Supplemental Information

Supplemental Information

Mechanistic Insight into a Novel Ultrasensitive Nicotine Assay Base

on High-Efficiency Quenching of Gold Nanocluster Cathodic

Electrochemiluminescence

Zhongnan Huang,† Zhenglian Li,† Luyao Xu,† Chaoguo Wei,† Chenting Zhu,† Haohua Deng,† Huaping Peng,*, † Xinghua Xia,‡ and Wei Chen*,†

†_{Higher Educational Key Laboratory for Nano Biomedical Technology of Fujian}

Province, Department of Pharmaceutical Analysis, Fujian Medical University, Fuzhou 350004, China

‡_{State Key Laboratory of Analytical Chemistry for Life Science and Collaborative}

Innovation Center of Chemistry for Life Sciences, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, China

Zhongnan Huang and Zhenglian Li contributed equally to this work.

*Corresponding author. E-mail address: [email protected] (H.P. Peng); [email protected] (W. Chen)

Table of contents

1. Synthesis of NAC-AuNCs ... S3 2. Fabrication of CR-AuNC/GCE ... S3 3. The mechanism of CR-AuNCs/K2S2O8 ECL system ... S3

4. The stability of CR-AuNC/GCE………..S4 5. UV-visible absorption spectrum of nicotine and ECL spectrum of AuNCs modified

GCE... S4 6. HPLC of standard niacin solutions and treated nicotine solution ... S4 7. Cyclic voltammogram of nicotine ... S5 8. Optimization of pH ... S6 9. Stability evaluation of the ECL sensor. ... S6 10. Table of analytical performance for the detection of nicotine. ... S7 11. References. ... S8

Synthesis of NAC-AuNCs. The NAC-AuNCs were prepared through a hydrothermal method according to our previous work.1 _{In brief, 0.6 mL of NaOH (0.5 M) and 0.4}

mL of HAuCl4 (48.56 mM) were added to 4 mL of 0.08 M N-acetyl-L-cysteine. The

mixture was incubated at 37 °C for 2.5 h, and the resulting solution was dialyzed for more than 24 h to remove all small-molecular impurities. The purified AuNCs were stored in the dark at 4 °C when not use.

Fabrication of CR-AuNC/GCE. The CR-AuNC/GCE was prepared through chemical reduction method according to our previous work.1 The glassy carbon electrode (GCE) was sequentially polished with 0.3 and 0.05 μm of alumina for 3 min and ultrasonically cleaned for 3 minutes in HNO3 solution (VHNO3:VH2O=1:1), ethanol

and deionized water, respectively. Then, NAC-AuNC/GCE was obtained by rapid drop-casting 5 μL NAC-AuNCs solution onto the surface of cleaned GCE and dried in air at room temperature. After that, the NAC-AuNC/GCE was immersed in a 0.1 M sodium borohydride (NaBH4) solution for 5 min to obtain chemically reduced (CR)

electrode, which was recorded as CR-AuNC/GCE. This resulting electrode was rinsed with water to remove the excess of the physical adsorption and stored at 4 °C for the further ECL detection.

The mechanism of CR-AuNCs/K2S2O8 ECL system. Based on the previous work

on CR-AuNCs/K2S2O8 ECL system,1 AuNCs were immobilized on the GCE and

converted to AuNC•− at negative potential (Eq. (S1)), while the peroxydisulfate could be electro-chemically reduced to sulfate radical anion (SO4•−) (Eq. (S2)). Then the

strong oxidant SO4•− undergoes an electron transfer reaction with the reduced species

of AuNCs (AuNC•−) to generate light (Eq. (S3)-(S4)).

AuNC + e− →AuNC•− (S1) S2O82− + e− → SO42− + SO4•− (S2)

AuNC•−+ SO4•− → AuNC* + SO42− (S3)

AuNC* → AuNC + hν (S4)

Figure S1. The stability of CR-AuNC/GCE at (A) 4 °C and (B) room temperature in K2S2O8 solution (0.1 M, pH 7.4), respectively.

Figure S2. UV-visible absorption spectrum of nicotine (red curve) and ECL spectrum of CR-AuNC/GCE in 0.1 M K2S2O8 (black curve).

Figure S3. HPLC of standard niacin solutions.

Figure S4. HPLC of nicotine standard sample treated with CR-AuNC/GCE by SP method in air-saturated aqueous solution without K2S2O8.

The E0(niacin/nicotine) was measured by cyclic voltammogram (CV) of bare GCE in 0.1 M PBS containing 1 mM nicotine with the potential cycled between 0 and +1.4 V at a scan rate of 100 mV⋅s−1. In this process, the conventional three-electrode system was used for detection: a bare GCE was used as the working electrode, Pt wire was used as the auxiliary electrode, and an Ag/AgCl (saturated KCl solution) was used as the reference electrode, respectively. As shown in Figure S5, the anodic peak at 0.93 V versus Ag/AgCl was corresponding to the oxidation of nicotine. Thus, the

Epa, nicotine was 0.93 V versus Ag/AgCl.

Figure S5. Cyclic voltammogram of 1 mM nicotine in 0.1 M PBS solution at a scan rate of 100 mV s-1.

Optimization of pH in the proposed ECL system. pH can not only interfere with the signal intensity of the CR-AuNCs/S2O82-system, but also can affect the ECL

inhibition efficiency of nicotine.With the aim of realizing the ultrasensitive detection of nicotine, the optimal pH of this system was explored. Taking the ECL intensity change of I0 - I as the function of the concentration of nicotine, I0 was the initial

intensity and I was the ECL intensities of CR-AuNC/GCE in K2S2O8solution in the

presence of nicotine, the nicotine-induced decrease in ECL intensity is given as I0 - I.

As shown in Figure S6, the ECL quenching effect elevated significantly with an increase in the pH value and the maximum value of ΔI appeared at pH of 7.4. With a further increase in the pH value, however, resulted in a dramatically decrease in the ECL quenching effect. Nicotine is a moderately strong base with pKa1=8.0 and

pKa2=3.0. When pH is more basic than 8.0, the neutral molecule in solution is the

predominant existence form of nicotine2_{. However, this molecule is fairly volatile}3

leads to decreased the concentration of nicotine and weaken the quenching effect. Thus, 7.4 was chosen as an optimal pH for nicotine detection and this pH was also favorable for the applications in biological sample analysis.

Figure S6. Effects of pH on ECL intensity difference (ΔI). (ΔI=I0-I, I0 stand for the

ECL intensity of CR-Au NCs/GCE, I stand for the ECL intensity of CR-Au NC/GCE in the presence of nicotine)

Figure S7. Stability evaluation of the purposed biosensor for three days.

Table S1. Comparison of the analytical performance of different methods in determination of nicotine.

Method Dynamic Linear range (mol/L) a_LOD (mol/L) Reference Electrochemistry 2.00 × 10-6–5.40 × 10-4 1.34 × 10-8 4 Electrochemistry 5.00 × 10-8–5.00 × 10-4 1.50 × 10-8 5 Electrochemistry 5.00 × 10-7–3.00 × 10-4 1.20 × 10-7 6 Raman spectroscopy 0.00 × 100_{–4.00× 10}-5 _{6.00 × 10}-8 ₇ Electrochemistry 1.00 × 10-7–5.00 × 10-4 5.00 × 10-9 8 Electrochemistry 5.00 × 10-5–7.00 × 10-4 1.20 × 10-5 9 Electrochemistry 1.00 × 10-5–1.00 × 10-4 1.46 × 10-7 10 Electrochemistry 1.00 × 10-4–1.00 × 10-3 1.00 × 10-6 11 Electrochemistry 4.00 × 10-6–5.00 × 10-4 9.43 × 10-8 12 Electrochemistry 8.00 × 10-7–8.00 × 10-4 3.60 × 10-9 13 Electrochemistry 1.00 × 10-9–1.00 × 10-7 3.50 × 10-8 14 Electrochemistry 0.00 × 100–2.00× 10-4 4.70 × 10-8 15 HPLC 7.70 × 10-8_{–3.08 × 10}-6 _{6.16× 10}-8 ₁₆ Fluorecscence 0.00 × 100_{–1.00× 10}-3 _{9.8× 10}-7 ₁₇ ECL 7.20 × 10-8_{–1.00 × 10}-4 _{7.20 × 10}-8 ₁₈ ECL 1.00 × 10-7_{–1.00 × 10}-4 _{6.90 × 10}-8 ₁₉ ECL - 1.60 × 10-6 ₂₀ ECL 2.00 × 10-6_{–3.00 × 10}-4 _{1.25 × 10}-9 ₂₁ ECL 5.00 × 10-7_{–5.00 × 10}-5 _{5.00 × 10}-8 ₂₂ ECL 1.00 × 10 -11_{–1.00 × 10}-5 1.00 × 10-4_{–1.00 × 10}-3 7.00×10 -13 _{This work}

a_{LOD, limit of detection (S/N=3). HPLC, high performance liquid chromatography; ECL,}

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