Density Control over MBD2 Receptor-Coated Surfaces Provides Superselective Binding of Hypermethylated DNA.

Using the biomarker hypermethylated DNA (hmDNA) for cancer detection requires a pretreatment to isolate or concentrate hmDNA from nonmethylated DNA. Affinity chromatography using a methyl binding domain-2 (MBD2) protein can be used, but the relatively low enrichment selectivity of MBD2 limits its clinical applicability. Here, we developed a superselective, multivalent, MBD2-coated platform to improve the selectivity of hmDNA enrichment. The multivalent platform employs control over the MBD2 surface receptor density, which is shown to strongly affect the binding of DNA with varying degrees of methylation, improving both the selectivity and the affinity of DNAs with higher numbers of methylation sites. Histidine-10-tagged MBD2 was immobilized on gold surfaces with receptor density control by tuning the amount of nickel nitrilotriacetic acid (NiNTA)-functionalized thiols in a thiol-based self-assembled monolayer. The required MBD2 surface receptor densities for DNA surface binding decreases for DNA with higher degrees of methylation. Both higher degrees of superselectivity and surface coverages were observed upon DNA binding at increasing methylation levels. Adopting the findings of this study into hmDNA enrichment of clinical samples has the potential to become more selective and sensitive than current MBD2-based methods and, therefore, to improve cancer diagnostics.

Controlled pharmacokinetic anti-cancer drug concentration profiles lead to growth inhibition of colorectal cancer cells in a microfluidic device.

We present a microfluidic device to expose cancer cells to a dynamic, in vivo-like concentration profile of a drug, and quantify efficacy on-chip. About 30% of cancer patients receive drug therapy. In conventional cell culture experiments drug efficacy is tested under static concentrations, e.g. 1 µM for 48 hours, whereas in vivo, drug concentration follows a pharmacokinetic profile with an initial peak and a decline over time. With the rise of microfluidic cell culture models, including organs-on-chips, there are opportunities to more realistically mimic in vivo-like concentrations. Our microfluidic device contains a cell culture chamber and a drug-dosing channel separated by a transparent membrane, to allow for shear stress-free drug exposure and label-free growth quantification. Dynamic drug concentration profiles in the cell culture chamber were controlled by continuously flowing controlled concentrations of drug in the dosing channel. The control over drug concentrations in the cell culture chambers was validated with fluorescence experiments and numerical simulations. Exposure of HCT116 colorectal cancer cells to static concentrations of the clinically used drug oxaliplatin resulted in a sensible dose-effect curve. Dynamic, in vivo-like drug exposure also led to statistically significant lower growth compared to untreated control. Continuous exposure to the average concentration of the in vivo-like exposure seems more effective than exposure to the peak concentration (Cmax) only. We expect that our microfluidic system will improve efficacy prediction of in vitro models, including organs-on-chips, and may lead to future clinical optimization of drug administration schedules.

Increasing the Sensitivity of Electrochemical DNA Detection by a Micropillar-Structured Biosensing Surface.

The available active surface area and the density of probes immobilized on this surface are responsible for achieving high specificity and sensitivity in electrochemical biosensors that detect biologically relevant molecules, including DNA. Here, we report the design of gold-coated, silicon micropillar-structured electrodes functionalized with modified poly-l-lysine (PLL) as an adhesion layer to concomitantly assess the increase in sensitivity with the increase of the electrochemical area and control over the probe density. By systematically reducing the center-to-center distance between the pillars (pitch), denser micropillar arrays were formed at the electrode, resulting in a larger sensing area. Azido-modified peptide nucleic acid (PNA) probes were click-reacted onto the electrode interface, exploiting PLL with appended oligo(ethylene glycol) (OEG) and dibenzocyclooctyne (DBCO) moieties (PLL-OEG-DBCO) for antifouling and probe binding properties, respectively. The selective electrochemical sandwich assay formation, composed of consecutive hybridization steps of the target complementary DNA (cDNA) and reporter DNA modified with the electroactive ferrocene functionality (rDNA-Fc), was monitored by quartz crystal microbalance. The DNA detection performance of micropillared electrodes with different pitches was evaluated by quantifying the cyclic voltammetric response of the surface-confined rDNA-Fc. By decrease of the pitch of the pillar array, the area of the electrode was enhanced by up to a factor 10.6. A comparison of the electrochemical data with the geometrical area of the pillared electrodes confirmed the validity of the increased sensitivity of the DNA detection by the design of the micropillar array.