boron-doped diamond film for electroanalysis of l -alanine

Accepted Manuscript Title: Amperometric biosensor based on nanoporous nickel/boron-doped diamond film for electroanalysis of L -alanine Author: Wei Dai Mingji Li Hongji Li Baohe Yang PII: DOI: Reference:

S0925-4005(14)00524-3 http://dx.doi.org/doi:10.1016/j.snb.2014.05.005 SNB 16879

To appear in:

Sensors and Actuators B

Received date: Revised date: Accepted date:

23-2-2014 25-4-2014 1-5-2014

Please cite this article as: W. Dai, M. Li, H. Li, B. Yang, Amperometric biosensor based on nanoporous nickel/boron-doped diamond film for electroanalysis of L -alanine, Sensors and Actuators B: Chemical (2014), http://dx.doi.org/10.1016/j.snb.2014.05.005 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. 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.

Highlights

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1. Self-supporting BDD films were prepared by EA-CVD. 2. Nanoporous-Ni coatings were fabricated by ultrasound-assisted electrodeposition at the surface of BDD electrode. 3. The nanoporous Ni/BDD electrode showed the best electrochemical activity. 4. The proposed system provides ultrahigh sensitivity and good selectivity for the determination of L-alanine.

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Amperometric biosensor based on nanoporous nickel/boron-doped diamond film for electroanalysis of L-alanine Wei Dai a, Mingji Li b, *, Hongji Li c, Baohe Yang a, b, * School of Precision Instrument and Optoelectronics Engineering, Tianjin University,

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a

Tianjin 300072, P. R. China

Tianjin Key Laboratory of Film Electronic and Communicate Devices, School of

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b

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Electronics Information Engineering, Tianjin University of Technology, Tianjin 300384, P. R. China

Tianjin Key Laboratory of Organic Solar Cells and Photochemical Conversion, School

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c

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of Chemistry & Chemical Engineering, Tianjin University of Technology, Tianjin 300384, P.R. China.

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*Corresponding author: Mingji Li; Baohe Yang

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Tel: +86 022 60215346

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E-mail: [email protected]; [email protected]

ABSTRACT

This paper presents an electrochemical biosensor for the detection of L-alanine based

on a nanoporous (NP) nickel (Ni)-modified boron-doped diamond (BDD) electrode. Self-supporting BDD films are prepared using an electron-assisted hot filament chemical vapor deposition method. Electrochemical studies show that increased surface concentration of redox moieties on the NP-Ni/BDD electrode leads to high electron transport and improved sensing performance. The electrochemical response of the NP-Ni/BDD electrode as a function of L-alanine concentration exhibits a linear range

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from 0.5 to 4.5 μM, and a detection limit of 0.01 μM at a sensitivity of 0.05 μA μM−1 cm−2 and a regression coefficient of 0.998. Furthermore, the proposed biosensor also

Boron-doped

diamond

electrode;

Nanoporous

nickel;

L-alanine;

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Keywords:

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showed high anti-interference ability, excellent stability and good reproducibility.

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Electrochemical sensor; Electrodeposition. 1. Introduction

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Quantification of L-amino acids for assessing the quality and genuineness of food,  drinks, and medicines [1] as well as their nutritional and physiological relevance is of

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importance [2]. There is an ongoing interest in the development of a reliable, rapid, and accurate method of L-amino acid analysis. Flow injection [3], chromatography [4],

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atomic absorption [5], and colorimetric [6] methods have been employed for the determination of L-amino acids. These methods have several disadvantages such as high

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cost, long analysis time, and low sensitivity, and have thus been unsuitable for routine analysis. Among these, electrochemical analysis has inherent advantages of simplicity, easy miniaturization, high sensitivity, and relatively low cost. Therefore, different chemically modified electrodes have been developed for the oxidation and detection of L-amino

acids [1, 7]. Over the last few years, the measurement of alanine has been a

focus for various purposes. For clinical analyses, the deficiency of alanine in blood has been implicated as a primary etiologic factor in ketotic hypoglycemia and the development of hypoglycemia associated with Sheehan’s syndrome and hypopituitarism [8, 9]. While L-amino acid biosensors capable of amperometric detection of cysteine [10, 11], lysine [12], tyrosine [13], and tryptophan [14, 15] are receiving much attention, Page 3 of 23

there are relatively few studies on alanine biosensors [9]. Boron-doped diamond (BDD) is one of the most promising new materials for electroanalytical measurements. Among the different electrode materials studied so far,

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BDD is distinct from conventional electrode materials because of several important technological properties such as biocompatibility, extremely wide electrochemical

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potential window, low and stable voltammetric background currents, corrosion stability

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in very aggressive media, an inert surface with low adsorption properties, very low double-layer capacitance, a strong tendency to resist deactivation, and robust oxidation

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capacity [16]. These attributes make the BDD electrode well suited for current-based

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electrochemical measurements. Literature studies have revealed that various nickel metal-, nickel hydroxide-, and nickel oxide- deposited electrodes show good

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electrocatalytic activity for the anodic oxidation of hydrogen peroxide [17],  L-histidine

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[18], tetracycline [19, 20], and glucose [21-23].

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In this study, two-dimensional nanoporous (NP)-Ni structures have been developed as a BDD electrode modifier for the first time. The resulting NP-Ni-modified BDD electrode was used for the sensitive determination of L-alanine, showing good results. 2. Experimental

2.1. Preparation of BDD electrode Self-supporting BDD electrodes were deposited on Mo substrates (50 mm in diameter

and 4 mm thick) using electron-assisted hot filament chemical vapor deposition Methane (CH4) and hydrogen (H2) were used as the reactant sources. Trimethyl borate (B(OCH3)3) was used as the source of boron and was dissolved in ethanol according to the preset [B]/[C] atomic ratio of 0.1% w/w. The BDD film thickness was

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approximately 140 μm after 144 h deposition. The resulting diamond film (Ф 50 mm × thickness 140 μm) was cut into several pieces (1 cm × 1.5 cm). A PTFE electrode holder was used to clamp the BDD film, and a platinum wire contact was formed between

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them. The exposed geometric area of the electrode was 1 cm2 (Fig. 1D). 2.2. Electrodeposition of nanoporous Ni layer

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NP-Ni coatings were fabricated by an ultrasound-assisted electrodeposition process at

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the surface of the BDD electrode. Prior to use, the BDD electrodes were sonicated in 2-propanol for 10 min. The electrochemical deposition of the NP-Ni layer was

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performed using cyclic voltammetry (CV) from −0.6 to 0.6 V with a scan rate of 100

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mVs−1 (80 segments) in an electrolyte composed of 0.1 M NaH2PO4 and 2 mM Ni(NO3)2. The experiments were carried out in an electrolytic cell at a temperature of

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298 ±2 K. Ultrasonic wave (50 W) irradiation was used during the deposition. The

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obtained NP-Ni/BDD electrode was washed carefully with redistilled water and then

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dried at room temperature.

2.3. Measurement and apparatus A field emission-scanning electron microscope (FE-SEM; JEOL JSM-6700F) was

used for morphological observations. The microstructure of the BDD was characterized by Raman spectroscopy (Raman, Thermo Scientific, DXR) using a YAG laser (excitation wavelength 532 nm) as the excitation source. The resistivity and Hall effect of the BDD film were determined using a Bio-Rad Microscience HL5000 Hall system. A conventional three-electrode system was employed. An Ag/AgCl (3 M KCl) and a Pt-wire electrode were used as reference electrode and counter electrode, respectively (insert of Fig.1D). Bare or modified BDD electrodes were used as the working electrode.

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Electrochemical impedance spectroscopy (EIS), CV, and differential pulse voltammetry (DPV) were used to characterize the fabrication of the electrode, using a IM6/Zennium (Zahner-Electrik,

Germany)

electrochemical

workstation

and

50

mL

of

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K3Fe(CN)6/K4Fe(CN)6 (1:1), NaOH, and L-alanine solution.

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3. Results and Discussion

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3.1. Morphological and structural characterization

Fig. 1A and B show SEM images of the bare BDD electrode. The typical size of the

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diamond crystals was between 10 and 20 μm. The BDD electrode consisted of randomly

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oriented crystallites with both cubic {100} and triangular {111} planes exposed on the surface. The grain size was evaluated by calculating the grain density based on the

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number of grains per surface unit. The growth surface exhibited 340 grains per square

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millimeter, indicating that the grain size was largest at the BDD surface.

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The quality of the BDD film was confirmed by Raman spectroscopy (Fig. 1C). As shown in Fig. 1C, four distinctive peaks around 586.3, 909.3, 1032.7, and 1327.8 cm−1 in the visible Raman spectrum were observed. The narrow peak located at around 1327.8 cm−1 is a characteristic diamond peak due to first order scattering on the diamond (sp3) crystal lattice. Three weak peaks located at 586.3, 909.3, and 1032.7 cm−1 were also observed. The 586.3 and 1032.7 cm−1 peaks were both superposed, including not only C vibrations but also B-B vibrations and B-C vibrations, respectively. The broad peak at 586 cm−1 is known to be related to phonon scattering by boron induced structural modifications. This proved that boron atoms had been incorporated into the diamond lattice. No sp2-bonded graphitic carbon at about (G peak) 1580 cm−1 and no

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graphitic carbon (D peak) at about 1350 cm−1 were found for this BDD film, which indicated that the non-diamond carbon content was very low. Hall effect measurements indicated clear p-type conductivity, with a resistivity of 520 mΩ cm and a hole carrier

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concentration equal to 1.8 × 1019 cm−3 at room temperature. The surface morphology of the as-prepared NP-Ni/BDD electrode was examined by

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SEM (Fig. 1E and F). As can be seen, the as-prepared electrode had thin

to form nanoporous structures (Fig. 1F).

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Fig. 1 here

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two-dimensional (2D) nanoplate-like morphology, and the nanosheets were interleaved

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3.2. Electrochemical characteristics of the electrodes

Cyclic voltammograms (CV) of the bare GC, Ni/GC, BDD and NP-Ni/BDD

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electrodes recorded in 0.1 M NaOH solution at a sweep rate of 10 mV s−1 are shown in

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Fig. 2A. In background voltammograms, the BDD electrode exhibited a cathodic and

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anodic potential limit near −1.33 and 0.65 V, respectively, whereas those of the GC electrode were at −0.32 and 0.56 V (vs. Ag/AgCl), respectively. The overpotentials for hydrogen and oxygen evolution were both qualitatively greater (wider window) at BDD and NP-Ni/BDD than at the GC and Ni/GC electrode. From this study, it is apparent that the BDD shows a higher cathodic limit, an important criterion for electrodeposition of metals, and lower background current than the GC. On the other hand, the NP-Ni/BDD electrode showed well-defined reduction and oxidation peaks at potentials of 0.412 and 0.508 V, respectively (Fig. 2B). In the blank NaOH solution, the NP-Ni/BDD showed a pair of redox peaks, assigned to the Ni(II)/Ni(III) redox couple in alkaline medium as follows [24]:

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Ni + 2OH− →Ni (OH)2 +2e−,

(1)

Ni (OH)2 +OH− →NiO(OH)+H2O +e−.

(2)

Fig. 2C presents the CVs of all four electrodes. While that of the Ni/GC and

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NP-Ni/BDD electrodes showed a pair of redox peaks, no peaks were observed for the GC or BDD electrodes. At the Ni/GC electrode, showed a weak electroactiviy of

. At the NP-Ni/BDD electrode, the anodic peak potential (Epa) and cathodic

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L-Alanine

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peak potential (Epc) were located at 549 and 369 mV (vs. Ag/AgCl) respectively, with a

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peak-to-peak separation (ᇞEp) of 180 mV. Because L-alanine has two acid-base

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equilibria, with pKa of 2.35 and 9.87, at 25 °C, the stabilities of its cationic and anionic forms in aqueous solution depend on the pH :

(3)

CH3CH(NH3+)COO−↔CH3CH(NH2)COO− + H+.

(4)

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CH3CH(NH3+)COOH↔CH3CH(NH3+)COO− + H+,

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Taking into account the pKa values and overlooking the buffer effect, more than

90% of L-alanine molecules in aqueous solution are anionic at 25 °C and above pH 11 [25].

The oxidation and reduction peaks of the composite in the absence of L-alanine were

attributed to the oxidation of Ni(OH)2 to NiO(OH) and successive reduction back. The catalytic oxidation of L-alanine to pyruvate by the high valent NiO(OH) occurred when L-alanine

was added. Besides, the oxidation of Ni(OH)2 to NiO(OH) could result in the

decrease of active sites for L-alanine adsorption. Along with a poisoning effect of the products or intermediates of the reaction, the overall rate of L-alanine oxidation could be decreased. All these effects could have caused the anodic peak shift for the L-alanine Page 8 of 23

oxidation. These processes can be explained by the following reaction: NiO(OH) + L-alanine+ OH−→Ni (OH)2 + pyruvate.

(5)

Fig. 2D shows the Nyquist plots of the bare GC, Ni/GC, BDD, and NP-Ni/BDD

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electrodes in 0.1 M KCl solution containing 5.0 mM K3Fe(CN)6/K4 Fe(CN)6 (1:1). The measured EIS data were simulated with a modified Randles equivalent circuit (EC) as

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shown in the inset of Fig. 3A. The EC consists of a serial resistance and two loops. The

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serial resistance Rs corresponds to the electrolyte resistance between the working electrode and the Ag/AgCl reference electrode. The first loop involves the ionic

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charge-transfer resistance at the film/electrolyte interface (Rint) and the constant phase

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element (CPE) [26]. The second loop, with the charge-transfer resistance (Rct) of the redox probe moving through the film, a Warburg element (Zw) and the double layer

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capacitance (C), could be considered to be due to diffusion phenomena. C appears in

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parallel with Rct and Zw, and is assumed to represent the capacitive contribution of the

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double-layer [26, 27]. The values of Rct were determined and found to be 177 Ω cm2 at the bare GC, 5.6 Ω cm2 at the Ni/GC, 98.2 Ω cm2 at bare BDD and 25.91 Ω cm2 at NP-Ni/BDD electrodes. The Rct of the NP-Ni/BDD electrode was smaller than those of the bare GC and BDD electrodes, which indicated the successful formation of a NP-Ni film, thus promoting the electron transfer of the redox probe to the BDD electrode surface.

Fig. 2 here

3.3. Effects pH and of scan rate

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Fig. 3A shows the DPV curves of the NP-Ni/BDD electrode in 0.1 M NaOH solution at different pH. The peak potentials and the magnitude of the current response were pH dependent (insets a and b). DPV curves with stable and well-defined peaks

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were observed in the pH range from 12.0 to 14.0, but both the anodic and the cathodic peak potentials were shifted negatively with increasing pH. This result indicates that

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solution pH has an impact on the electrochemical behavior of L-alanine because protons

L-alanine

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participate in the electrode process. The linear equations relating Epa and pH for are Epa = −0.116 pH + 1.9 (R = 0.998) and Epc = −0.112 pH + 1.9 (R = 0.993)

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(inset a). The shift in Ep depended on pH, suggesting that the redox reaction was

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accompanied by the transfer of proton. All the values of the slopes (116 mV / pH for Epa and 112 mV / pH for Epc) are larger than the theoretically expected value, 59 mV/pH for

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one electron and one proton reaction. The reason might be the influence of the

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deprotonation and de-amine of L-alanine. Therefore, the mechanism of the

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electro-oxidation of L-alanine can be proposed as follows [28]: Ni(OH)2 +OH− ↔NiO(OH)+H2O +e−,

(6)

CH3CH(NH2)COOH↔CH3CH(NH2)COO−+H+,

(7)

CH3CH(NH2)COO− ↔ CH3COCOO−+NH3.

(8)

CH3CH(NH2)COO− +2OH− ↔ CH3CNHCOO- + 2H2O + e− CH3CNHCOO− + H2O ↔ CH3CHNH2+HCO3−

(9) (10)

Inset (b) in Fig. 3A illustrates the variation in the redox peak current of L-alanine with pH. It can be seen that the oxidation peak currents of L-alanine slightly decrease as the pH is increased from 12.0 to 14.0. The reduction peak currents increase as the pH is increased from 12.0 to 13.5, presumably owing to instability in the alkaline media. Electrostatic repulsion between the analytes and the NP-Ni/BDD electrode might also Page 10 of 23

contribute to the reduced peak current [29]. The effect of scan rate (ν) on the CV response was also investigated. From Fig. 3B, we can observe the redox peak currents and peak potentials of L-alanine on the

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NP-Ni/BDD electrode. The plots of the anodic and cathodic peak currents (Ipa and Ipc) were linearly dependent on the square root of scan rate (ν1/2) in the range 10–280 mV s–1

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Fig. 3 here

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NP-Ni/BDD electrode were diffusion controlled processes [30].

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(Fig. 5B), indicating that the redox reactions of the analyte at the surface of the

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3.4. Amperometric determination of L-alanine by NP-Ni/BDD electrode Fig. 4A shows the amperometric response of the NP-Ni/BDD electrode at 0.55 V

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with successive additions of L-alanine. The calibration curve for the NP-Ni/BDD

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electrode is shown in the inset of Fig. 4A. A good linear relationship (I (μA) =0.05 C

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(μM)+1.348, R=0.998) was exhibited and the concentration was in the range of 0.5–4.5 μM, with a detection limit of 0.01 μM based on S/N=3. The detection limit was less than the previously reported values of 29.67 μM for NiO nanoparticle-modified GC [9] and 7.2 μM for a salicylate hydroxylase/L-alanine dehydrogenase/pyruvate oxidase trienzyme system [8]. The capabilities of the present NP-Ni/BDD electrodes are superior to those of other materials developed. These are absolutely caused by the synergistic effect of NP-Ni and BDD, which facilitated high catalytic activity towards L-alanine

and large active surface area. Fig. 4 here

3.5. Effect of interfering species

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The measured effects of different interferents with L-alanine at 0.55 V are shown in Fig. 5. A substance was considered to be an interfering substance when the relative error (Er) exceeded 5%. It was found that high concentrations of most ions and common

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substances only caused a negligible change: Na+, K+, Cl−, Cu2−, Ca2+, Zn2+, Mg2+, Ni2+, NO3−, SO42−, CH3COO− (500-fold); hydroquinone, catechol, resorcinol (5-fold); and

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glucose, L-lysine, L-serine, L-glycine, L-cysteine (10-fold). The amperometric responses

L-glutamic

L-phenylalanine,

acid,

L-arginine,

L-aspartic

L-histidine,

acid,

L-valine,

L-methionine,

L-cystine,

L-proline,

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L-threonine,

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at the NP-Ni/BDD electrode to the addition of thirteen kinds amino acids (L-leucine, L-tyrosine,

L-isoleucine)

as

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interfering substances (4-fold) in 0.1 M NaOH at 0.55 V were shown in Fig. 5d. A notable response was produced immediately when 100 μM L-alanine was added. There

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was no significant change of the response current to be observed after the additions of

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these amino acids. The further addition of 100 μM L-alanine produced a noteworthy

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response, revealing the excellent selectivity of the proposed sensor. These results indicate that the present NP-Ni/BDD electrode has an excellent anti-interference ability for L-alanine detection.

Fig. 5 here

3.6. Stability, repeatability and reproducibility studies The repeatability of one NP-Ni/BDD electrode was examined by CV measurements

using 100 μM L-alanine in 0.1 M NaOH (pH 13). The electrode exhibited relative standard deviations (RSDs) of 1.80% and 3.10% for 100 successive cycles at an electrode modified once (Fig. S3A) and for five different measurements at an electrode modified five times (Fig. S3B). The reproducibility of the NP-Ni/BDD electrode was

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also examined by CV measurements. Five electrodes were fabricated based on the same process to examine their CV responses, and the RSD was 4.76% (Fig. S3C). The satisfactory RSD values obtained at the NP-Ni/BDD electrode validated their good

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repeatability and reproducibility for the determination of L-alanine. Finally, the storage stability of the NP-Ni/BDD electrode was investigated by measuring its sensitivity (S)

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over three weeks under ambient conditions, during which the sensor retained 74.38% of

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its original sensitivity (Fig. S3D). 4. Conclusions

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The BDD electrode consisted of randomly oriented crystallites with both cubic {100}

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and triangular {111} planes exposed on the surface. We have found that BDD exhibits negligible background capacitive currents from charging–discharging of the electrode

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surface. NP-Ni coatings were fabricated using ultrasound-assisted electrodeposition at

through NP-Ni/BDD film is demonstrated. The results indicate that the NP-Ni

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L-alanine

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surface of the BDD electrode. An electrochemical biosensor for the detection of

layer provides a suitable microenvironment for the adsorption of L-alanine, leading to improved sensing parameters. The fabricated sensor exhibited a sensitivity of 0.05 μA μM−1cm−2 at a detection limit of 0.01 μM. Acknowledgements: This work was supported by the Hi-tech Research and

Development Program of China (863 Program, 2013AA030801), the National Nature Science Foundation of China (no. 61301045, 61106007), the Natural Science Foundation of Tianjin (no. 13JCZDJC36000), the Excellent Young Teachers Program of Tianjin. References

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Biographies

Wei Dai was born in 1982 in Tianjin (China). He received his B.S. degree from the Bio-medical

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Engineering School in Tianjin Medical University in 2006. Later, he received his M.S. degree from the School of Electronics Information Engineering in Tianjin University of Technology. He is currently

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pursuing his Ph.D. in the School of Precision Instrument and Opto-electronics Engineering in Tianjin University. His research interests include Diamond, Ni nano-materials and their applications for

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electrochemical sensors.

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Mingji Li received his B.Sc. degree in physics from the Department of Physics of Jilin University in 2001, and received a Ph.D. degree in the National Laboratory of Super-hard Materials from the Jilin University in 2006. Currently, he works as a professor at Tianjin University of Technology. His research interests

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include nanomaterials, carbon-based thin films, and electrochemical sensors and biosensors.

Hongji Li received her B. Eng. degree in material science from the College of Material Science and

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Engineering of Jilin University in 2001. She received her doctoral degree from the College of Material Science and Engineering of Jilin University in 2006. Currently, she is a lecturer at Tianjin University of Technology, China. Her research interests include electrochemical sensors and related nanomaterials.

Baohe Yang received his Ph.D. in the Department of Microelectronic and Solid Electronic from Hebei University of Technology in 2003 and is currently professor in the School of Electronics Information Engineering, Tianjin University of Technology. His research interests focus on microwave communication devices, high-frequency surface acoustic wave devices, and thin film electronic devices. He is the Director of the Tianjin Key Laboratory of Film Electronic and Communicate Devices.

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Figure Captions

Fig. 1. SEM images showing a (A) top view and (B) side view of the BDD film. (C)

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Raman spectrum of the BDD electrode. (D) Cyclic voltammogram (CV) for the BDD electrode recorded in 0.1 M NaH2PO4 containing 2 mM Ni(NO3)2 at a scan rate of 100

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electrode. (E,F) SEM images of the NP-Ni/BDD electrode.

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mV s−1. Insets show photos of the electrolytic cell and nanoporous nickel (NP-Ni)/BDD

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Fig. 2. (A) CVs of bare GC, Ni/GC, BDD and NP-Ni/BDD electrodes recorded in 0.1 M

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NaOH at a scan rate of 10 mV s−1. (B) High-magnification of the rectangular area in (A). (C) CV curves of bare GC, Ni/GC, BDD and NP-Ni/BDD electrodes in 0.1 M NaOH

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(pH 13) solution containing 0.1 mM L-alanine at a scan rate (v) of 100 mV s−1.

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(D) Nyquist plots of EIS at a bare GC, Ni/GC, BDD and NP-Ni/BDD electrodes in 0.1

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M KCl solution containing 5.0 mM K3Fe(CN)6/K4 Fe(CN)6 (1:1). Inset is the equivalent circuit applied to model impedance spectra data in the presence of the redox probe.

Fig. 3. (A) DPVs recorded on NP-Ni/BDD electrode in 0.1 mM L-alanine solution adjusted to pH of 12.0, 12.5, 13.0, 13.5 and 14.0. (Inset a) Linear dependence between peak potential and pH. (Inset b) Linear dependence between peak current and pH. (B) CVs of the NP-Ni/BDD electrode exposed to 0.1 mM L-alanine in 0.1 M NaOH at different scan rates: 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260 and 280 mV s−1. (Inset) Plots of peak current vs. ν1/2.

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Fig. 4. Amperometric responses of NP-Ni/BDD electrode at 0.55 V, in stirred 0.1 M NaOH with successive additions of L-alanine. The inset is the plot of electrocatalytic current vs. L-alanine concentration.

μM

L-alanine

and other interferents as indicated. (a1–a10):

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Fig. 5. Interference test of the NP-Ni/BDD electrode in 0.1 M NaOH at 0.55 V with 10 L-alanine,

Na2HPO4,

KH2PO4, FeCl3, Zn(NO3)2, Cu(CH3COO)2, CaCl2, NiSO4, Mg(NO3)2, Na2S; (b1–b7):

phenol, aniline, hydroquinone, catechol, resorcinol, uric acid; (c1–c8):

L-alanine,

glucose, L-lysine, L-serine, L-glycine, L-cysteine, ascorbic acid, dopamine;

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L-alanine,

(d1-d14): L-alanine, L-leucine, L-threonine, L-glutamic acid, L-aspartic acid, L-valine,

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L-cystine, L-tyrosine, L-phenylalanine, L-arginine, L-histidine, L-methionine, L-proline,

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L-isoleucine.

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Figure(s)

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Fig. 2.

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Fig. 3.

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Fig. 4.

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Fig. 5.

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