Single copy-sensitive electrochemical assay for circulating methylated DNA in clinical samples with ultrahigh specificity based on a sequential discrimination–amplification strategy† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c7sc01035d Click here for additional data file.

A sequential discrimination–amplification (SEDA) electrochemical strategy was constructed for the identification of single-copy circulating methylated DNA.


Sample preparation
In vitro methylated DNA was obtained with M.SssI enzyme. Briefly, aliquots of 2 μl of 10× NEBuffer 2, 2 μl of S-adenosylmethionine (SAM, 3.2 mM), 4 U of M.SssI and 2 μg of DNA extracted from healthy volunteers were added to a final volume of 20 μl, followed by incubation at 37 ºC for an hour. The enzyme was then inactivated at 65 ºC for 20 min, and the solution was directly used for bisulfite treatment with an EZ DNA Methylation-Gold Kit (ZYMO Research) in accordance with the manufacturer's instructions. Final elution was performed with 20 μl of M-elution buffer. These bisulfite-treated DNA was aliquoted and stored at -20 ºC until ready for use. Blood samples from NSCLC patients and healthy volunteers were obtained from The Second Affiliated Hospital of Xi'an Jiaotong University with written informed consent for DNA isolation and employment. All experiments were performed in compliance with the relevant laws and institutional guidelines.Blood samples were firstly centrifuged at 2000× g, and then supernatant plasma was carefully removed and collected. 200 μl of plasma was used for DNA extraction with a Serum/Plasma Circulating DNA Kit (Tiangen Biotech, Beijing, China). A final elution volume of 20 μl was used, followed by sodium bisulfite conversion as mentioned above. In order to yield more concentrated DNA, 10 μl of elution volume was recommended for circulating DNA from this real sample.

Self-Assembly of the DNA Tetrahedral Probes at Gold Electrode Surface.
The interfacial DNA nanostructured probes was derived from the previous published protocol. 1 We kept the sequence of the bottom three strands (Tetra-B, C and D, in Table 1) and lengthened Tetra-A sequence, yielding an appended pendant ssDNA probe to gain high hybridization selectivity. The lengthened 34 nt probe (Tetra-A highlighted in red) span the region (AT highlighted in green) which is directly extended by the labeled reverse primer with a 2 nt spare. 2.5 μl of four nucleotides (Tetra-A, B, C, D, each 20 μM initial concentration in TE buffer) were mixed in 5 μl of TM buffer with 5 μl of TCEP (30mM) to a final volume of 50 μl (30 μl water added), yielding the tetrahedral concentration of 1 μM. The solution was heated to 95 ºC for 5 min, then quickly cooled down to 4 ºC over 30 s and incubated until use. The gold electrodes (Au, 2 mm in diameter) were cleaned according to the reported protocol. After drying with N 2 gas, 3 μl of the as-prepared tetrahedral solution were added to the surface and incubated overnight at room temperature. (Cap the electrodes with plastic electrode caps in order to prevent solutions from dying up.)

Primer design
This assay contraposed tumor-associated aberrant p16 methylation. The p16INK4a tumor suppressor gene that maps to chromosome band 9p21, is linked to the earliest stages of respiratory carcinogenesis. 2 This gene's hypermethylation status has been studied many times. [3][4][5] Thus, taken together these primers used, we designed the asymmetric MSP primers, 150F and LR (LR was rationally labeled with a biotin unit in its 5' terminal). According to the known sequence of p16 gene promotor obtained from NCBI and the predicted bisulfite converted sequence through the bioinformatics program 6 (http://www.urogene.org/cgibin/methprimer/methprimer.cgi), we fixed the reverse primer at the original place (LR) and moved the forward primer closer step by step while considering the primer designing criteria in Oligo 7. Eventually we obtained three more forward primers (125F, 102F, 81F). The template strand M was 58nt and the 3' dA terminal of M is a spacer. So, if not consider the dA overhang at the 3' end generated by the Hot Start Taq DNA Polymerase 7 , the target amplicon is 57nt.

PCR condition
All PCR were run on a LightCycler 480 (Roche). To a 20 μl reaction volume, 7 μl of water, 2 μl of 10 × Standard Taq Reaction Buffer, 2 μl of dNTP (2mM), 2 μl of forward primers (200nM), 2 μl of reverse primers (2 μM), 1 μl of DNA and 2 μl of Hot Start Taq Polymerase were added and mixed well. Asymmetric PCR procedures (92 ºC for 15 sec, 64 ºC for 20 sec and 72 ºC for 15 sec) were performed for 50 cycles. After the PCR amplification, certain volume was taken out to mix with the H-buffer and the final volume was 5 μl for an electrode to hybridize with the probe. For non-tail ssDNA target obtaining, 10 nM of strand M was added as the template with 200 nM of reverse primer (LR) in a 20 μl of PCR reaction system and thermo cycling on the LightCycler 480 with a normal APCR program.
Hybridization based AMSP product detection AMSP product was mixed in a certain volume or by a certain dilution with the Hbuffer. The electrode was incubated with 5 μl of the mixture for an optimized time at the room temperature and then rinsed by W-buffer. After dried with N 2 gas, the electrode was then incubated with 5 μl of avidin-HRP (1:1000 dilution in 1× PBS containing 1% BSA) for 15 min, followed by washing with W-buffer. After that, each electrode was detected by cyclic voltammetry (CV) and subsequent chronoamperometric measurement in the substrate of TMB.        When more template DNA was added, the primer-dimer reduced and the target amplicon increased.