Abstract
Standard protocols for the analysis of circulating tumor DNA (ctDNA) include the isolation of DNA from the patient’s plasma and its amplification and analysis in buffered solutions. The application of such protocols is hampered by several factors, including the complexity and time-constrained preanalytical procedures, risks for sample contamination, extended analysis time, and assay costs. A recently introduced nanoparticle-enhanced surface plasmon resonance imaging-based assay has been shown to simplify procedures for the direct detection of tumor DNA in the patient’s plasma, greatly simplifying the cumbersome preanalytical phase. To further simplify the protocol, a new dual-functional low-fouling poly-l-lysine (PLL)-based surface layer has been introduced that is described herein. The new PLL-based layer includes a densely immobilized CEEEEE oligopeptide to create a charge-balanced system preventing the nonspecific adsorption of plasma components on the sensor surface. The layer also comprises sparsely attached peptide nucleic acid probes complementary to the sequence of circulating DNA, e.g., the analyte that has to be captured in the plasma from cancer patients. We thoroughly investigated the contribution of each component of the dual-functional polymer to the antifouling properties of the surface layer. The low-fouling property of the new surface layer allowed us to detect wild-type and KRAS p.G12D-mutated DNA in human plasma at the attomolar level (∼2.5 aM) and KRAS p.G13D-mutated tumor DNA in liquid biopsy from a cancer patient with almost no preanalytical treatment of the patient’s plasma, no need to isolate DNA from plasma, and without PCR amplification of the target sequence.
Highlights
Standard protocols for the analysis of circulating tumor DNA include the isolation of DNA from the patient’s plasma and its amplification and analysis in buffered solutions
To demonstrate that the PLL-mal(26%)-peptide nucleic acid (PNA)-CEEEEE surface layer combined with nanoparticle-enhanced surface plasmon resonance imaging (SPRI) detection of mutated and wild-type DNAs in human plasma provides a new platform for the direct analysis of plasma samples from cancer patients, we adsorbed PLL-mal(26%) on the SPRI gold surface and immobilized PNA probes (0.1 μM in PBS, flow rate 10 μL min−1) as already described (Figures 3 and 1a)
SPRI responses detected after the adsorption of plasma samples (300 μL, 10 μL min−1) spiked with p.G12D-mutated gDNA on PLL-mal(26%)-PNA-CEEEEE layers bearing PNAWT and PNA-G12D (Figure S13) confirmed the antifouling properties of the surface layer and provided no helpful signal to identify the specific interactions between p.G12D-mutated gDNA and the complementary PNA-G12D probe
Summary
CTACGTCACCAGCT-Gly-NH2 G13D aSPDP, N-succinimidyl 3-(2-pyridyldithio)propionate; PEG, poly-. To demonstrate that the PLL-mal(26%)-PNA-CEEEEE surface layer combined with nanoparticle-enhanced SPRI detection of mutated and wild-type DNAs in human plasma provides a new platform for the direct analysis of plasma samples from cancer patients, we adsorbed PLL-mal(26%) on the SPRI gold surface and immobilized PNA probes (0.1 μM in PBS, flow rate 10 μL min−1) as already described (Figures 3 and 1a). SPRI responses detected after the adsorption of plasma samples (300 μL, 10 μL min−1) spiked with p.G12D-mutated gDNA on PLL-mal(26%)-PNA-CEEEEE layers bearing PNAWT and PNA-G12D (Figure S13) confirmed the antifouling properties of the surface layer and provided no helpful signal to identify the specific interactions between p.G12D-mutated gDNA and the complementary PNA-G12D probe. Article adsorption as a consequence of the surface fouling resistance introduced by PLL-mal(26%)-PNA-CEEEEE
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