Electrokinetic Scanning Probe.

The theoretical analysis and experimental demonstration of a new concept are presented for a non-contact scanning probe, in which transport of fluid and molecules is controlled by electric fields. The electrokinetic scanning probe (ESP) enables local chemical and biochemical interactions with surfaces in liquid environments. The physical mechanism and design criteria for such a probe are presented, and its compatibility with a wide range of processing solutions and pH values are demonstrated. The applicability of the probe is shown for surface patterning in conjunction with localized heating and 250-fold analyte stacking.

Real-Time Monitoring of Fluorescence in Situ Hybridization Kinetics.

We present a novel method for real-time monitoring and kinetic analysis of fluorescence in situ hybridization (FISH). We implement the method using a vertical microfluidic probe containing a microstructure designed for rapid switching between probe solution and nonfluorescent imaging buffer. The FISH signal is monitored in real time during the imaging buffer wash, during which signal associated with unbound probes is removed. We provide a theoretical description of the method as well as a demonstration of its applicability using a model system of centromeric probes (Cen17). We demonstrate the applicability of the method for characterization of FISH kinetics under conditions of varying probe concentration, destabilizing agent (formamide) content, volume exclusion agent (dextran sulfate) content, and ionic strength. We show that our method can be used to investigate the effect of each of these variables and provide insight into processes affecting in situ hybridization, facilitating the design of new assays.

Focused upon hybridization: rapid and high sensitivity detection of DNA using isotachophoresis and peptide nucleic acid probes.

We present a novel assay for rapid and high sensitivity detection of nucleic acids without amplification. Utilizing the neutral backbone of peptide nucleic acids (PNA), our method is based on the design of low electrophoretic mobility PNA probes, which do not focus under isotachophoresis (ITP) unless bound to their target sequence. Thus, background noise associated with free probes is entirely eliminated, significantly improving the signal-to-noise ratio while maintaining a simple single-step assay requiring no amplification steps. We provide a detailed analytical model and experimentally demonstrate the ability to detect targets as short as 17 nucleotides (nt) and a limit of detection of 100 fM with a dynamic range of 5 decades. We also demonstrate that the assay can be successfully implemented for detection of DNA in human serum without loss of signal. The assay requires 15 min to complete, and it could potentially be used in applications where rapid and highly sensitive amplification-free detection of nucleic acids is desired.