DETECTION OF FORENSIC TRACES USING
GRAPHENE QUANTUM DOTS
Supervisor: Dr. Threes Smijs (Netherlands Forensic Institute) Examiner: Prof. Dr. Fred Brouwer
(University of Amsterdam)
Period:1st February – 25th August 2017
Credits: 36EC
Research Institute: University of Amsterdam Journal: Forensic Chemistry
Presenting Date: 24th August 2017
Aishwarya Nagarajan Student no: 11106484 MSc. Forensic Science University of Amsterdam
2 1. INTRODUCTION
The detection of forensic traces is an important aspect encountered at a crime scene. Every trace obtained at the crime scene must be identified before further analysis. Methods for the detection of traces must be sensitive, cheap and non-destructive for subsequent analysis. The common traces encountered at a crime scene are blood, fingerprints, saliva, semen etc. these are biological traces. Most of these traces cannot be visually observed and distinguished immediately, hence need a visual confirmation. Specifically, blood is one among the most commonly used trace for the identification of suspects. The detection of blood is frequently carried out with luminol [1,2] visualised with a chemiluminescence [3]. Although this is a fast method, the lifetime of the chemiluminescence is very short, usage during daylight is difficult and its sensitivity and specificity is far from optimal [4]. Even with the continued use of luminol, there is a need for an improved blood detection method. Prolonged research on the use of nanotechnology in forensic trace identification along with the optimization of older techniques is taking place. Studies have shown that identification of forensic traces can be achieved with the use of fluorescent biosensors such as quantum dots (QDs) [5–7]. QDs are semiconductor nano crystals that exhibit size-dependent optical and electrical properties. However, commonly used QDs contain heavy metal such as cadmium and thus possess risk to human health and the environment [8]. Hence the necessity of the production of efficient, stable and environmentally friendly QDs is desired. Graphene quantum dots (GQD) can be an attractive alternative, that is more environment friendly and less toxic [9,10]. Graphene’s structure can be tuned by varying the sp2 core and
hence influencing its size, chemical functionality and the edge structure [11]. The GQDs possess excellent photoluminescence, good stability, and low cytotoxicity [12,13]. These unique properties of the GQDs have been explored for various applications in electronics, bio-sensing, drug delivery, storage media etc [14–16]. A forensic relevant application has not yet been investigated.
The synthesis of the GQDs can be achieved by various processes. They are widely classified as top-down and bottom-up approaches. The top-down methods are based on electron beam lithography, chemical vapour deposition, C60 transformation [17–20] . The bottom-up
methods consist of conversion of organic precursors into GQDs through thermal pyrolysis, microwave, conversion of graphene sheets and carbon fibres into GQDs [21–24].
The bottom-up approach using a simple precursor like citric acid is one among the simplest and environmentally friendly synthesis route. The citric acid undergoes pyrolyzation at high temperature (200°C) and regulated time (figure 1). The control of the temperature and reaction time allows control over the morphology and size of the GQDs. The lower quantum yield (QY) resulting from the bottom-up process can be increased by doping the GQDs with sulphur or nitrogen [25].
3
Blood has various components, among which iron plays a crucial role in the oxygen transport, cellular metabolism and other physiological process. The haemoglobin in blood are carriers of oxygen and iron ions. When the haemoglobin is oxygen saturated and forms met-haemoglobin producing ferric ions. Blood detection can be achieved using functionalized GQDs, for instance, by coupling the dopamine (DA) to the carboxylic groups in the surface of GQDs [26]. In the presence of Fe3+ the DA as present in the
DA-functionalized GQDs (DA-GQDs) will be oxidized leading to quenching of the initial DA-GQDs photoluminescence. The oxidation of DA-GQDs mediated by Fe3+ may be used for the
detection of blood [27].
The here presented study focuses on the synthesis of GQDs and DA-GQDs, their physical and chemical characterization, the quantum yield (QY) related to the usefulness at a crime scene. Additionally, the Fe3+ sensing ability of the GQDs was studied in vitro.
4 2. MATERIALS AND METHODS
2.1 Raw Materials
99% Citric acid (CA), N-Hydroxysuccinimide (NHS) and Dopamine hydrochloride (DA. HCl) were purchased from Sigma Aldrich (Zwijndrecht, the Netherlands). Sodium Hydroxide pellets obtained from EMSURE Millipore (North-Holland, the Netherlands) and 1-Ethyl-3-(3’-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) from Novabiochem (North-Holland, the Netherlands). Sodium monobasic and dibasic phosphate and ferric chloride powder were purchased from Sigma Aldrich (Zwijndrecht, the Netherlands). For all solutions milli-Q was used.
2.2 Synthesis of GQDs
Two grams of CA was weighed in a round bottom flask, inserted in an oil bath that was pre-heated to 200°C. The CA liquified after 5 mins and the heating was continued for 20 mins. The total reaction time of 25 mins [10,28]. Further, NaOH (0.25 N) was used to adjust the pH to 7. The final product a yellow coloured liquid was stored at 4°C for further use.
2.3 Functionalization of GQDs with dopamine
To 20 ml of as prepared GQD solution at pH6, 0.3 g of EDC, 0.4 g NHS were added under vigorous stirring (see figure 2 for the reaction scheme). An incubation period of 15 minutes for the activation of the carboxylic groups in the GQD was maintained [28]. Later 0.2 g of DA. HCl was added under vigorous stirring. This reaction was continued at room temperature with rapid stirring for 24 hours [29].
5
The reaction mechanism of the carbodiimide mediated coupling of the -COOH group of the GQDs with DA to functionalize the GQDs is given in figure 3 where the primary amine represents the DA with its free NH2 group and the carboxylic group represents the COOH
groups in the edge of the GQDs. The GQD is functionalized with DA in a one-step EDC/NHS reaction. This reaction helps to form the amide bond between carboxyl group on the surface of the GQDs and the amine group in the dopamine. The EDC along with the carboxyl group forms an unstable O-acyclic urea ester and upon adding NHS, a semi stable NHS ester is formed with a better leaving group than the original OH- group present in the COOH. Hence the amide bond between the dopamine and the GQD is formed obtaining Dopamine functionalised GQDs (DA-GQDs)
The resultant solution was dialysed to remove unreacted chemicals, in a 1KDa dialysis membrane (Spectrum labs) for 48 hours with a change in membrane after 24 hours. The dialysis was carried out against milli-Q and the dialyser was changed thrice in 24 hours.
Figure 3: Carbodiimide mediated coupling, (1) is the carboxylic group, (2) EDC, (3) is the O-acylisourea ester, (4) is NHS, (5) semi stable NHS ester, (6) the amine and (7) formed product with amide bond.
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2.4 Physical and chemical particle characterization
For further characterization, a small portion of the product was dried in a rotary evaporator (BUCHI Rotavapor R-3 with CVC2 vacuubrand) for 30 mins at 40 mbar pressure and further dried in the oven (Memmert UF 55, Schwa Bach, Germany) at 85°C for 3.5- 4 hours.
2.4.1 Characterization
UV-Visible absorption spectra were recorded using a UV-2700 SHIMADZU (s-Hertogenbosch, the Netherlands) spectrometer. Fluorescence spectra were recorded with Flurolog Jobin Yvon-SPEX (Son, the Netherlands) spectrophotometer and the Gilden photonics M-series (Gaslow, UK) fluorescence spectrometer along with the quantum yield of the GQDs and DA-GQDs measurement using quinine sulphate solution (0.1M HClO4, Φ=0.59) as reference.
Particle size was analysed first using Dynamic light scattering (DLS) Dynapro Nanostar, (Hochstraße, Dernbach) and Atomic force microscope (AFM, Bruker, Leiderdorp, Nederland.)
To investigate the elemental composition of the GQDs and DA-GQDs the Fourier transform infrared spectroscopy (FTIR) Bruker (Leiderdorp, Nederland) FTIR spectrometer and X-ray photoelectron spectroscopy (XPS) and elemental analysis to investigate the presence of nitrogen introduced by the amide bond after GQD functionalisation.
2.4.2 Quantum yield calculation
Fluorescence quantum yield for the products is measured to understand the number of photons emitted. For which the relative measurement of quantum yield is performed using quinine sulphate as the standard to compare the sample. The standard quinine sulphate was chosen based on the absorbance (270-400nm) and emission (385-700nm) range similar to the synthesized product and the quantum yield of the standard is 0.59. The solvent used for the preparation of the standard is perchloric acid (0.1M HClO4) [30].
The formula used for the calculation of the QY is
where
• x denotes the sample and st the standard • F is the integral photon flux
7 • IC is corrected spectrum
The corrected spectrum is directly obtained from the spectrometer. • f is the absorption factor
• n is the refractive index • Φ is the quantum yield
2.5 Sensing function of DA-GQD
The sensing of Fe3+ ions with the DA-GQD was performed in vitro using phosphate buffer (10
mM) at pH 6, the mild acidic pH was used to prevent self-oxidation of dopamine. Briefly, 4 ml of DA-GQD in phosphate buffer were mixed with 10 μl of FeCl3 solution prepared in
phosphate buffer and incubated for 5 mins with stirring. The final FeCl3 concentrations were
between 1 – 20 μM. The quenching of the DA-GQD fluorescence was measured at 360 nm and performed in duplicates.
8 3. RESULTS AND DISCUSSION
3.1 Synthesis of GQDs and DAGQDs
From several syntheses performed it was observed that two factors; temperature and reaction time have a profound effect on the characteristics of the synthesized GQDs. The GQDs were synthesized by maintaining 200°C and for 25 mins, while increasing the reaction
time ≥ 30 mins the product showed more structural similarities to graphene oxide than GQD (based on FTIR spectra). With the above-mentioned reaction time and temperature, the colour of the final product was pale orange an increase in reaction time ≥ 30 mins changes the colour of the product to dark orange (supplementary A).
The synthesis of functionalized DA-GQDs is dependent on its precursor, the ratio of chemicals and the duration of stirring. The number of carboxylic groups decides the ratio of chemicals used. The DA.HCL carbodiimide-mediated linkage needs tremendous stirring for the reaction to complete. The EDC/NHS reaction also depends on the solvent; in case of water the reaction takes 24 hours to complete.
3.2 Characterization
3.2.1 Fluorescence properties.
Investigation of the photoluminescence properties was examined with absorption and fluorescence spectroscopy. A representative absorption spectrum is given in figure 4b. In this figure, the absorption band located at 360 nm likely originates from n π* transition resulting in blue fluorescence. Apart from this, a similar synthesis also leads to GQDs with absorption at 345 nm. This change in absorption was observed to be consistent with the increase in the temperature during the synthesis. This temperature difference might have caused by use of a different temperature controller. The higher temperature may have contributed in the synthesis of smaller particles which can be evident with respect to the absorption band of 345 nm [31]. A study by S. Zhu et.al [32] mention that the core and the surface of the GQDs has a considerable influence on the photoluminescence property. Smaller GQDs have higher energy and their PL is thus more blue shifted.
The fluorescence emission of the synthesized GQDs is constant at 460 nm. The synthesized GQDs, when excited at various excitation wavelengths the intensity increases between 345-370 nm and then decreases when the excitation wavelength is 375-400 nm (figure 4a). It gives a maximum fluorescence at 360nm.
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Figure 4: a) Fluorescence spectra of GQD of various excitation wavelengths, b) absorption spectra of GQD and c) GQD solution and water under UV.
Similarly, the functionalised DA-GQDs in figure 5b shows an absorption shoulder at 340 nm, while the emission is predominant at 480 nm excitation independent. The intensity increases between 345-370 nm and a decrease in the intensity is observed between excitation wavelengths 375 – 400 nm (figure 5a). The DAGQD also give an optimal fluorescence intensity when excited at 360nm.
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Figure 6: a) Fluorescence spectra of GQDs with respect to time at 360nm and b) line graph showing the reduction in fluorescence
3.2.1.1 Stability of GQDs and DA-GQDs
In a forensic perspective, the stability as well as fluorescence stability of the GQDs and the GQDs is of prime importance. To be implemented at a crime scene, the GQDs and DA-GQDs must be stable for a certain duration of time where even with repeated illuminations the fluorescence intensity must remain the same. Figure 6a shows the spectra measured at 360 nm at time intervals 24 hours, 48 hours, and 1 week. Figure 6b shows that the GQDs are stable upto 7 days with very less amount of decay in fluorescence.
Figure 5: and b) Fluorescence spectra of DA-GQD of various excitation wavelengths, b) absorption spectrum of DA-GQD and c) DA-GQD and water under UV.
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Figure 7: a) Fluorescence spectra of DA-GQDs with respect to time at 360nm and b) line graph showing the reduction in fluorescence
DA-GQDs are not as stable as GQDs (figure 7a); after 48 hours, the intensity is seen to reduce. The stability of the DA-GQD might be affected due to the presence of unreacted chemicals or dopamine reacting with oxygen causing a reduction in fluorescence. This could be prevented if it is stored under inert gas or Nitrogen.
3.2.2 Quantum yield
The quantum yield of GQDs using quinine sulphate as reference was calculated to be 0.04±0.02 (n=5). Reports of the quantum yield with this route of synthesis is varied. Dong et.al [10] obtained a QY of 0.09. Another study using the similar route obtain 0.10 as their QY [28]. Other study described a QY to be 0.04 [33]. In general, the QY is based on the parameters of the synthesis; temperature and reaction time and N or S doping [26].
The obtained DA-GQDs have showed a lower QY compared to the GQDs, using same quinine sulphate as the refence it was calculated to be 0.025 (n=2). The reduction in QY might be due to the presence of dopamine attached to the GQDs surface.
3.2.3 Size of the synthesized particles
The size determination was first carried out with DLS. As it is based on the light scattering of particles, the obtained results were not very consistent. The inconsistency in the results may be due to shadowing of the smaller particle scattering by the scattering of the larger particles. The other reason for improper results is the shape of the particles. Aimed synthesized particles are not spherical in shape but appear as sheets, while the DLS model considers only spherical structures for the analysis. Nevertheless, the DLS results have been included in supplementary B.
12 3.2.3.1 AFM
The investigation of the particle size of the GQDs and DA-GQDs was done with AFM in tapping mode. It was performed with a silicon tip (at a frequency of 320kHz and of spring constant 19N/m) to obtain the height images of the samples.
The figure 8a is a representative height image depicting the topography of the substrate’s surface and figure 8b giving a roughness profile of the substrate and showing the presence of GQDs. From which the GQDs have an estimated height of 1-3 nm. The size of the particles is roughly found to be between 12-25 nm. But this estimation is based on a small scale of data, for an exact determination of the particle size more height images must be analysed.
Figure 8: a) The height image of the GQDs, b) the roughness profile where the peaks show the GQDs and space between the peaks depicting the flatness of the substrate mica.
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Similarly, for the DA-GQDs figure 9a shows the height image and 9b the roughness profile. The estimated height of the particles is 1-2 nm. The size of the DA-GQDs roughly estimated to be 10-30 nm. More precise estimation of the size of the particles can be obtained with more height image data.
Figure 9: a) The height image of the DA-GQDs, b) the roughness profile where the peaks show the DA-GQDs and space between the peaks depicting the flatness of the substrate mica.
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3.2.4 Structural properties
The structural properties of the GQDs were determined by analysing the core and the surface. The core is composed of carbon lattice and the functional groups are present on the surfaces of the GQDs.
3.2.4.1 XPS
A representative XPS spectrum of the GQDs is shown in figure 10. The spectrum shows that the GQDs contain C, O and Na. The peaks at 285eV and 535 eV are C1s and O1s respectively. The presence of these elements shows that the GQDs surface has functional groups which correlates well with other studies [26,28]. The XPS for the DAGQD was unable to be performed, due to the unavailability of the instrument.
15 3.2.4.2 FTIR
The FTIR is used along with the XPS to confirm the structural composition of the GQDs and DAGQDs. In figure 11, a representative GQD FTIR spectrum is given showing characteristic peaks at 1558 cm-1 and 1381 cm-1 that corresponds to C=O stretching and C=C vibrations
respectively. The peak at 3200-3500 cm-1correspond to hydroxyl groups (-OH) from water
molecules and carboxylic groups.
In contrast to most literature, the carboxyl stretching peak is in a range 1850-1650 cm-1 [34],
instead of 1558 cm-1. To investigate this the pH of GQDs was varied and the result of these
studies is given in figure 12. This figure shows the FTIR spectra of GQDs at pH4, pH6 and the pyrolyzed product (pH 1.5-2). This shows the carbonyl peak at 1703 cm-1 corresponding to
C=O stretching as given in literature. With an increase in pH the peak at 1700 cm-1diminishes
to a shoulder, and peaks at 1570 cm-1 and 1556 cm-1 appear corresponding to C=O
stretching. This shift in the peaks can probably be explained by the fact that at lower pH the carboxyl group is present as COOH while a raise in pH causes this group to be present as COO- thatis stabilized by resonance energy. This structural difference may have caused the
shifts observed in the FTIR spectra.
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Figure 12: The FTIR spectra of GQDs of varied pH, the spectra are artificially inset for clarity.
Figure 13 shows the FTIR of DA-GQD with the peaks at 1571 cm-1 and 1387 cm-1 identified as
C=O stretching and C=C vibrations respectively. The peaks between 3000-3500 cm-1
attributes to the -OH but may also correspond to the -NH bending. The small peak at 1703 cm-1 can attribute to C=O stretching. Additionally, the DA-GQD has peaks at 1233 cm-1 and
1120 cm-1 which can correspond to bound NH bending and C-N stretching respectively.
These peaks corroborate the successful binding of the dopamine to the GQDs.
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3.3 Sensing Function with DA-GQD
The detection of Fe3+ ions can be achieved by both GQD and the DA-GQDs [35]. Considering
the sensitivity and specificity for the detection, the DA-GQD is preferred over the GQD for the Fe3+ ion detection [24,25]. Upon addition of Fe3+ ions to DA-GQDs, the catechol from
the dopamine in the DA-GQDs gets oxidised to a semi quinone leading to a decrease in fluorescence intensity.
The decrease of fluorescence intensity is observed as the concentration of Fe3+ increases
(figure 14). The quenching effect is not drastic, but there is a reduction in the fluorescence intensity. A 73% of decrease in fluorescence intensity was observed in the lowest concentration of Fe3+. As this experiment was performed only twice, the significance of
these results is low. Due to the presence of noise or background in the fluorescence spectra it is difficult to make a conclusion (supplementary C). As the DA-GQD sensing function is influenced by factors such as pH and molarity of the buffer, additional research is required to assure effectiveness in trace detection.
Figure 14: An artificial representation of the fluorescence spectra showing the decrease in fluorescence intensity with the increase in concentration of Fe3+. Original spectra
18 4. CONCLUSION
Detection of forensic traces such as blood using GQDs and DA-GQDs is a promising research for the forensic field. However, it should be noticed that even with a simple synthesis route for the GQDs and functionalised DA-GQDs, the characterization of these are time-consuming. Specifically, the size determination of the GQDs requires extensive research and dedicated time to obtain reliable data.
The in vitro detection of Fe3+ ions with DA-GQDs has been partially successful in a small
scale. For the sensitivity and specificity further work is required. As prepared GQDs and the DA-GQDs are efficient, sensitive and stable. Hence the manipulation/functionalization of the surface of the GQD can be used to target other forensic traces like finger mark or semen if it is possible to chemically link them to specifically functionalized GQDs.
FUTURE WORK
• The sensing ability of the DA-GQD with real blood stains including the sensitivity and specificity studies.
• A study on the effect of various backgrounds that can be encountered at a crime scene with respect to the sensing ability of the DA-GQDs.
• Photo stability of the GQD and DA-GQDs as a function of time.
• A comparative study between luminol and GQDs based on environment, luminescence lifetime and selectivity of luminol and sensitivity.
• Extensive studies on the toxicity of the GQDs and DA-GQDs.
• Using upconversion, to adjust the fluorescence colour and wavelength to prevent the blood from distributing light absorbtion.
ACKNOWLEDGMENTS
I would like to thank Netherlands forensic institute for the funding of the project. I thank my supervisor Dr. Threes Smijs for her guidance in the project and to be available all the time and for her enormous support throughout the 6 months.
I would like to thank Prof. Dr. Fred Brouwer for his guidance and advice in the project. A special thanks to Hans Sanders (MSc) for clearing my doubts whenever I asked and Dong Dong for assisting with QY measurements. The whole molecular photonics group for being supportive and friendly throughout the duration of the project.
19 SUPPLEMENTARY INFORMATION
A) Image of GQD synthesis; with different reaction time
Reaction time 55 minutes Temp was 200°C
Reaction time 25 minutes Temp was 200°C
20
B) DLS data
From the DLS, the GQD has larger number of particles above 100nm, and a few around 10nm in size. As mentioned previously the scattering by the larger particles has a higher intensity than the small particles. The similar sized particles are also observed in DA-GQDs.
Figure 1: A histogram representing the size of GQD particles. Three population of particles is represented. The 100nm particles are of the highest intensity compared to the others
21 C) Spectra of the Sensing function with DA-GQD
A decrease in fluorescence intensity can be observed with an increase in the Fe3+
concentration. There is a sharp decrease in fluorescence between the 0μM and the 5μM, but between the 10-20μM the reduction in fluorescence intensity is difficult to distinguish with the background disturbance.
The fluorescence spectra of the DA-GQD with the Fe3+ ions, at different
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