Potential forensic applications of carbon nanodots

Nanotechnology is a flourishing branch of research due to the incredible properties nanomaterials exhibit over their massive counterparts. Carbon nanodots are the members of the carbon nanoparticle family with prominent properties like hydrophilicity, low toxicity, biocompatibility, increased stability and ease of functionalization. They are excellent replacement to metal based quantum dots which are highly toxic. Carbon nanodots are being used in the fields of medicine, environmental science, drug delivery and forensic science. Applications of carbon nanodots can be seen in bio imaging, bio sensing, cancer diagnosis and treatment, biological activities, solar cells and as photo catalysts. Forensic science is the science behind crime investigation and evidence analysis. Development of novel, rapid, on site testing materials for accurate, specific and sensitive detection of forensic evidences is required for delivery of justice to the victims of crime. Carbon nanodots have shown promising applications in the field of forensic science as well. Use of carbon nanodots as invisible ink for anti-counterfeiting application of currency and security documents and use of these nanomaterials in development of latent fingerprints are some forensic applications. Carbon nanodots based detection of food contaminants, heavy metal poisons, pesticides, illicit drugs and explosives are other forensic applications. Carbon nanodots are showing a future direction in development of on field testing kits for forensic evidences.


Introduction
Nanotechnology is a flourishing and an on-going field of science and engineering which is focussed on design, synthesis, characterization and applications of structures whose size and shape can be manipulated at the nanometre scale.Manipulating materials to nano size customizes the materials to display properties which are different and more potential than their larger counterparts.In the dimensions of 1-100nm, materials start showing novel and unique properties which can be tuned by altering the size and shape of the particles.In the nano size range particles have a large surface to volume ratio and display quantum effects which enable the particles to exhibit different properties than their massive counterparts.Nanomaterials can be classified into zero dimensional, one-dimensional, two-dimensional and three dimensional nanomaterials.Zero dimensional nanomaterials have all the dimensions within the nano range.One-dimensional nanomaterials have one dimension out of nano size and two dimensions within nano range.Two-dimensional nanomaterials show two dimensions out of nano range while one dimension is within the nano size.Three dimensional nanomaterials possess all the three dimensions out of the nano range.Nanodots, nanorods, nano films and nano crystals are the examples of zero dimensional, one-dimensional, two-dimensional and three-dimensional nanomaterials respectively [1].
Nanodots are nanomaterials in the size range of 2-20nm with remarkable photo luminescent and plasmonic properties [2].Nanodots of different varieties have been manufactured in due course of research with unique and novel optical, electronic and chemical properties [3].Of the various nanodots being synthesized, carbon nanodots (CNDs) also referred to as carbon dots have gained significance due to their ease of synthesis, unique optical, electronic and luminescent properties.CNDs belong to the carbon nanoparticle family usually smaller than 10nm and are the latest version of quantum nanodots [4].CNDs have dragged the attention of researchers due to their incredible properties like hydrophilicity, low toxicity, biocompatibility, chemical inertness, increased stability, and ease of surface modification or functionalization [5].These advantages of CNDs find applications in the field of medicine [6], chemistry [7], electronics [8] and sensing [9].The astounding applications these nanomaterials offer in various fields of science, engineering and technology have grabbed the focus of researchers to work with CNDs in other emerging fields.
Forensic Science helps in investigation of crimes.It gives a scientific aid to investigation through evidence analysis.Several evidences found at the crime scene such as drugs of abuse, explosives, poisons, etc. are analysed using sophisticated instrumental procedures in the Forensic Science Laboratory.The procedures are tedious, time consuming, require costly equipment and considering the enormous case work the Forensic Science laboratories handle each day, they require much simpler, easy, sensitive, selective on field testing kits for analysis of such physical evidences recovered from a crime scene.As CNDs have proved to be useful sensors, the same have also been explored for forensic applications.In this paper, an attempt has been made to present a recent review on the forensic applications of carbon nanodots.

Carbon nanodots (CNDs)
CNDs are zero dimensional fluorescent nanomaterials with a potential for application in various fields of science.Purification of single walled carbon nanotubes led to the discovery of carbon nanodots.These are quasispherical or spherical in shape with a size range of 2-20nm [10].CNDs exhibit excellent luminescent properties in contrast to the traditional carbon which cannot emit any light [11].The incredible properties exhibited by CNDs include strong photoluminescence, biocompatibility, hydrophilicity, chemical inertness, increased stability, low toxicity, and ease of surface functionalization as seen from Figure 1.

Chemical inertness and enhanced stability
CNDs exhibit fluorescent properties due to presence of fluorophores which are the functional groups like amino, carboxyl groups present on their surface.CNDs exhibit broad emission spectrum ranging from UV to near Infrared regions.They exhibit optical properties which are size tuneable and their absorption properties also depend on the surface modification of CNDs [10].These optical properties make CNDs suitable for applications as sensors and in bioimaging [12].The presence of several groups on the surface of CNDs such as carboxyl, amine, carbonyl, and amide groups contribute to the biocompatibility and hydrophilicity of the CNDs.The presence of oxygen containing groups on the surface of CNDs makes these nanomaterials hydrophilic in nature.However, the CNDs can be made hydrophobic to act as organic synthesis catalysts by customizing the surface moieties with nonpolar solvents.The carbon core along with the surface modification help in synthesizing CNDs with controlled activity and versatility [10].CNDs are known to be highly stable under various environmental conditions making them more advantageous over their other nano counterparts.The fluorescence properties of the CNDs are not affected on exposure to light exhibiting photo stability [13].Owing to these properties, CNDs have been in the core area of research in the recent past.

Synthesis of CNDs
The properties and applications of the CNDs depend on the synthetic methods used.Size tuneability of the CNDs, surface passivation and preventing agglomeration of particles are important while choosing the suitable synthesis methods [10].Conventionally, there are two important strategies for synthesis of CNDs, Top down and Bottom up approaches as demonstrated in Figure 2. In the top down approach, the macroscopic carbon clusters are broken down into CNDs.Top down approaches are well suited for bulk production of CNDs.Some methods grouped under top down strategy are ultrasonic synthesis [14], arc discharge [15], and laser ablation [16].However, these methods are tedious, time consuming, require extreme reaction conditions and costly equipment [17].

Figure 2. Synthesis methods of Carbon nanodots
A few other methods of synthesis of CNDs include solvothermal method [18], microwave synthesis [19], and pyrolysis method [20].These methods make use of carbon precursors which combine into CNDs.CNDs synthesized by these methods display fewer defects and are highly controllable in their properties and applications [17].These conventional methods are being replaced by newer and greener procedures of synthesis.Replacing the pricy and toxic precursors used in the conventional methods with natural, cost efficient precursors is the need of the hour for synthesis of biocompatible, low toxic and environmental friendly CNDs.Use of apple peels [21], orange and banana peels [22] for synthesis of CNDs are some studies reported.To obtain CNDs with desired properties and applications, post synthetic modifications by doping with heteroatoms or functionalizing the surface with moieties targeted for specific applications needs to be carried out [10].

Forensic applications of Carbon nanodots (CNDs)
CNDs have shown applications in the field of medicine, chemistry, drug delivery, etc. Due to the availability of CNDs for surface modification, they can be used for specific applications.Forensic Science is the investigative science behind solving crimes.It provides a scientific value to evidence recovered in a crime.Evidences found at the crime scene can range from biological such as blood, semen, saliva, hair, etc. to physical evidences such as fingerprints, gunshot residues to chemical evidences which include drugs of abuse, poisons, explosives, etc. and also document evidences.There are conventional methods of analysis of evidences which are tedious, time consuming, cost intensive and require use of sophisticated instruments.However, CNDs can be used for selective, specific, stable, reproducible analysis of forensic evidences.Figure 3 displays the potential forensic applications of CNDs.

Application of CNDs in fingerprint development
Fingerprint science helps in identification of suspects in crimes, victims in mass disaster cases or cases where bodies have been destroyed beyond identification.Latent fingerprints are invisible fingerprints found at the crimes scene which need to be developed and then compared with the suspects or victims.There are methods like dusting with fingerprint powders, iodine fuming method, ninhydrin method, cyanoacrylate fuming methods, etc. for development of latent fingerprints used in the labs.However, nanotechnology has opened new avenues for development of methods with much accuracy and precision.CNDs exhibit fluorescence when exposed to light.This enhances the fingerprint characteristics and reduces background interferences [23].CNDs also prove useful in development of aged fingerprints.
Rhodamine 6G and Rhodamine B mediated hydrothermally synthesized carbon dots adsorbed onto diatomite were used for development of latent fingerprints.When enhanced with green light in the visible region, Rhodamine 6G mediated carbon dots displayed green fluorescence while the Rhodamine B mediated carbon dots showed yellow fluorescence.These nanocomposite powders can be applied for latent fingerprint development on patterned and multicolored surface eliminating background interference [24].Magnolia grandiflora flower mediated hydrothermal synthesis of carbon dots doped with hydrogen sulfide were in combination with corn starch used for the minutiae detection in latent fingerprints on non-porous surfaces such as glass, aluminium foil and iron surface.When exposed to UV light, a blue fluorescence enhanced the minutiae pattern making it easy to observe the latent fingerprints.On storage, minutiae could be detected up to 120 hours at room temperature [25].Carbon dots synthesized hydrothermally from coconut water, ethanol and luminol also displayed potential applications in enhancement of latent fingerprints.Luminol CNDs mixed with commercial starch with a size of 12.9 nm had blue fluorescence under UV illumination at 395 nm.These particles were used for detection of latent fingerprints on non-porous surfaces such as plastic, metal, glass and wooden surface.Addition of luminol increase chemiluminescence which is useful in forensic detection of latent fingerprints.At room temperature, the minutiae in the latent fingerprint were detected up to 28 days [26].
Hydrothermally synthesized orange emitting CNDs were used for latent fingerprint detection at a high speed.These CNDs worked well for development of aged latent fingerprints of even 120 days.They showed clear results on problematic surfaces such as multicolored, curved surfaces and also blue fluorescent backgrounds [27].Yellow green fluorescence emitting CNDs synthesized by microwave assisted method using phthalic acid and piperazine as precursors were used in quick detection of latent fingerprints on different substrates [28].N-doped CNDs modified with Zinc coated on hydrotalcite support showed physical adsorption with the components of the latent fingerprint residue and exhibited green fluorescence when excited with 365 nm UV light.The nanocomposite powder was highly sensitive and could provide a good contrast from the background interferences with a clear demarcation of ridge patterns on porous and non-porous substrates [29].

Application of CNDs in detection of drugs of abuse
Drugs of abuse are drugs used for the purpose of pleasure or euphoria.In the recent times, there has been an increase in the use of illicit drugs all over the world.Drugs can range from pain killers to sleeping pills, date rape drugs to stimulants.Drugs in the illicit drug market when recovered as evidence need to be analyzed qualitatively and quantitatively.Development of nano based simple on field testing kits can help solve illicit drug cases at a faster pace.CNDs have been used for detection of certain drugs of abuse.
Hydrophobic CNDs synthesized hydrothermally from D-phenylalanine have been used to detect nitro substituted benzodiazepines used as date rape drugs.These CNDs after liquid-liquid extraction of the sample to reduce matrix interference were used for quantitative estimation of nimetazepam.The method was found to be selective to nitro substituted benzodiazepines over other drugs of abuse.Using a UV lamp and a smartphone camera, a selective, rapid and cost efficient sensing system was developed for analyzing beverages in drug facilitated crimes [30].L-arginine mediated hydrothermal synthesis of CNDs was used for quantitative estimation of psychoactive substance, 4chloroethcathinone in aqueous medium and on CND modified paper.At pH 7, abused drugs, cocaine, heroin and cathinones were screened but at pH 11, the probe becomes selective to cathinones alone.Using a UV lamp and smartphone, on site screening of drugs at crime scenes can be carried out [31].

Applications of CNDs in explosive detection
Explosives are evidences recovered in cases where there is a threat to security of public.Explosives and explosion residue analysis forms a major work in Forensic Chemistry laboratory.Low and high explosive detection is carried out using colour tests and sophisticated instruments such as HPTLC, HPLC are used for confirmatory analysis.However, on site testing of explosive samples using kits can prove beneficial in the forensic analysis.
CNDs with photoluminescence property have been used for detecting a dangerous explosive, 2,4,6 -trinitrophenol (TNP) selectively with a detection limit of 0.2M [32].Environmental detection of TNP in tap and lake water samples was carried out using on field portable sensing gadget.Hydrothermally synthesized CNDs could specifically differentiate TNP from other nitro aromatic explosives.The detection limit of TNP using this method was 0.48 M [33].Solvothermally prepared fluorescent CNDs were used as selective and sensitive detection system for visual detection of TNP.This method was employed for on field testing of TNP in environmental samples [34].Nitrogen and sulphur doped CNDs synthesized using L-cysteine and citric acid were used as fluorescent detection probe for TNP.The probe exhibited a detection limit of 43 nM with good selectivity and sensitivity for environmental samples [35].Co-doped CNDs with nitrogen and phosphorus with average diameter of 5.7 nm were found to be sensitive and selective for TNP.The detection limit of TNP for this method was 23 M [36].A novel portable combination of wood with CNDs was used for detection of nitro aromatic explosives.The porous nature of wood prevents agglomeration of CNDs.This method displayed high sensitivity and selectivity for TNP with a visual detection mechanism [37].

Applications of CNDs in detection of poisons
Toxicology is a major branch of Forensic Science which deals with identification and quantification of poisons.Poisons include metallic poisons, pesticides, food poisons, etc. CNDs have been explored for their application in detection of poisons.
Cauliflower mediated CNDs have been used for specific and sensitive detection of diazinon, amicarbazone and glyphosate with detection limits of 0.25, 0.5 and 2 ng/mL respectively.This nano sensor could not detect bromacil and dialen super pesticides [38].Phenylalanine synthesized CNDs could be used for sensing dimethoate, DDVP, and parathion-methyl organophosphorus pesticides with a sub-nM level of detection limit in serum and several fruit samples [39].1,4-dihydroxyanthraquinone carbon dots were used for development of a highly sensitive nanosensor for detection of glyphosate with a lowest value of detection limit reported so far.This sensor was used for identification of glyphosate on the surface of vegetables [40].Carbon dots with 5,5-dithiobis-(2-nitrobenzoic acid) were used for fluorometric and colorimetric dual detection method for detecting organophosphorus pesticides.The method was highly sensitive, selective and accurate [41].
Oyster mushroom synthesized blue-green fluorescent carbon dots were used as colorimetric sensor for detection of toxic metal ions such as lead ions [42].Blue emitting CNDs synthesized from apple peels were used for detection of chromium (VI) ions based on fluorescence intensity quenching with a detection limit of 0.73 M [21].Red Fuji apple peels were used as precursors for synthesis of carbon dots with bright blue fluorescence.These carbon dots quenched the fluorescence intensity in presence of Fe +3 with a detection limit of 1.56 M in tap water and pork liver samples [43].Magnetic functionalized carbon dots fluorescent sensor has been used for detection of Cu +2 [44].

Anticounterfeiting applications of CNDs
Anticounterfeiting application adds features to prevent duplication of documents.Natural biomass was used as precursor for synthesis of fluorescent carbon dots.These were used for multisignal anti-counterfeiting applications [45].Hydrothermal synthesis mediated yellow fluorescent carbon dots were used for fluorescence anti-counterfeiting applications [46].The bio waste from monkey orange fruit was used for the synthesis of carbon dots exhibiting blue fluorescence.The carbon dots thus synthesized were made into a gel ink which was printed on currency notes.These showed good chemical stability even in presence of water, ethanol and acetone.These carbon dots also exhibited photo stability when exposed to UV light [47].
A summary of studies discussed above have been briefly presented in Table 1.

Hydrothermal synthesis
Rhodamine 6G and Rhodamine B CNDs adsorbed on diatomite.Used for latent fingerprint development on [24] Hydrothermal synthesis

Magnolia grandiflora flower
CNDs doped with hydrogen sulphide and mixed with corn starch.Minutiae were detected up to 120 hours on storage at room temperature on nonporous surfaces such as glass, aluminium foil and iron surface. [25]

Hydrothermal synthesis
Coconut water, ethanol and luminol Latent fingerprints were detected on nonporous surfaces such as plastic, metal, glass and wood surface.Minutiae could be detected up to 28 days at room temperature [26] Hydrothermal synthesis ---Development of aged latent fingerprints of 120 days old.Clear results on multicolored surfaces and curved surfaces [27] Microwave synthesis

Phthalic acid and piperazine
Yellow green fluorescence emitting CNDs.Used for development of latent fingerprints on different substrates [28] N-doped CNDs modified with Zinc and coated on hydrotalcite ---Highly sensitive and gives good contrast from background interferences with clear ridge patterns on porous and non-porous surfaces. [29]

Detection of drugs of abuse
Hydrothermal synthesis D-Phenylalanine Used for detection of nitro substituted benzodiazepines used as date rape drugs.Quantitative estimation of nimetazepam [30] Hydrothermal synthesis L-Arginine Quantitative estimation of 4chloroethcathinone [31]

[33]
Solvothermal ---Selective and sensitive on field testing of [34] synthesis TNP in environmental samples N and S doped CNDs

L-Cysteine and citric acid
Detection probe with good selectivity and sensitivity with a detection limit of 43 nM in environmental samples [35] N and P doped CNDs ---Sensitive and selective method for TNP with a detection limit of 23 M [36] Wood with CNDs ---Visual detection mechanism for TNP [37] Detection of pesticides ---Cauliflower Specific and sensitive detection of diazinon, amicarbazone and glyphosate with detection limits of 0.25, 0.5 and 2 ng/mL respectively.This nano sensor could not detect bromacil and dialen super pesticides [38] ---Phenylalanine Used for sensing dimethoate, DDVP, and parathion-methyl organophosphorus pesticides with a sub-nM level of detection limit in serum and several fruit samples [39] ---1,4dihydroxyanthraquinone Used as sensor for detection of glyphosate on surface of vegetables [40] CNDs with 5,5-dithiobis-(2nitrobenzoic acid) ---Dual detection method for detecting organophosphorus pesticides. [41] ---Water hyacinth Fluorescent sensor for detecting herbicide, Pretilachlor with detection limit of 2.9 M

Hydrothermal synthesis
Oyster mushrooms Blue-green fluorescent CNDs used as colorimetric sensor for detection of Lead ions [42] Hydrothermal synthesis Apple peels Blue fluorescent CNDs used for detection of chromium(VI) ions with detection limit of 0.73 M [21] Hydrothermal synthesis Red Fuji apple peels Blue fluorescence CNDs used for detection of Fe +3 with detection limit of 1.56 M in tap water and pork liver samples [43] Magnetic functionalized CNDs ---Fluorescent sensor for detection of Cu +2 [44] Hydrothermal synthesis

Red lentils
Strong sensor for Fe +3 with detection limit of 0.10 M [49] Hydrothermal synthesis

Prosopis juliflora
Fluorescent sensor for Hg +2 with a very low detection limit of 1.26 ng/mL [50] Hydrothermal synthesis Hongcaitai (vegetables) Fluorescent sensor for Hg +2 with a detection limit of 0.06 M When printed on currency notes, CNDs displayed chemical stability and photo stability [47] Table 1: Studies on potential forensic applications of CNDs

Conclusion
Carbon nanodots have been explored for various potential forensic applications.However, converting the technology for real on field testing has to be given a serious thought and consideration by researchers and forensic scientists as well.Development of onsite testing kits using the fluorescent technology of carbon nanodots can prove a breakthrough in forensic science.CNDs have been explored mostly for development of latent fingerprints owing to the excellent fluorescent properties of CNDs.The drugs detected using CNDs were mostly methamphetamine and benzodiazepines.TNP was the only explosive material which was detected using CNDs.CNDs were also used for identification of poisons such as pesticides and heavy metal ions and have also been applied for anticounterfeiting applications.CNDs can further be used for identification of biological fluids in crime scene samples, detection of alterations in questioned documents, as molecular sensors for identification of a wide variety of forensic evidences ranging from narcotic drugs to explosive samples.The scope for further research in the area of exploring forensic applications of CNDs is very vast and is open to researchers to find better detection systems for identification of potential forensic evidences for delivery of timely justice.