A Nitrite Biosensor Based on Coimmobilization of Nitrite Reductase and Viologen‐Modified Polysiloxane on Glassy Carbon Electrode

Abstract

Copper containing nitrite reductase (Cu-NiR) and viologen-modified sulfonated polyaminopropylsiloxane (PAPS-SO 3 H-V) were co-immobilized on glassy carbon electrode (GCE) by hydrophilic polyurethane (HPU) drop-coating, and the electrode was tested as a reagentless electrochemical biosensor for nitrite detection. The newly synthesized PAPS-SO 3 H-V as an electron transfer (ET) mediator between electrode and NiR was effective, and could be effectively immobilized in HPU membrane. The NiR and PAPS-SO 3 H-V co-immobilized GCE used as a nitrite biosensor showed the following performance factors: sensitivity ¼ 12.0 nA mM À1, limit of detection (LOD) ¼ 60 nM (S/N ¼ 3), linear response range ¼ 0 -18 mM (r 2 ¼ 0.996) and response time (t 90% ) ¼ 60 s, respectively. LineweaverBurk plot shows that apparent Michaelis -Menten constant (K app M Þ is 101 mM. Storage stability of the sensor is 51 days (80% of initial activity) in condition of storing in ambient air at room temperature. The sensor showed a relative standard deviation (RSD) of 3.2% (n ¼ 5) even in condition of injection of 1 mM nitrite. Interference study showed that common anions in water sample such as chlorate, chloride, sulfate and sulfite do not interfere with the nitrite detection. However, nitrate interfered with a relative sensitivity of 80% due to inherent character of the enzyme used.

A Nitrite Biosensor Based on Co-immobilization of Nitrite Reductase and Viologen-modified Chitosan on a Glassy Carbon Electrode

“…This pH profile is in accordance with that we reported previously [17,22], and this optimal pH is also very comparable to those for free Cu-NiRs purified from different sources, generally at pH 6.0−6.2 [25,30,31]. On the other hand, it was reported that the solubility transition point of chitosan is ∼6.3 [39–42], and this may be partially responsible for the sharp decreases of response in the 6.5−8.0 pH range.…”

“…Stable successive CV scans showing ΔE p = 0.159 V and i p,a/ i p,c = 0.657 were obtained. A similar result was also obtained in the case where NiR and PAPS-V were co-immobilized on a GCE by drop-coating of HPU [22]. Figure 5 shows CVs recorded with the co-immobilized NiR and CHIT-V GCE at a scan rate of 0.002 V/s.…”

“…This is a typical EC’ process [47] and the sigmoid shape of the CV corresponds to the mediated catalytic reduction scheme shown in Figure 1. Similar electro-enzymatic catalytic reductions of nitrite by NiR and poly-viologen co-immobilized electrodes were also reported [15,22]. Control experiments were also conducted under similar conditions with a co-immobilized CHIT-V and bovine serum albumin (BSA) electrode (no NiR, data not shown), and those results showed that no catalytic reduction current could be observed, as reported [15,17–19].…”

“…In the case reported here, 6 μg of CHIT-V resulted in immobilization of 8.3 × 10 −10 mol of viologen (see discussion in Section 3.2), which equals 9.2 × 10 −11 mol viologen·μg −1 CHIT-V. Similarly, a value of 2.3 × 10 −10 mol viologen·μg −1 PAPS-V was obtained for the reported case [22]. Very interestingly, the optimal mass ratio obtained in this study is in good accordance with that reported in the other one, i.e., 3.0 μg NiR:5.5 × 10 −10 mol viologen (this study) vs. 2.5 μg NiR:5.8 × 10 −10 mol viologen (reported).…”

“…A similar profile was also obtained with NiR and polyviologen co-immobilized on a GCE [15], and in another similar study with NiR and PAPS-V co-immobilized on a GCE, a mass ratio of 1:1 (2.5 μg:2.5 μg) was obtained [22]. In the case reported here, 6 μg of CHIT-V resulted in immobilization of 8.3 × 10 −10 mol of viologen (see discussion in Section 3.2), which equals 9.2 × 10 −11 mol viologen·μg −1 CHIT-V.…”