Electronic Supplementary Material

Pt@Pd nanowire and horse radish peroxidase dual-amplified DNA based electrochemical biosensor for the detection of Mycoplasma pneumoniae

Linlin Liu, Guiming Xiang, Dongneng Jiang, Chang Liu, Weiwei Huang, Xiaoyun Pu*

Department of Clinical Laboratory, XinqiaoHospital, ThirdMilitaryMedicalUniversity, Chongqing, China

The optimization of analytical conditions for target DNA detection

In order to improve the analytical performance of the DNA sensor, the capture DNA1 concentration, incubation time of M. pneu. target DNA and H2O2 concentration were optimized in our experiment. The results were shown in Fig. S1A, S1B and S1C, respectively. As shown in Fig. S1A, the electrochemical signal decreased with the concentration capture DNA1 concentration ranged from 0.5 μM to 1.5 μM, and reached plateau regions at the concentration of 2 μM. Thus, the optimal concentration of capture DNA1 was 2 μM.

The incubation time for the interaction between the capture DNA1 and M. pneutarget DNA was also studied. Fig S1B shows the electrochemical signal of the modified electrode for M. pneu. (1 nM) in 1 mL of 5 mM [Fe(CN)6]3−/4− solution under different incubation time (from 60 min to 140 min). From Fig. S1B we can see that the electrochemical signal decreased with the increasing of the incubation time of M. pneutarget DNA and reached level off at 120 min. Therefore, 120 min is optimal for the biosensor.

The concentration of H2O2 in the detection cell played a very important role in the signal amplification process, which further influenced the sensitivity of the biosensor. Therefore, the optimization of H2O2 concentration was also investigated. As shown in Fig. S1C, the reduction peak current change increased with the increasing of H2O2 concentration and tended to reach saturation at the concentration of 1.6 mM. Thus, 1.6 mM was chosen as the optimal H2O2 concentration in the whole measurement process.

Fig. S1.The optimization of (A) capture DNA1 concentration and (B) M. pneu. related DNA incubation time in 5 mM Fe(CN)6]3−/4− solution and the optimization of (C) H2O2 concentration in 1 mL phosphate buffer (pH 7.0). Scan rate: 100 mV s-1.

Fig. S2The stability of the proposed DNA biosensor with CV technique in 1 mL PBS (pH 7.0)at a scan rate of 100 mV s-1.

Comparative analysis with PCR

All experiments were performed in compliance with the relevantlaws and institutional guidelines, and the appropriate authoritieskindly approved the experimental work.All the clinical samples (human nasopharyngealwash specimens) werecollected from XinqiaoHospital.Total sixty clinical samples(thirty positive, thirty negative)were identified by the electrochemical detection measurement and the standard PCR method.

First, clinical samples were decontaminatedusing 1M NaOH at 95°C for 10 min and subsequentlyconcentrated by centrifugation. DNA of sampleswas purified using theTaKaRa MiniBEST Bacterial Genomic DNA extraction kit(Takara Bio, Shigo, Japan), according to the manufacturer'sinstructions. The extracted DNA samples were retained at -20°C for further use.

Then, the PCR assay of extracted DNAs,initial amplificated at 95 °C for 120 s, followed by 40 cycles each at 94 °C for 15 s , 50 °C for 30 sand 72°C for 45 s, and concluded at 72 °C for 10 min after the final cycle.Each PCR well had a final volume of 25 μL and contained 0.4 μmol·mL-1 of each primer, 2.0 μL extracted DNA，1.0 μL Taqman DNA polymerase，and12.5μL Taqman Fast Universal Real-Time Master Mix. The products were resolved using 2% agarose gel and visualised using a Gel Logic 212 Imaging system (Kodak, Japan). The extracted DNAs were then detected by the electrochemical method at the same time.

Last, the results were compared with difference (χ2 test), accuracy,sensitivity, and specificity by SPSS 18.0 software (IBM, USA).The results of comparison between the electrochemical detection measurement andPCRidentification (Table S1) suggested that there was no significant difference between the electrochemical detection measurement and PCR identification (P > 0.05). The electrochemical detection measurement had high accuracy (95.00%), sensitivity (93.33%), and specificity (96.67%)compared to the standard PCR method.a