Electronic Supplementary Material

Screening for cystic fibrosis via a magnetic and microfluidic immunoassay format with electrochemical detection using a copper nanoparticle-modified gold electrode

Maria Luz Scala Benuzzi, Sirley V. Pereira, Julio Raba, Germán A. Messina*

INQUISAL, Department of Chemistry.National University of San Luis, CONICET. Chacabuco 917. D5700BWS San Luis, Argentina

*Author to whom correspondence should be addressed: ()

(Tel.) +54-266- 425385; (Fax) +54-2652-43-0224.

Electrochemical study of catechol on Au electrode

The study of catechol behavior on Au electrode was carried out by Cyclic Voltammetry. A Q solution in 0.1 M phosphate–citrate buffer (pH 5.0) was evaluated scanning in the potential range of -200 to 550 mV. The cyclic voltammogram obtained showed a good definition of the anodic and cationic peaks, which correspond to the transformation of Q to o-benzoquinone and vice versa. This is a quasi-reversible procedure in which 2 electrons are transferred. The relation of the current peaks, close to one, particularly when a repetitive cycling was performed, can be inferred as a stability criterion of the Q produced on the electrode surface in the work conditions employed.

Fig. S1 Cyclic voltammogram of catechol in aqueous solution containing 1mM of Q in
0.10M phosphate/citrate buffer, pH 5.0 with a Gold electrode. Scan rate: 100mVs−1.

Amount of incorporated MNPs

The amount of MNPs to be incorporated in the central channel of the device was studied in the range of 0.1-0.4 mg, employing a 282 ng mL-1 IRT standard. The optimal amount of MNPs was 0.4 mg, due to the fact that higher amount of particles produces channel obstruction, impeding the normal flow of solutions in the microfluidic system.

Effect of the variation of anti-IRT-Ab concentration

The optimal concentration of anti-IRT-Ab to be immobilized on MNPs surface was evaluated using different concentrations of anti-IRT-Ab (1.0-14.0 μg mL-1). Then a constant and saturating concentration of HRP (1 mg mL-1 of enzyme prepared in PBS) was adsorbed in unoccupied sites (saturation method with peroxidase). The enzymatic activity was studied in Batch by addition of 0.001 M H2O2, 0.001 M Q solution (pH 5.0). Electrochemical reduction was detected at a potential of -150 mV. The generated current was inversely proportional to the amount of immobilized anti-IRT-Ab. The optimal anti-IRT-Ab concentration to MNPs modification was 10 μg mL-1 (Fig. S2a).


Effect of flow rate

In a microfluidic immunosensor, the flow rates of the transported solutions from the channel input to the detection system play an important role in the detection procedure.

For the determination of IRT, the flow rate was analyzed evaluating the current generated employing a 282 ng mL-1 IRT standard at different flow rates: 1, 2, 3, 4, 5 and 7 μL min-1 (Fig. S2b). The signal obtained was constant in the flow range 1-3 μL min-1and it was significantly reduced when the flow rate exceeded 4 μL min-1. Therefore, a flow rate of 2 μL min-1 was used for samples, reagents, and washing solutions.

Fig. S2 Optimization of experimental conditions. The effect of the variation of anti-IRT-Ab concentration (a), effect of flow rate (b), evaluation of the sample volume (c).

Optimal ranges of sample volume, temperature and pH

The sample volume was evaluated in the range of 1-15 μL, using a 282 ng mL-1 IRT standard (Fig. S2c). The response rate increased linearly when the sample volume grew between 1-10 μL, insignificant differences were obtained for greater sample volume. As a result, the sample volume employed for IRT determinations was 10 μL.

A study of the enzymatic response under flow conditions was performed in the pH range of 3.0 to 8.0 and exhibited a maximum value of activity at pH 5.05. Moreover, the best enzyme activity is shown over the temperature range of 20 to 25 °C [40].

Effect of reaction time

The analysis time represent a relevant factor in the microfluidic immunosensor performance. For this reason, we evaluated the incubation time, the minimum time required for IRT binding to anti-IRT-Ab-MNP, as a strategy to minimize the total analysis time.

Fig. S3 shows how the current generated varies for different incubation times. The concentration 53, 154, 282 and 580 ng mL-1 IRT standards were used. The current intensity increased when the IRT concentration grew. The intensity of the current increased with the reaction time, however, did not increase considerably until 5 min had passed, which was likely due to saturation of the specific antibody sites. Therefore, the optimal reaction time was 5 min.

Fig. S3 Current intensity as a function of reaction time for 53, 154, 282 and 580 ng mL-1 IRT standards. 0.1 M phosphate–citrate buffer, pH 5.0, containing 0.001 M H2O2 and 0.001 M Q were injected at 2 µL min−1, and the enzymatic product was measured by amperometry at -150 mV.

Effect of the variation of catechol and H2O2 concentration

The H2O2 concentration was varied from 0.1 to 10 mM, while Q concentration was 10 mM and the current response was studied. On the other hand, the Q concentration was varied in the range of 0.15-25 mM for a 10 mM H2O2 solution and the signal response was studied. According to the obtained results, the optimal concentrations of H2O2 and Q were 1 mM.

Amplification effect of the obtained signal

With the purpose of evaluate the amplification effect resulting from the incorporation of the MNPs, the signal intensity obtained using the sensor was compared with a signal obtained from a similar sensor in which the immunoreagents were incorporated by a direct modification of the central channel with –APTES.

For these experiments, the central channel was pretreated-treated according to the following steps: 15 min washed with 1 M HCL, 15 min rinsed with deionized water and 15 min with methanol at 2 µL min-1. After preconditioning procedure, the 2.5 % (v/v) APTES in methanol solution was introduced into the glass channel inner surface at 2 µL min-1 for 30 min and left to stand filled with the silanization solution for 2 h. Then, the channel was rinsed with methanol and dried under nitrogen flow. Later, aqueous solution of 5 % w/w glutaraldehyde in 0.20 M CO32-/ HCO3- buffer at pH 10.00 was injected to induce the formation of aldehyde groups at 25 °C for 2 h. After that, it was left to stand filled with the same solution solutions for 60 min and finally rinsed with phosphate buffer at pH 7.2 for 15 min. Finally, a solution of 10 µg mL-1 anti-IRT-Ab in 0.10 M phosphate buffer of pH 7.2 was injected through the central channel of the modified chip and left to react at 25 °C for 2 h. Finally, the central channel was rinsed with phosphate buffer at pH 7.2 to remove the unbound anti-IRT-Ab and stored in the same buffer at 4°C.