the light beam at the filter fibers, (iii) and a false response from non-CPs, which can lead to the false estimation of these measurements [176,181].
Thermal radiation techniques work on the principle of measuring the mass concentrations of CPs based on heating the light-absorbing carbonaceous aerosols followed by the analysis of emitted radiation [182]. Laser-induced incandescence (LII) is a powerful thermal radiation technique in which the emission signal from the particles is generated by the absorption of high-intensity laser radiation, and the thermal emission from the heated particles is detected in the selected detection window [175,183]. Depending on the laser's wavelength, LII can identify different fractions of CP in gaseous samples [182]. It can also quantify the mass concentrations (based on the mass per volume) of CPs, as well as measure the particle size based on the temporal decay of the LII signal [184]. It is used extensively for environmental applications such as ambient air quality or source monitoring [182]. There are several limitations of this technique; for example: (i) the instrument response depends on the type of CP, (ii) proper calibration is required to convert the intensity of the thermal radiation to the CP mass, and (iii) no established reference materials are available for calibration [174,181].
Furthermore, the combination of LII and light scattering from particles has also been employed to identify CP, as well as to measure their mass concentrations and particle size. This method is used in Single Particle Soot Photometers (SP2) for single-particle analysis [185,186]. In SP2, LII measures CPs such as BC, whereas single-particle light scattering is employed to measure the particle size and mass concentrations [186]. This instrument can also measure the content of CPs without interference from semi-volatile materials or mineral dust particles [175,186]. As SP2 works based on LII and light scattering, the limitations of this method in addition to LII's drawbacks include (i) the requirement for prior information about the refractive index and shape of the particles, and (ii) the necessity of calibrating the instrument for the accurate determination of the mass concentrations and particle size [175].
Other common optical techniques used for atmospheric CP measurements include cavity ring-down spectroscopy (CRDS) and multi-angle absorption photometer (MAAP) [187][188][189]. In addition, high-performance liquid chromatography (HPLC), as well as mass spectrometry (MS), are used for the characterization of chromophores in BrC samples from different emission sources [58,190]. The above-mentioned techniques are briefly discussed for atmospheric samples; however, the detailed discussion of atmospheric CP characterization exceeds the scope of this work. There is an evident need for the characterization of CPs in biological samples in order to gain insights into the toxicity of CPs towards humans.

Characterization Techniques for CPs in Biological Samples
Currently, the epidemiological research studying the toxic effects of CPs in biological samples is hampered by the scarcity of analytical tools that function in biological samples [171]. Most of the available techniques for the detection of CPs cannot be used because of their limited biocompatibility and the interference of background signals from biological samples [171]. Indirect measurements can make use of radiolabeled CPs to study the toxic effects of CPs in model organisms after deliberate exposure. Further mass spectrometry, electron microscopy, and optical techniques can-to some extent-also be employed for studying CPs from natural exposure.

Isotope Tracing/Radiolabeling Detection of CPs
Isotope tracing is a useful technique for tracking carbonaceous nanomaterials in different biological systems [191]. This approach has been used to trace CPs in lab animals as well as in clinical studies [191][192][193]. In animal studies on mice, the translocation of ultrafine carbon particles (elemental 13 C) from the respiratory tract, via the bloodstream, to the liver was observed [191,192]. Human respiratory detection and clearance studies use scintigraphy methods such as CPs labeled with gamma-ray-emitting radioisotopes, and gamma-ray emission can be used for the localization and quantification of CPs in the lungs as well as the clearance and translocation of these particles [194,195].
Among the different clinically employed radiolabeling techniques, the most predominant is the "Technegas approach", which uses ultrafine CP suspensions labeled with Technetium-99m ( 99m Tc) [196]. The preparation of Technegas is carried out in a special machine at 2550 • C in an atmosphere of 100% argon gas and carbon, resulting in a thin layer of technetium encapsulating the carbon nanoparticles with a typical size of 30 to 60 nm [196,197]. The limitations of the employed isotopes in the Technegas technique are as follows: (i) 99m Tc has a short physical half-life (~6 h); (ii) 99m Tc leaches from the CPs, which limits this technique's use for clearance studies; and (iii) the hygroscopic properties of the particles as well as the presence of free pertechnetate can cause chemical instability of the generated particles [193,196,198]. Therefore, many modifications to this technique are used such as labeling with an indium isotope ( 111 In) or 68 GaCl 3 , which can extend the investigation time from a few hours to a few days [198][199][200]. Although these labeling methods offer sensitive localization and quantification of carbon nanoparticles in human studies, they have limitations, such as exposing the subject to radiation; the use of labeled carbon nanoparticles, which can modify the properties of the nanoparticles; and the limited post-administration follow-up, as radionuclides decay after few days [171,196].

Mass Spectrometry Detection of CPs
Laser desorption/ionization mass spectrometry (LDI-MS) is widely used for the characterization of CPs in environmental samples, and has been recently employed for the identification of CPs in biological samples such as cells and those in animal studies [86,201]. LDI-MS works on the principle of the ionization of the analyte using a laser beam, thereby removing the molecules from the surface of the material and ionizing them [202]. Afterwards, the ionized molecules are characterized based on their mass-to-charge ratio (m/z) in the analyzer through the time-of-flight (TOF) measurements [203,204]. Lin et al. used LDI-MS to study the biodistribution of soot particles in mice after their inhalation of PM 2.5 [201]. The samples with CPs were ionized under laser excitation in a vacuum atmosphere resulting in the formation of carbon cluster structures and characteristics peaks (C n − ) with repeated mass units [201]. The identification and quantification in biological samples were performed using murine lung samples and consistent anionic carbon cluster peaks were observed, making this technique feasible for measurements in biological samples [201]. The use of mass spectroscopy to identify and quantify CPs (soot) in biological samples is very promising; however, there are limitations of this approach, for instance: (i) the removal of the impurities can make sample preparation very complicated, and (ii) mostly biological samples contain carbon; hence, mass spectrometry can result in the detection of carbon cluster peaks from biological samples instead of soot particles [205,206].

Electron Microscopy Detection of CPs
Electron microscopy (EM) is a conventional technique that can provide good spatial resolution to analyze the cellular uptake of nanoparticles [207][208][209]. By using EM, CPs are characterized based on their typically black aggregates in the cells or tissue samples [210,211]. Scanning electron microscopy (SEM) scans the surface of a sample using a focused electron beam and can provide images of the sample in 3D with a very high resolution (3-20 nm) [212,213]. The detailed visualization and internalization of the nanoparticles by a cellular organism can be performed by transmission electron microscopy (TEM), in which a beam of electrons is transmitted through a thin specimen [213]. TEM analysis also provides information about different parameters of the nano-particulates such as their size distribution, shape, and aggregation [213]. Owing to its sub-nanometer resolution, it can reveal the fine relationships between the nano-particulates and the cellular/tissue components [210,213]  TEM can provide a very high resolution; however, some of the limitations of this technique are as follows. (i) CPs can only be identified based on their dark appearance in a bio-context, hence, it is difficult to discriminate different CPs. (ii) Biological samples need to be prepared carefully by skilled persons and sliced into very thin sections for analysis using TEM. (iii) The small field of view of TEM can hamper the imaging of large biological specimens such as entire organisms. (iv) Finally, TEM can only provide a static snapshot of the sample; thus, the investigation of dynamic cellular processes is limited [215,216]. Although conventional EM is widely used in NP research in biological systems, it cannot be used as the only tool for the qualitative or quantitative evaluation of the cellular uptake of NPs [217]. Hence, to obtain more abundant and accurate information, it is mostly combined with other methods such as optical microscopy techniques, electron energy loss spectroscopy (EELS), or energy dispersive X-ray (EDX) [218]. Another drawback of EM is that it involves the use of a very expensive instrument compared to most optical microscopes [213].

Optical Techniques for the Detection of CPs
Optical techniques can offer the capabilities of the non-destructive and label-free detection of CPs in their biological context. These techniques work based on different phenomena, including the optical absorption or scattering of light, molecular vibrational fingerprinting, or exploiting the non-linear optical behavior of nanoparticles.

•
Absorption-and scattering-based microscopy Absorption-based microscopy is one of the most straightforward forms of optical microscopy techniques and can be implemented in a bright-field microscopy system [219,220]. It works on the principle of the transmission of light through the sample and the generation of contrast based on the changes in the absorption of light in the denser regions of the sample [219]. Therefore, CB/BC NPs will appear darker compared to BrC NPs against the transparent biological media in the bright-field images [39,219]. In comparison, scatteringbased microscopy works on the principle of the detection of Rayleigh scattering from the specimen, in which contrast between different materials is generated based on the scattering intensity [219,221]. The scattering intensity from the particles in a biological context depends not only on their size but also on their refractive index, the refractive index of the surroundings, and the optical configuration of the setup in which the scattered light is detected [222]. Scattering-based microscopy images can be obtained using a conventional dark-field microscope [219]. Hence, both of these optical techniques can be applied for the label-free observation of CPs based on their light absorption or scattering in biological fluids, as well as their uptake by cells or tissues [171].
You et al. observed the nanoparticulate CB in the lung CD11c + cells from mice deliberately exposed to CB using bright-field and dark-field microscopy [223]. In addition, Modrzynska et al. observed the black aggregates in the liver sections of mice exposed to CB NPs using bright-field microscopy [224]. Absorption-and scattering-based microscopy are simple yet effective approaches to the visualization of CPs in biological samples, but they are limited by: (i) low resolution; hence, the detection of very small CPs below 200 nm is challenging and only large aggregates can be detected; (ii) visualization at a low concentration is difficult; and (iii) the identification of different types of CPs is not possible [171,225].

• Raman spectroscopy
Raman spectroscopy is a valuable technique for the label-free quantitative and qualitative characterization of CPs in biological samples [226,227]. It works on the principle of detecting inelastically scattered light to study the vibrational fingerprinting of different materials for their characterization [228]. Raman spectroscopy is used to obtain detailed information on different types of carbonaceous materials and their degrees of structural disorder [229]. Different types of CPs are distinguished based on their degree of graphitization using Raman spectroscopy [226,230,231]. The features of the first-order Raman spectra of carbonaceous materials are in the region from 1200 cm −1 to 1700 cm −1 , whereas second-order features are around 2700 cm −1 and can range up to 3500 cm −1 [226,232]. The first-order Raman transitions provide information about carbon material with a longrange order (graphitic carbon) or without a long-range order (amorphous carbon) [226]. Amorphous carbon is mostly an unknown mixture of sp 2 -and sp 3 -bonded carbon [226]. The most important Raman features observed for carbonaceous materials are around 1580 cm −1 , representing an ideal graphitic lattice (G-band), and at~1350 cm −1 , representing a disordered graphitic lattice from graphene layer edges (D1-band) ( Figure 5) [232,233]. Some smaller features are also observed at~1620 cm −1 (D2-band),~1500 cm −1 (D3-band), and~1200 cm −1 (D4-band) [232,233]. The D2-, D3-, and D4-bands represent the disordered graphitic lattice at the surface graphene layers, amorphous carbon, and a disordered graphitic lattice due to polyenes or ionic impurities, respectively [233][234][235]. Although Raman spectroscopy is mostly used for the characterization of CPs in environmental samples, few studies have reported the use of Raman spectroscopy for characterizing carbon materials in biological samples [223,[236][237][238]. For example, Knief et al. studied the suitability of Raman spectroscopy to determining the toxicity of carbon nanotubes in human epithelial cells (A549) [236]. In addition, the Raman fingerprints of CPs in the lungs of mice exposed to cigarette smoke for four months were observed [223]. Furthermore,Čabanová et al. detected the Raman fingerprints of amorphous carbon in the mucosa and hypertrophic tissue samples of patients with chronic rhinitis, which indicates the potential of Raman spectroscopy to identify CPs in real exposure measurements [237,238].
Raman spectroscopy has the potential to characterize CPs based on their vibrational fingerprint; however, the application of Raman spectroscopy in most biological samples is limited by the strong autofluorescence background signal (from PAHs in BrC) [227,228]. In addition, to achieve a good signal-to-noise ratio (SNR), long acquisition times are needed, which greatly slows the associated measurements [227]. Hence, acquiring Raman measurements of CPs in (bio)liquid samples is not a simple task due to the long measurement times. A very weak Raman-scattering signal can be compensated with higher laser power densities, which can be detrimental to biological samples [171,228].

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Photothermal pump-probe microscopy Pump-probe microscopy is a label-free optical technique used for the characterization of different types of nanoparticles in biological samples [239,240]. The basic idea behind the pump-probe approach is that the pump field excites the sample to a higher energy state or perturbs the electronic states of the materials, whereas the probe field determines the changes in the electronic states [240]. The probe field's absorption is either transiently enhanced or reduced [240,241]. In transient absorption microscopy, the absorption of the probe beam is transiently altered by the pump field, resulting in short-term changes in the probe field's intensity [240,242]. Ground-state depletion microscopy is another type of pump-probe microscopy approach, in which the population of the ground state of a molecule is altered and hence results in the reduced absorption of the probe field [243]. The photothermal approach to pump-probe microscopy also uses a pump field to heat the specimen in focus and induce changes in the refractive index of the surrounding medium, and modifications in the probe beam are observed due to the induced heat [244].
Steuwe et al. used photothermal pump-probe microscopy to identify CPs in different biological samples based on the contrast generation due to changes in the probe field intensity [245]. In addition to visualizing CB NPs in an automated fashion in a flow cell, CB NPs were also detected inside the human lung fibroblasts and spiked urine samples. The nonlinear nature of the signal provides 3D sectioning as well as large imaging depths due to the use of longer laser wavelengths.
This technique successfully detected CPs in different biological samples; however, some of the limitations of this approach are as follows: (i) the non-specific nature of the signal makes it difficult to distinguish different types of CPs; (ii) typically, the pump field can excite several molecular states simultaneously and result in the generation of spurious signals from non-CPs; and (iii) pump-probe microscopy requires the use of two ultrafast light sources mainly comprising a laser and an Optical Parametric Oscillator (OPO), hence making it an expensive approach [239,246]. Pump-probe approaches have also been proposed using cheaper diode laser; however, this can compromise the resolution [247,248].

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Femtosecond-pulsed laser microscopy (non-incandescence-related white light generation) for CP detection Femtosecond-(fs) pulsed laser microscopy (FPLM) is an innovative approach for the label-free and non-invasive characterization of CPs in fluids, cells, and tissue samples [249]. This approach can be implemented using a multiphoton microscopy setup available in most biomedical research labs [250]. Some of the non-linear optical processes observed using a multiphoton microscope include two-photon-excited autofluorescence (TPAF), secondharmonic generation (SHG), third-harmonic generation (THG), etc. [250][251][252][253]. These nonlinear processes work based on the simultaneous absorption of two or more photons by the specimen [250,254]. The multiphoton approach has the advantages of imaging living tissue samples with greater penetration depth and lower radiation damage due to the use of nIR laser wavelengths [250,255].
Recently, Bové et al. developed a novel biocompatible approach based on fs-pulsed laser microscopy for the label-free identification of CPs in biological media (Figure 6a) [249]. This approach works on the principle of non-incandescence-related white light (WL) generation from CB NPs when illuminated using a fs-pulsed laser at nIR wavelengths. The light emitted by the CB NPs covers the whole visible spectrum, and based on this spectral information, the emitted light can be detected in different spectral windows compatible with most multiphoton microscopes [256]. By using this approach, BC particles were detected in urine samples, human placenta samples (Figure 6b), and on plant leaves [257][258][259]. Further, the relationship between the WL emission intensity and particle size was also reported in aqueous suspensions [260]. Using FPLM, Bongaerts et al. recently revealed the presence of ambient BC particles in fetal blood and organs [261]. Table 2 provides a detailed overview of the techniques for the detection of CPs.   [171,[194][195][196]198] Mass spectrometry

Conclusions
CPs, being a major fraction of ultrafine PM, are abundant in the atmosphere and originate from different anthropogenic and natural sources. Their abundant presence in the atmosphere not only contributes to environmental impacts such as global warming (by their absorption of solar radiation) but also presents a likely pathway for their inhalation by humans. The inhalation of CPs by humans results in a wide range of adverse effects, ranging from respiratory to carcinogenic effects, as ultrafine CPs can translocate through the bloodstream to different organs. The qualitative and quantitative detection of CPs in atmospheric and biological samples is very important to understand their adverse effects. Different analytical techniques such as laser-induced incandescence and optical absorption are used for CPs' detection in gaseous samples. However, CPs' detection in biological samples is very challenging due to their heterogeneous nature and complexity associated with biological samples. The different analytical techniques used for CPs' detection in model studies in biological samples include radiolabeling detection, electron microscopy, mass spectrometry, etc. Whereas Raman spectroscopy and FPLM are employed for CP detection in models and real-exposure samples.

Challenges and Outlook
Different techniques such as FPLM or Raman spectroscopy are very sensitive for the qualitative and quantitative detection of CPs in real-exposure biological samples; however, these microspectroscopy techniques are very labor-intensive and time-consuming due to the related sample preparation, microscopy measurements, and data analysis. Therefore, to detect CPs in biological samples for clinical applications, there is still a need for the development of robust, sensitive, and time-effective analytical techniques. There is also growing evidence that the harmful effects of PM pollutants are size-dependent, in wherein ultrafine particles are the most harmful [262]. Hence, the size determination of CPs in biological samples can provide additional information about the toxicity of CPs related to their size. The techniques discussed in this review, e.g., FPLM and Raman spectroscopy, cannot detect very small particles (<100 nm) in biological samples; hence, high-resolution techniques are needed to detect particles below 100 nm.
Although BC and CB constitute a major fraction of CPs, recently, BrC has also received significant attention from scientists. The complexity and heterogeneity of CPs, the variability of their molecular properties, and the nature of their emission sources pose great challenges to their detection and discrimination in biological samples. Hence, there is a need for the development of advanced and sensitive analytical tools for the qualitative and quantitative detection and discrimination of different components of CPs to understand their adverse effects on human health. The currently available approaches will require optimization to not only detect very small particles (<100 nm) but to also discriminate between different CPs for diagnostic purposes.
In addition to understanding the adverse effects of CPs on human health, their atmospheric concentrations from different anthropogenic sources need to be controlled by better policy making (and implementation) at the regional and national levels. Recently, the WHO released new Global Air Quality Guidelines intending to save millions of lives from the harmful effects of air pollution. Among the different constituents of air pollution, particulate matter (PM) is on the top of the agenda of the WHO guidelines. In addition, the EU key directive (2008/50/EC) on ambient air quality and cleaner air for Europe also stressed reducing air pollution levels to minimize the adverse effects on human health.