Nitrite amperometric sensor for gunshot residue screening

Journal Pre-proof Nitrite amperometric sensor for gunshot residue screening Kiattisak Promsuwan, Proespichaya Kanatharana, Panote Thavarungkul, Warakorn Limbut PII:

S0013-4686(19)32181-4

DOI:

https://doi.org/10.1016/j.electacta.2019.135309

Reference:

EA 135309

To appear in:

Electrochimica Acta

Received Date: 13 September 2019 Revised Date:

10 November 2019

Accepted Date: 14 November 2019

Please cite this article as: K. Promsuwan, P. Kanatharana, P. Thavarungkul, W. Limbut, Nitrite amperometric sensor for gunshot residue screening, Electrochimica Acta (2019), doi: https:// doi.org/10.1016/j.electacta.2019.135309. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.

Graphical abstract

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Nitrite amperometric sensor for gunshot residue screening

Kiattisak Promsuwana,b,c, Proespichaya Kanatharanaa,b,c, Panote Thavarungkula,b,d, Warakorn Limbuta,b,e*

a

Center of Excellence for Trace Analysis and Biosensor, Prince of Songkla University, Hat

Yai, Songkhla 90112, Thailand. b

Center of Excellence for Innovation in Chemistry, Faculty of Science, Prince of Songkla

University, HatYai, Songkhla 90112, Thailand. c

Department of Chemistry, Faculty of Science, Prince of Songkla University, Hat Yai,

Songkhla 90112, Thailand. d

Department of Physics, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla

90112, Thailand. e

Department of Applied Science, Faculty of Science, Prince of Songkla University, Hat Yai,

Songkhla 90112, Thailand

*Corresponding author at:

Department of Applied Science, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand. Tel.: +66 74 288563; Fax: +66 74 446681

E-mail addresses:

[email protected] (W. Limbut)

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Abstract A novel electrochemical sensor, a glassy carbon electrode modified with a composite of palladium (Pd) particles and glassy carbon microspheres (GCMs), has been developed to detect gunshot residue via nitrite determination. Pd-GCMs was synthesized through a simple electroless deposition method and characterized by scanning electron microscopy and energydispersive X-ray spectroscopy. The electrocatalytic response of nitrite on Pd-GCMs/GCE is greatly enhanced relative to that on GCMs/GCE and bare GCE. The applied potential, flow rate and sample volume of flow injection amperometric detection were optimized. The sensor displayed a linear response to nitrite between 0.10 µmol L-1 and 4.0 mmol L-1, with a limit of detection and quantification of 0.030 and 0.11 µmol L-1, respectively. It exhibited very good performance with excellent electrocatalytic activity and high sensitivity (500±5 µA mM-1 cm-2). In addition, the electrochemical sensor presented good repeatability (RSD < 1.5%, n=20), reproducibility (RSD = 1.4%, n=6), operational stability (RSD = 2.8%, n=268) and high sample throughput (165 samples h-1). The results for detecting nitrite in gunshot residue with this sensor were in good agreement with those obtained with the Griess method (P>0.05). With its good performance this sensor could be a viable alternative method for detecting nitrite to identify gunshot residue in forensic evaluations via nitrite detection.

Keywords: Pd particle-glassy carbon microspheres (Pd-GCMs), nitrite, gunshot residue, flow injection amperometric detection

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1. Introduction The detection and identification of gunshot residue (GSR) can provide very important evidence in forensic investigations for cases related to firearm use. GSR can be used to identify suspects, differentiate between a homicide and suicide, evaluate the firing distance and indicate a bullet hole [1-3]. To date, the morphology and elemental composition (focuses on Pb, Ba and Sb) for the confirmation of GSR in forensic evaluations are mostly based on scanning electron microscopy coupled with energy dispersive X-ray analysis (SEM-EDX)[4], atomic absorption spectroscopy (AAS) [5], inductively coupled plasma-mass spectrometry (ICP-MS) [6] and inductively coupled plasma-optical emission spectroscopy [7]. Although these methods are sensitive and accurate, they require sophisticated equipment and skilled operators. The analysis is expensive and takes a relatively long time [8, 9]. Thus, a simple and rapid screening method with high sensitivity and selectivity, i.e., the ability to identify all cases that are positive for the presence of GSR, before applying more specific techniques is potentially useful. Nitrite ions, an inorganic component of GSR, are generated after shooting from primer detonation and gunpowder combustion [10-12]. Their determination can be a useful preliminary analysis for the presence of GSR on a shooter or suspect, target, and other surfaces exposed to cartridge discharge products, and it can provide investigators key evidence against perpetrators. Thus, the detection of nitrite can be used as a screening tool to investigate the presence of GSR. Analytical methods that have been developed to detect nitrite include ratiometric fluorescent [13], spectrophotometry [14], ion chromatography [15], electrophoresis [16]

and

electrochemical

methods

[17-19]. Among

these

methods,

electrochemical detection is widely used owing to its simple operation, sensitive response, cost effectiveness and rapid analysis. In cases that involve many suspects and a large number of samples, rapid screening for GSR is required before applying more specific techniques.

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Thus, to provide continuous, high-throughput and rapid analysis of nitrite in GSR samples, flow injection analysis coupled with an electrochemical detection method can be used to provide low sample contamination, good precision, acceptable accuracy and low consumption of reagent and sample. However, for electrochemical detection of nitrite, conventional electrodes are not suitable because of their poor catalytic properties, and the nitrite oxidation process requires a very high working potential that may result in the lack of selectivity. To overcome the aforementioned problems, electrochemical nitrite sensors have recently been fabricated based on chemical modification of electrodes to improve the sensitivity and lower the potential necessary for nitrite detection. Noble metal particles, such as platinum (Pt) [20], gold (Au)[21] , silver (Ag) [22] and palladium (Pd) [23-25] on the nano- and microscales are widely used because they enhance the surface active area and electrocatalytic features of an electrode. For nitrite oxidation, Pd particles are attractive due to their excellent electrical conductivity and catalytic activity, nontoxicity and good chemical stability [23, 26]. The catalytic performance of Pd particles may also be further enhanced by the synthesis of these metal nanoparticles on carbon materials, such as carbon nanotubes (CNTs) [25, 27], graphene (Gr) [26, 28] and carbon microspheres (CMs) [29]. Among these materials, CMs, which are inexpensive and easy to prepare and modify [29, 30], have attracted much attention. They offer high electrochemical reactivity, a wide potential window with a low background current, good particle size and shape tunability, size homogeneity, and high specific surface area for high metal nanoparticle loading. The electrical conductivity and chemical stability of CMs are satisfactory compared to those of CNTs and graphene [31, 32]. This report presents a simple, cost-effective and environmentally friendly method for the preparation of Pd particles on glassy carbon microspheres (Pd-GCMs) via an electroless deposition method using hydrazine as the reducing agent. The morphology of the Pd-GCMs

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was characterized and compared with those of GCMs using scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDX). The electrochemical behavior of nitrite was evaluated by cyclic voltammetry (CV), and the analytical measurements were performed by flow injection amperometry (FI-amp). Experimental parameters were optimized and applied for the detection of GSR. To date, this is the first report on the preparation of Pd particles on a carbon microsphere modified glassy carbon electrode (PdGCMs/GCE) with a flow injection system as a facile approach to screen and identify GSR via nitrite determination for forensic evaluation. 2. Experimental methods 2.1. Materials Palladium chloride (PdCl2, 99.9%) and hydrazine (N2 H4, 35 wt% solution in water) were from Sigma-Aldrich (Louis, USA). Glassy carbon microspherical powder was from SPI-Chem (West Chester, USA). Ethylenediaminetetraacetic acid disodium salt (EDTA) was from BDH (Poole, UK). Ammonium hydroxide (NH4OH, 28-30%) was from J.T. Baker (Phillipsburg, NJ, USA). Hydrochloric acid (HCl, 37%) was from Merck KGaA (Darmstadt, Germany). Sodium nitrite (NaNO2) was from Ajax Chemical Finechem Pty Ltd. (Taren Point NSW, Australia). A 0.10 mol L-1 phosphate buffer solution (PBS) with a pH of 5.00 was prepared by mixing a solution of K2HPO4 and KH2PO4 and used as the supporting electrolyte. All solutions and subsequent dilutions were prepared using deionized water from a Barnstead TM Easy Pure TM II water purification system with a resistivity of no less than 18.2 MΩ cm (Thermo Scientific TM, USA).

2.2. Instrumentation and apparatus Electrochemical experiments were carried out using an Autolab 910 PSTAT mini controlled by PSTAT software (Metrohm, Herisau, Switzerland). The electrochemical cell

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consisted of a glassy carbon electrode (GCE, 3 mm diameter) modified with a Pd-GCMs as a working electrode, an Ag/AgCl (3 M KCl) as a reference electrode and a platinum wire as a counter electrode. The SEM and EDX analysis, the sample of GCMs and Pd-GCMs were drop casted on a glass slide and coated with gold before characterization by SEM with EDX (Quanta 400, FEI, USA). 2.3. Pd-GCMs composite preparation The Pd-GCMs composite was prepared following the method described by Sirisaeng et al. [33] with some modification. Briefly, 0.2000 g of glassy carbon microspherical powder was dispersed in 80.0 mL of deionized water and sonicated for 1 hour in an ultrasonic bath. Then, 545 µL of 2.82 mol L-1 PdCl2 (0.2730 g of PdCl2 dissolved in 545 µL of 37% HCl) and 3.500 g of EDTA were added under stirring. The mixture was adjusted to a pH of 10.00 with NH4OH. Next, 1.0 mL of hydrazine was slowly added into the mixture under stirring. Then the mixture was continuously stirred at 45 ºC for 2 hours. The solid in the mixture was then filtered, washed with deionized water until the pH was neutral and dried overnight at 60 ºC to obtain the palladium particle decorated glassy carbon microspheres (Pd-GCMs). 2.4. Electrode modification A GCE was polished with 0.50 and 0.050 µm alumina slurries until a smooth mirrorlike surface was obtained. Then, the material was rinsed with doubly deionized water, sonicated for 5 min, and then dried by pure nitrogen gas. Pd-GCMs suspension (2.0 mg mL-1) was prepared by dispersing 4.0 mg of the Pd-GCMs in 2.0 mL of N, N-dimethylmethanamide and sonicating the mixture for 30 min. Then, 10.0 µL (20 µg) of the suspension was dropcasted onto the cleaned GCE surface and allowed to dry at 60 ºC to obtain the Pd-GCMs modified GCE. For comparison GCMs/GCE without Pd was also prepared by the same procedure as described above.

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2.5. Electrochemical measurements Electrochemical measurements were carried out by an Autolab 910 PSTAT mini controlled by PSTAT software. Electrochemical characterization of the GCE, GCM/GCE and Pd-GCM/GCE toward nitrite was performed by cyclic voltammetry (CV) in a batch system from +0.30 to +1.10 V with a scan rate of 100 mV s−1 in 5.0 mL of 0.10 mol L-1 phosphate buffer (pH 5.00). A chronoamperometric study was performed in a batch system by applying a constant +0.85 V to solutions containing various concentrations of nitrite in 5.0 mL of 0.10 mol L-1 PBS (pH 5.00). Flow injection amperometric detection was carried out at a constant potential using a lab-built flow cell (10 µL) consisting of a modified GCE working electrode, a Ag/AgCl reference electrode and a stainless-steel tube counter electrode. A peristaltic pump (Miniplus 3, Gilson, France) and a six-port injection valve (Valco Instrument, USA) were used to drive the carrier buffer solution (0.10 mol L-1 PBS, pH 5.00) and to control the sample volume, respectively. Current responses for the electrocatalysis of nitrite were obtained with a series of nitrite standard concentration solutions (in 0.10 mol L-1 PBS pH 5.00) injected into the flow system by the carrier stream passed over the Pd-GCMs/GCE (detector). 2.6. Optimization Optimization of the sensor fabrication (i.e., amount of Pd-GCMs) and operational conditions (i.e., pH of the buffer solution, working potential, flow rate and sample volume) was performed to obtain the best electrocatalysis of Pd-GCMs/GCE for nitrite oxidation. CV was performed between +0.30 and +1.10 V at a scan rate of 100 mV s-1 with 0.20, 0.40, 0.60, 0.80 and 1.00 mmol L-1 of nitrite in 0.10 mol L-1 PBS (pH 5.00). For the operational conditions, FI-amp was used to improve the limit of detection and the sensitivity of PdGCMs/GCE for nitrite analysis. Factors affecting the nitrite current signal, i.e., the applied

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potential, flow rate and sample volume, were investigated. In all FI-amp experiments, 0.10 mol L-1 PBS (pH 5.00) was used as the carrier solution. Optimization of the applied potential was performed using a series of standard nitrite solutions (0.10, 0.20, 0.30, 0.40 and 0.50 mmol L-1, six replicates for each concentration) with a flow rate of 0.50 mL min-1 and a sample volume of 250 µL, and the potential that provided the highest sensitivity was considered optimal. The flow rate (0.50 to 3.00 mL min-1) and sample volume (150 to 400 µL) were optimized together by changing the sample volume while keeping the flow rate constant and then injecting three replicates of a 0.10 mmol L-1 standard nitrite solution for each flow rate and sample volume. The optimal flow rate and sample volume were those that provided in the highest current response and a shot analysis time. 2.7. GSR sample collection and analysis The nitrite collection and extraction processes of GSR samples were adapted from the method described by Erol et al. [34]. The shooting test was performed using a 9 mm semiautomatic firearm and bullets (9 mm Paralead round nose 135 gram, Bullet Master Co., Ltd). GSRs were collected by swiping with a cotton swab (cotton swab,15 cm sized L, United Medicine Instruments Co., Ltd.) on the shooter’s hands and clothes (chest area) before and after shooting 1 and 3 shots. Each cotton swab was cut and placed in a 50 mL centrifuge tube before adding 30.0 mL of 0.10 mol L-1 PBS (pH 5.00) and extracting in an ultrasonic bath for 30 min at room temperature. The extract was passed through a 0.45 µm pore paper filter. The filtrated extract samples were analyzed using the FI-amp system with the PdGCMs/GCE and the Griess spectrophotometric method. In the Griess method, the extract sample was mixed with Griess reagent, which is a mixture of equal volumes of N-(1naphthyl) ethylenediamine and sulfanilic acid [35]. This mixture was incubated for 30 minutes at room temperature and absorption was measured at 548 nm by UV-Vis

9

spectrophotometry. The results from the FI-amp system and the standard Griess method were statistically compared by the Wilcoxon signed rank test.

3. Results and discussion 3.1. Characterization of the GCMs and Pd-GCMs Fig. 1A shows the SEM micrograph of spherical GCMs with smooth surfaces. The particle sizes ranged between 1 and 15 µm. The particles provide a large surface area when deposited on an electrode surface for the attachment of Pd particles. The synthesized Pd particles are distributed on the smooth surface of GCMs (Fig. 1B) with an average diameter of 0.23±0.08 µm (Fig. 1C) (n=100, 100 Pd particles from SEM image were randomly selected). The Pd-GCMs on the electrode surface resembled a three-dimensional porous structure. The EDX technique was carried out to confirm Pd-GCMs synthesis. The EDX spectrum of the GCMs (Fig. 1D) showed no palladium element. Pd particles were synthesized, and palladium element was observed in the EDX spectrum (Fig. 1E). These results confirmed that Pd-GCMs were successfully synthesized.



3.2. Electrochemical characterization of the modified electrode 3.2.1. Electrochemical behavior toward nitrite

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The electrocatalytic activity of different modified electrodes for the oxidation of 1.0 mmol L-1 nitrite was evaluated by CV as shown in Fig. 2A. Without nitrite, only background current was observed for all electrode modifications, i.e., bare GCE (a'), GCMs/GCE (b') and Pd-GCMs/GCE (c'). The interface properties and electron-transfer resistance of the surfacemodified electrodes were investigated by electrochemical impedance spectroscopy. Supplementary data Fig. S1A exhibited the Nyquist plot with the charge transfer resistance (Rct). Fig. S1(a) displays the EIS spectrum of GCE with a large semicircle (Rct = 1,888 Ω), indicating that the bare GCE had relatively low conductivity. In the case of the GCMs/GCE, the semicircle diameter (Rct = 679 Ω) was smaller than that of the bare GCE (Supplementary data, Fig. S1(b)), suggesting that GCMs/GCE enhances the conductivity of the electrode. After modification of the GCE surface with the Pd-GCMs, a further decrease in the semicircle diameter (Rct= 198 Ω) was observed (Supplementary data, Fig. S1(c)). The decrease in Rct indicated that Pd-GCMs composite has superior conductivity and can obviously promote the electron transfer efficiency. Upon addition of 1.0 mmol L-1 nitrite, the peak response of the bare GCE (Fig. 2A(a)) toward nitrite oxidation appeared at +1.06 V. The GCMs/GCE (Fig. 2A(b)) showed a slightly lower current response at + 0.98 V. The peak current was decreased and the peak potential was negatively shifted relative to those obtained with the bare GCE. These results illustrated that the GCMs/GCE have low electrocatalytic activity towards nitrite oxidation in this situation while it has good electrical conductor (Supplementary data, Fig. S1(b)). A well-defined peak at +0.85 V of the Pd-GCMs/GCE (Fig. 2A(c)) was 1.2 and 1.9 times of the GCE and GCMs/GCE peak current, respectively. This increase indicated the high electrocatalytic activity and fast electron transfer ability of the Pd for nitrite oxidation, which may be attributed to the synergistic effect of the GCMs and Pd particles that increased the number of catalytic sites and the rate of electron transfer. Fig. 2B shows the sensitivities (slope of the calibration curve) of the different electrodes; as

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expected, the Pd-GCMs/GCE exhibits 1.2- and 1.9-fold higher sensitivity than the bare GCE and GCMs/GCE, respectively. In the presence of different concentrations of nitrite, the peak currents of the Pd-GCMs/GCE gradually increased with nitrite concentrations (Fig. 2C), where a linear relationship between the peak current and nitrite concentration was obtained from 0.20 to 1.00 mmol L-1 with a correlation coefficient of 0.9999 (Fig. 2D). 3.2.2. Effect of scan rate The kinetics of the Pd-GCMs/GCE reactions were studied by testing the effect of the scan rate on the anodic peak current and nitrite potential. The CVs for oxidation of 2.0 mmol L-1 nitrite with Pd-GCMs/GCE at scan rates between 20 and 220 mV s‒1 in 0.10 mol L-1 PBS (pH 5.00) showed increased peak current with the scan rate (Supplementary data, Fig. S2A). The anodic peak current vs. square root of the scan rate was fitted with a linear regression equation of Ipc (µA) = (8.247±0.004) ν1/2 (mV/s)1/2 + (10.29±0.04), R2 = 0.9992 (Supplementary data, Fig. S2B). The linear correlation of anodic peak current vs. square root of the scan rate indicated that the mass transport for nitrite oxidation with PdGCMs/GCE is controlled by diffusion. Moreover, the plot of Ep (V) versus ln ν gave a linear relationship with a regression equation: Ep(V) = (0.0257±0.0003) ln ν (Vs-1) + (0.904±0.001), R² = 0.9977 (Supplementary data, Fig. S2C). According to this linear equation, the number of transferred electrons can be calculated based on the following equation [36]: Epa = K + (RT /2(1 − α)nαF) ln ν where α is the electron transfer coefficient and nα is the number of electrons in the ratedetermining step. Other symbols have their common definitions. The value of (1-α) nα can be calculated from the slope of the linear plot of Ep versus ln ν. In this work, the slope is 0.0257±0.0003. The value of (1-α)nα is 0.498±0.005. Normally, the electron transfer

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coefficient (α) is assumed to be 0.5 in a completely irreversible electrode process [37]. The number of electrons transferred (n) in the electrooxidation process of nitrite is 0.99±0.01 (n~1). Thus, the oxidation of nitrite on Pd-GCMs/GCE has a one-electron transfer process as the rate-determining step, which is in agreement with previous literature [38, 39]. Thus, the mechanism of the electron transfer process for the electrocatalytic oxidation of nitrite on the Pd-GCMs/GCE can be expressed as reported previously [40, 41] as follows. -

Nitrite forms a complex with Pd-GCMs on the electrode, i.e., Pd-GCMs(NO2 ) (reaction 1). This complex is then electrochemically oxidized to produce NO2 intermediates by losing one electron (reaction 2), followed by the rapid disproportionation of NO2 to give NO2− and NO3− (reactions 3 and 4); the final product of nitrite oxidation process is nitrate. -

Pd-GCMs + NO2-

[Pd-GCMs(NO2 )]

[Pd-GCMs(NO2-)]

Pd-GCMs +NO2 + e

2NO2 + H2O -

NO2 + H2O

(1) -

-

2H+ + NO3 + NO2 -

-

-

2H+ + NO3 + 2e

(2) rate-determining step (3) (4)

3.2.3. Diffusion coefficient (D) and standard heterogeneous rate (ks) The diffusion coefficient of nitrite on Pd-GCMs/GCE was investigated by chronoamperometry with different concentrations of nitrite (0.2-1.2 mmol L-1) in 0.10 mol L1

PBS (pH 5.00) at a working potential of +0.85 V (Supplementary data, Fig. S3). The

current response of an electroactive material (nitrite in this case) under diffusion control is described by the Cottrell equation:

I = nFACD1/2/π1/2t1/2

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where n is the total number of electrons transferred (n = 1 in this study), F is the Faraday constant (96,487 C mol-1), A is the real surface area of the electrode (0.11 cm2), C is the bulk concentration (mol cm−3), D is the diffusion coefficient (cm2 s−1) and t is time (s). Experimental plots of I vs. t

−1/2

were used, with the best fit for various concentrations of

nitrite (Supplementary data, Fig. S3A). The slopes of the resulting straight lines were then plotted vs. the nitrite concentration (Supplementary data, Fig. S3B). The resulting slope (26.3±0.1 µA s1/2 mM-1 = nFACD1/2/π1/2 in the Cottrell equation) was used to estimate the mean value of D to be (1.93±0.03) ×10−5 cm2 s−1, which was higher than that with Fe3O4/carbon ionic liquid electrode (0.42×10–7 cm2 s−1) [42], AuNF/Cys/Au electrode (7.36×10−6 cm2 s−1) [43], nano-Pt/P3MT/GCE (1.16×10−5 cm2 s−1) [44], nano-Al2O3/GCE (1.09×10−5 cm2 s−1) and bare GCE (4.15×10−6 cm2 s−1) [45]. The standard heterogeneous rate constant (ks) of nitrite on Pd-GCMs/GCE was estimated based on the following equation [46]:

ks = 2.4⋅e (−0.02F/RT)⋅D1/2⋅(Ep−Ep/2)−1/2⋅ν1/2 where Ep is the peak potential and Ep/2 is the potential when the current is half of the peak current of the electrochemical response. In this work, Ep−Ep/2 (850-790 = 60 mV), D = (1.93±0.03)×10−5 cm2 s−1, ν = 100 mV s−1 and T = 298 K. Thus, ks is (6.25±0.04)×10-3 cm s−1, illustrating a relatively fast sensing process and explaining the sharp feature of the catalytic peak observed for the catalytic oxidation of nitrite at the surface of Pd-GCMs/GCE. 3.3 Optimal conditions 3.3.1. The amount of Pd-GCMs

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The amount of Pd-GCMs on the surface of the electrode played a crucial role in the electrocatalytic activity of the electrode for nitrite oxidation. The effect of 5, 10, 15, 20, 25 and 30 µg of Pd-GCMs was studied by CV. As shown in Fig. 3A, the sensitivity initially increased with the amount of Pd-GCMs from 5 to 20 µg due to the increase in catalyst on the electrode surface, which enhanced the electrocatalytic activity of the electrode. However, at larger amounts, sensitivity decreased, possibly because the increased resistance of a thicker Pd-GCMs layer reduced the electron transfer efficiency of the electrode [24, 26]. Therefore, 20 µg of Pd-GCMs was selected for electrode modification. 3.3.2. Buffer pH The electrocatalytic activity for nitrite oxidation is affected by the pH of the supporting electrolyte. The effect of pH in the range from 3.00 to 8.00 in a 0.10 mol L-1 PBS on the sensitivity of Pd-GCMs/GCE for nitrite detection is shown in Fig. 3B. The maximum value was at a pH of 5.00. The low sensitivity at pH < 5.00 is likely due to the instability of nitrite anions, which are easily decomposed to nitrate ions in strong acidic media [26] as shown in reaction 5: 3NO2− + 2H+ → 2NO + NO3− + H2O

(5)

However, in alkaline solutions, the decrease in sensitivity of Pd-GCMs/GCE for nitrite detection may be caused by the oxide layers which hindered the adsorption of nitrite on the electrode surface [41, 47]. 3.3.3. Working potential To obtain the best electrocatalytic activity for nitrite oxidation on the Pd-GCMs modified GCE, potentials covering the nitrite oxidation peak (0.75, 0.80, 0.85, 0.90, 0.95 and 1.00 V) were investigated by FI-amp. Fig. 3C shows the sensitivity of Pd-GCMs/GCE after injection of nitrite (0.10-0.50 mmol L-1) into the flow system using 0.10 mol L-1 PBS (pH

15

5.00) as the carrier stream at a flow rate of 0.50 mL min-1 with a sample volume of 250 µL. The maximum sensitivity was at 0.90 V, consistent with the CV result (Fig. 2A(c)). Therefore, an applied potential of 0.90 V was selected for further experiments. 3.3.4. Flow rate and sample volume The influence of the flow rate and sample volume was also investigated to improve the sensitivity and detection limit of Pd-GCMs/GCE for nitrite. Flow rates of 0.50, 1.00, 1.50, 2.00, 2.50 and 3.00 mL min-1 and sample volumes of 150, 200, 250, 300, 350 and 400 µL were tested by measuring the current response to 0.10 mmol L-1 standard nitrite, as shown in Fig. 3D. For every sample volume, as the flow rate increased, the current response increased from 0.50 to 2.50 mL min-1, and then slightly decreased at 3.00 mL min-1. The increase in current with the flow rate is due to a higher analyte mass transfer rate to the surface of the modified electrode [48, 49]. When the flow rate is too fast, the contact time between the analyte and modifier is too short [50, 51], resulting in the decrease in response. Moreover, a flow rate that is too high generates back pressure and bubbles, leading to errors in the results. For each flow rate, the current response increased with the sample volume from 150 to 300 µL before reaching a stable current. Considering the highest reproducible current, shortest analysis time, highest sample throughput and lowest chemical consumption, a flow rate of 2.50 mL min-1 and a sample volume of 300 µL were chosen as the optimal conditions.



3.4 Analytical features of Pd-GCMs/GCE for nitrite 3.4.1 Linear dynamic range, limit of detection and limit of quantitation

16

The analytical performance of Pd-GCMs/GCE toward nitrite was determined under the FI-amp optimal conditions. Fig. 4A shows typical flow injection amperometric signals upon successive injections of standard nitrite solutions. The nitrite peak currents are linearly related to the concentration in the range from 0.10 µmol L-1 to 4.0 mmol L-1 (Fig. 4B) with a regression equation of Ipa/µA = (35.0±0.2)Cnitrite/mmol L-1 + (0.4±0.2) (r = 0.9998). The sensitivity (slope of the calibration curve against the electrode surface area, 0.070 cm-2) is 500±5 µA mM-1cm-2. The limit of detection (LOD) and quantitation (LOQ) are 0.030 and 0.11 µmol L-1, respectively (LOD = 3σ/S and LOQ = 10σ/S, where σ and S are the standard deviation of the blank (n=20) and the slope of the calibration curve). These figures of merit were compared with those of other electroanalyses for nitrite determination (Table 1). The Pd-GCMs/GCE sensor has the best sensitivity and LOD with a wide linear range, comparable to that of most sensors. When compared with those applied in forensic science, the figures of merit of this inexpensive electrochemical sensor are much better; this sensor also represents a faster and simpler method that requires less chemical consumption. The sample throughput at the upper limit of linearity, 4.0 mmol L-1, was 165 samples h-1 (the analysis time was 21.8±0.8 s (n=6)). The good performance may be attributed to the distribution of Pd particles on the large surface area of the GCMs. A large amount of Pd-GCMs were present on the electrode resembling a three-dimensional porous structure, resulting in a large surface area, fast electron transfer capability and excellent electrocatalytic activity for the oxidation of nitrite.

< Table 1. >

17

3.4.2 Repeatability Repeatability was evaluated from the current responses of 20 injections of standard nitrite solutions at 0.010, 0.050 and 0.10 mg L-1, which cover the concentration range found in the real samples; the relative standard deviations (RSDs) of the current response are 1.0%, 1.5% and 1.1%, respectively (Supplementary data, Fig. S4). These values were acceptable according to the AOAC guideline, which is less than 7.3% for 10 mg L-1 (0.10 mmol L-1 = 4.6 mg L-1) [52], indicating good repeatability. 3.4.3 Reproducibility The reproducibility was tested by six Pd-GCMs/GCEs prepared at different times to detect a series of nitrite concentrations of 0.010, 0.025, 0.050, 0.075 and 0.100 mmol L-1 under the same conditions. The RSDs of each concentration for the six electrodes are in the range from 1.2 to 3.1% (Supplementary data, Fig. S5A), which is acceptable according to the AOAC recommendation [52]. The sensitivities of the six electrode were 38.7±0.5, 38.7±0.4, 39.7±0.3, 39.9±0.3, 39.5±0.5 and 39.9±0.7 µA mM-1 (Supplementary data, Fig. S5B), which are not significantly different (P > 0.05), and the RSD of the average sensitivity is 1.4%, indicating good reproducibility for different preparations. 3.4.4 Operational stability The operational stability of the Pd-GCMs/GCE was evaluated by measuring and comparing the current response after continuous injection of 0.050 mmol L-1 standard nitrite (the middle value of the series concentration that tested the repeatability and reproducibility) under optimal conditions. The Pd-GCMs/GCE has a good operational stability and can be used up to 268 injections with the average current response of 101±3% and an RSD of 2.8 % (Supplementary data, Fig. S6). After 268 injections, the current response remained at 95% and gradually decreased to reach 90% at 275 injections. A decrease in the catalytic area was

18

confirmed by the smaller catalyst surface coverage (ᴦ, mol cm-2) of the electrode calculated from the area under the reduction peak of palladium using the equation ᴦ = Q/nFA, where Q is the charge gain from integration of the reduction peak of palladium, n is the number of electrons in the reaction (n = 2), F is Faraday’s constant (96,487C) and A is the geometric area of GCE (0.070 cm2) [48]. The catalyst surface coverage after measuring nitrite was (4.11±0.08) ×10-9 (n = 3) mol cm-2 while that of before was (6.00±0.06) ×10-9 mol cm-2 (n = 3). That is, after measuring nitrite, the catalyst surface was 69±1% (n=3) of the original value. 3.4.5 Interference study The developed sensor will be applied to screen GSR via the detection of nitrite; thus, some of the electroactive interferences that may be found in GSR were studied. These interferences include some organic compounds (dinitrobenzene (DNB), dinitrotoluene (DNT), trinitrotoluene TNT), cyclonite (RDX) and urea) and some inorganic components of GSR (Ni2+, Mg2+, Fe3+, Ba2+, Sb2+, Pb2+, Cu2+, Zn2+, NO2- NO3- and SO42-)[53]. The effect of each interference was evaluated using the tolerance limit value, which is defined as the highest concentration in a mixture of nitrite that produces a change in the current response with a relative error of less than ±5.0% [54] compared to the current response to 10.0 µmol L1

nitrite under the optimal conditions. No interference could be detected in the presence of the

organic compounds in GSR, such as 50-fold DNB, 100-fold TNT, DNT and RDX, and 250fold urea concentrations. For inorganic components in GSR, such as 50-fold Ni2+, Mg2+, Fe3+, Ba2+, and Sb2+, 100-fold Pb2+ and Cu2+, 250-fold Zn2+ and 1000-fold NO3- and SO42-, there was no influence on the nitrite determination (Supplementary data, Table S1). These results indicate that Pd-GCMs/GCE can be successfully used to determine the nitrite concentration in the presence of different organic and inorganic compounds in GSR.

19

3.5 Analysis of GSR samples The ability of the developed sensor to measure nitrite in GSR samples was evaluated. The matrix effect was first studied. The slopes of the calibration curves obtained from standard nitrite solutions of 5.0, 10.0, 15.0, 20.0 and 25.0 µmol L-1 were compared to that of the spiked samples. Spiked samples were prepared by spiking the standard nitrite solution at the same concentration into extract GSR samples and were analyzed without dilution under optimized conditions. The slope of the spiked samples was compared with that of standard nitrite by two-way ANOVA. There was no significant different at a 95% confidence level (P > 0.05) between the two slopes, indicating that the matrix effect is negligible. Thus, the measured nitrite concentration in the extracted GSR sample without dilution was directly calculated using the calibration equation. The results from the developed method were compared with those from the spectrophotometric method (Griess method [35]). The results in Table 2 show no significant difference between the two methods. To confirm the accuracy, the recovery was also tested by spiking known nitrite concentrations (5.0, 10.0, 15.0, 20.0 and 25.0 µmol L-1) into the extracted GSR samples. The recoveries for all nitrite concentrations are acceptable and in the range from 80-110% [52], i.e., between 85±3% and 105±1% (Table 2). Thus, the sensor provides accurate results for real sample analysis.

< Table 2. >

4. Conclusions Glassy carbon microspheres decorated with Palladium particles (Pd-GCMs) were successfully synthesized using a simple electroless deposition method and used to fabricate a

20

novel electrochemical sensor for nitrite detection to screen for the presence of GSR. Under optimal conditions, the Pd-GCMs/GCE shows excellent electrocatalytic activity toward nitrite together with a high sensitivity (500±5 µA mM-1cm-2), wide linear range (0.10 µmol L1

to 4.0 mmol L-1) and low limit of detection (0.030 µM). Furthermore, the electrochemical

sensor provides good operational stability (268 injections with RSD = 2.8%), excellent repeatability (RSD < 1.5%, n=20) and reproducibility (RSD =1.4%, n=6), and high sample throughput (165 samples h-1). The sensor was successfully applied for the detection of nitrite in GSR samples. This novel Pd-GCMs/GCE is a potential alternative tool for investigating GSR via nitrite detection for forensic applications, and it can be possibly applied to detect nitrite in environmental and food samples.

Acknowledgments This work was supported by the Thailand Research Fund (TRF) and Prince of Songkla University (grant no. RSA 6280081). The financial support for Kiattisak Promsuwan from the Royal Golden Jubilee Ph.D. program (RGJ) was supported by the Thailand Research Fund (3.C.PS/59/J.1). The Center of Excellence for Innovation in Chemistry (PERCH-CIC), the Office of the Higher Education Commission, the Center of Excellence for Trace Analysis and Biosensor (TAB-CoE) and Graduate School and Faculty of Science, Prince of Songkla University, Hat Yai, Thailand are gratefully acknowledged.

References

21

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25

Table captions Table 1. Comparison with other reported nitrite detection methods Table 2. Determination of nitrite in GSR samples by the proposed method and the Griess method and the recovery test (n = 3)

26

Sensitivity

Linear range

LOD

(µA mM-1cm-2)

(µmol L-1)

(µmol L-1)

417

2–1230

DPV

308.57

Amp

d

Pd/graphite/GCE

e

Modified electrodes

Techniques

Applications

Refs.

a

r

0.25

-

[55]

1-1000

0.23

-

[41]

23.28

500-25500

0.8

Food

[56]

Amp

290

0.3–50.7

0.071

Environmental, Food

[26]

Pd/Fe3O4/polyDOPA/RGO/GCE

Amp

219.57

2.5–6470

0.5

Environmental, Food

[23]

NGE/PdNC/GCE

Amp

342.4

0.5-1510

0.11

Food

[57]

g

Amp

142.86

0.2-5000

0.1

Food

[58]

h

Amp

265

4 - 1440

0.40

Environmental, Food

[59]

SWV

414.51

1 - 10

1.00

-

[60]

DPV

253.23

4 - 2000

0.48

Environmental

[61]

CV

226

5-65000

1.00

-

[62]

NGQDs@NCNFs/GCE

DPV

141

5-3000

3.00

Environmental, Food

[28]

m

Pt/Ni(OH)2/MWCNTs/GCE

Amp

145

0.4–5670

0.13

Food

[17]

n

PtNPs/P3MT/GCE

DPV

222.82

8–1,700

1.5

-

[44]

o

Amp

-

1-1000

0.2

Environmental

[18]

FI Amp

469.51

2-800

4.50

Food

[22]

FI Amp

500±5

0.1-4000

0.030

Forensic science

(This work)

Capillary Electrophoresis

-

-

0.36-72

2.45

Forensic science

[34]

Ion chromatography

-

-

2.17-4348

6.5

Forensic science

[63]

Pd/SWCNT electrode

b

Pd/RGO/GCE

c

Amp

s

np-PdFe/GCE

f

Pd/CoPc/GCE Ag-PAMAM/GCE

i

AgNPs/TPDT–SiO2/GCE

j

Ag/CNC/GR/GCE

k

Au/Zn-MOF-5/GCE

l

Ag/Cu/MWNTs/GCE

p

AgMCs-PAA/PVA/SPCE

q

Pd-GCM/GCE

t

u

v

Other method

Table 1.

27

a

b

Pd/SWCNT electrode: Urchin-like palladium nanostructures on carbon nanotube thin film electrodes. Pd/RGO/GCE: Palladium and reduced graphene oxide

nanocomposites modified glassy carbon electrode. cnp-PdFe/GCE: Nanoporous PdFe alloy modified glassy carbon electrode. dPd/graphite/GCE: Graphitesupported Pd nanoparticles modified glassy carbon electrode. ePd/Fe3O4/polyDOPA/RGO/GCE: Pd/Fe3O4 composite based on poly DOPA functionalized reduced graphene oxide modified glassy carbon electrode. fNGE/PdNC/GCE: Palladium nanocubes/nitrogen-doped graphene nanocomposites modified glassy carbon electrode. gPd/CoPc/GCE: Cobalt phthalocyanine-supported Pd nanoparticles modified a glassy carbon electrode. hAg-PAMAM/GCE: Silver nanoparticles-polyamidoamine

modified

(trimethoxysilyl)propyl]diethylenetriamine-amine/

glassy silica

carbon spheres

electrode.

modified

glassy

i

AgNPs/TPDT–SiO₂/GCE:

carbon

electrode.

Ag

j

Ag/CNC/GR/GCE:

nanoparticles/N-[3Silver/carbon

nano-

composite/grapheme modified glassy carbon electrode. kAu/Zn-MOF-5/GCE: Gold nanoparticles incorporated zinc based metal-organic framework modified glassy carbon electrode. lNGQDs@NCNFs/GCE: N-doped graphene quantum dots decorated N-doped carbon nanofibers modified glassy carbon electrode. m

Pt/Ni(OH)2/MWCNTs/GCE: Pt nanoparticles loaded Ni(OH)2/multi-walled carbon nanotubes nanocomposites modified glassy carbon electrode.

n

PtNPs/P3MT/GCE: Pt nanoparticles and poly(3-methylthiophene) nanorods at a glassy carbon electrode.

o

Ag/Cu/MWNTs/GCE:

Ag/Cu nanoclusters and

multi-walled carbon nanotubes modified glassy carbon electrode. pAgMCs-PAA/PVA/SPCE: Silver microcubics-poly (acrylic acid)/poly (vinyl alcohol) Palladium -glassy carbon microspheres modified glassy carbon electrode. rAmp:

modified screen printed carbon electrode. qPd-GCM/GCE:

Amperometry. sDPV: Differential pulse voltammetry. amperometry.

t

SWV:

Square wave voltammetry. uCV: Cyclic voltammetry. vFI Amp: Flow injection

29

Table 2. Propose method Griess method

% Recovery of propose method (n=3)

Gunshot residue 5.0

10.0

15.0

20.0

25.0

N.D.

N.D.

87±2

96±4

102±3

97±1

99±1

N.D.

N.D.

89±2

102±3

98±2

102±2

98±1

N.D.

N.D.

100±1

103±3

103±2

100±2

102±1

Right hand

3.7±0.2

3.9±0.2

85±3

95±2

93±2

96±1

96±1

Left hand

2.0±0.2

2.2±0.1

89±3

93±3

101±1

98±4

101±1

Cloth

8.8±0.4

8.6±0.1

95±3

96±3

97±1

98±4

100±1

Right hand

7.1±0.2

7.2±0.1

95±2

98±2

98±2

99±1

101±3

Left hand

4.2±0.2

4.0±0.1

90±1

100±2

100±3

97±1

101±1

Cloth

14.0±0.3

14.2±0.1

97±3

100±3

98±2

101±3

105±1

Right hand Before shooting

After shooting (1 shot)

After shooting (3 shot)

Left hand a

Cloth

a

Cloth (Chest area)

b

N.D. = Not detected

Found (µM) (n=3)

Concentration of spiking (µM)

Found (µM) (n=3)

samples

b

30

Figures captions Fig. 1. Electrode characterization showing SEM images of (A) GCMs and (B) Pd-GCMs. (C) Histogram of the Pd particle size on GCMs. EDX spectra of (D) GCMs and (E) Pd-GCMs. Fig. 2. Electrochemical behavior. (A) CVs of the bare GCE (a), GCMs/GCE (b), PdGCMs/GCE (c) in 0.10 mol L-1 PBS (pH 5.00) with 1.0 mmol L-1 nitrite at a scan rate of 100 mV s−1 compared to those without nitrite (a', b', c'). (B) Sensitivities of different modified electrodes for 0.2-1.0 mmol L-1 nitrite. (C) CVs from the Pd-GCMs/GCE in the presence of 0.2-1.0 mmol L-1 nitrite. (D) The relationship between oxidation peak current and nitrite concentration. Fig. 3. The nitrite detection sensitivity at concentrations from 0.10 to 0.50 mmol L-1 in 0.10 mol L-1 PBS (pH 5.00), (A) the influence of the amounts of Pd-GCMs/GCE, (B) the effect of solution pH. The effect of (C) the applied potential on nitrite detection sensitivity (0.10-0.50 mmol L-1), 0.10 mol L-1 PBS (pH 5.00) carrier stream, (D) the flow rate and sample volume on the current response of Pd-GCMs/GCE for nitrite detection at a concentration of 0.10 mmol L-1. Fig. 4. Analytical responses: (A) Flow injection amperograms of nitrite from 0.1 to 4000 µmol L-1 (n=6) with Pd-GCMs/GCE under optimal conditions using 0.10 mol L-1 PBS (pH 5.00) as the carrier stream and (B) the corresponding calibration graph for the peak current of nitrite.

Highlights


Novel nitrite amperometric sensor for gunshot residue screening
Pd-GCMs were prepared by a simple electroless deposition method
Pd-GCMs/GCE exhibited excellent electrocatalytic nitrite oxidation performance
FI amperometric nitrite sensor shows high sensitivity, low LOD and wide linear range
High-throughput screening (165 samples per hour)
This sensor successfully detected nitrite in gunshot residue

Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

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Manuscript title: Nitrite

amperometric sensor for gunshot residue screening

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