Figure 1 Regulation effect of NarP and NarL on the expression of nrfA and nirB nitrite reductase genes

Figure 2 Overall workflow of our cell experiment

Figure 3 Three validation methods of constructed E. coli strain with enhanced nitrite-digesting ability

For point-of-care testing, our sensor design could be generally divided into Arduino-based electrochemical detection system and screen-printed electrode sensing system.

Figure 4 Overall workflow of our sensor experiment

Figure 5 shows the result of we use different concentration of GelMA (15%, 20%, 30% (W/V)) to test immobilizing E. coli on cell orifice plate. According to the result, the 15% concentration of GelMA could perfectly meet our requirements fabricating GelMA coating on SPE surface.

Figure 5: (A) Use UV light to treat GelMA;
(B) Use GelMA lysis buffer to lysis for 5 mins;
(C) GelMA in 15%, 20% and 25% (w/v); (D) Coating GelMA on SPE surface.

Cyclic voltammetry was conducted to investigate the electrochemical performance of the SPE in the presence of 1.0 mM [Fe (CN)6]3-/4- containing 0.1 M KCl at a scan rate of 100 mV s−1. In the absence of electroactive species, the cyclic voltammogram showed a low background current without an oxidation or reduction peak. This indicates that graphite ink components are not electroactive in the CVs potential range. However, a pair of redox peaks appeared in the presence of 1.0 mM [Fe (CN)6]3-/4-. This suggests that [Fe (CN)6]3-/4- has a quasi-reversible behavior. As shown in Figure 6, the cyclic voltammetry results of the screen-printed electrode indicated that the electrode worked ideally.

Figure 6: (A) 1st circle of Cyclic Voltammetry using [Fe (CN)6]3-/4-as the standard probe;
(B) Cyclic Voltammetry using [Fe (CN)6]3-/4-as the standard probe for 1st to 20th circles.

Using standard nitrite solution [0 mM, 1 mM, 3 mM, 4 mM, 6 mM] as sample solution, we applied cyclic voltammetry method with E initial=-1.2V, E final=0.6V, scan rate of 100mV s-1. The results of the experiment demonstrate that the sensor system successfully detected the signal of the nitrite solution as indicated by the current response wave crest at -1.05V. Although the characteristic peak didn’t show at -0.8V as expected, from the experiment we have conducted, we assume the cell contents have interfere with the signal readout. The overall characteristic shape of the current-potential diagram is in consistence with the reference article (Monteiro, 2015). Hence it can be concluded that the sensor system did function. However, as we vary the concentration of nitrite in the tested solution, the curves of the current-potential diagram, which is shown below, didn’t changes accordingly as indicated in the refence article. Although the result shows a positive linear relationship between the nitrite concentration in the sample solution and the peak value of the current response, the range of response variation is too small, indicating that our sensor needs further improvement in the detection sensitivity. And according to our preliminary judgment, it may be the mixture of cell contents/lysis buffer that interferes enzymatic reaction. This behavior exhibits the need for us to improve the sensitivity of the sensor system in the future.

Figure 7:
(A) CV result of 0 mM Nitrite standard solution at a scan rate of 100 mV s−1;
(B) CV result of 1 mM Nitrite standard solution at a scan rate of 100 mV s−1;
(C) CV result of 3 mM Nitrite standard solution at a scan rate of 100 mV s−1;
(D) CV result of 4 mM Nitrite standard solution at a scan rate of 100 mV s−1;
(E) CV result of 6 mM Nitrite standard solution at a scan rate of 100 mV s−1. All the potential E (V) is vs pseudo-Ag/AgCl;
(F) linear correlation between the ΔIcat at −1.05 V (slope 120 mA M −1cm−2, R2 0.919) and NO2− concentration.

Hardware

Based on our experiments, our sensor and the detecting system have equipped fundamental selectivity and sensitivity for nitrite detection and could eliminate some of the preliminary interferences from samples. The testing samples include drinking (mineral) water, milk, plasma, natural water, etc. Also, selectivity, repeatability, and reproducibility are examined. For repeatability, the electrochemical response of a single electrode does not change significantly during consecutive runs. To test reproducibility, we have made sure that the results of electrochemical responses of seven different electrodes fabricated in the same batch are similar. For selectivity, we have continued to test the samples with common interfering ions like uric acid, ascorbic acid and CN- in the future.

Click here to see specific measurement ability of our sensor.