Abstract
Single cylindrical submicron pores in PMMA polymer membranes are micropatterned by electron beam lithography and integrated into all PMMA-based electrophoretic flow detector systems. Pore dimensions are 450 nm in diameter and 1 μm in length. The pores are electrically characterized in aqueous KCl electrolyte, exhibiting a stable time-independent ionic current through the pore with a noise level of less than 1% of the open-pore current. The current-voltage curves are linear and scale with electrolyte concentration. The negative surface charge of the membrane over-proportionally decreases pore conductance at low electrolyte concentrations (≤0.1 M) that are still beyond those typically applied in biological experiments. Pores do not exhibit rectification of current flowing through them, allowing for operation with either polarity. To allow for detection of yet much smaller particles, the described PMMA-based system also was successfully equipped with pores of 1.5 nm instead of 450 nm in diameter. This was achieved by introducing naturally occurring biological protein pores of α-hemolysin on a lipid bilayer into the prepatterned PMMA membrane of an assembled PMMA-based electrophoretic flow detector system. Characteristics of translocation events of single-stranded linear plasmid DNA molecules through the pores were recorded, and ionic current deductions during biomolecule translocation were clear and distinguished. Based on the presented submicron scale open pore ionic current transport properties, as well as the observed passage of DNA molecules through protein pores inserted into PMMA membranes, our current research proposes that all PMMA electrophoretic flow detectors exhibit an excellent potential for future use as biomedical resistive-pulse sensors, as long as pore dimensions match those of biomolecules to be detected.
Highlights
Resistive-pulse sensing (Coulter counting) is a powerful method for detection and characterization of micro- and nano-scale particles and biomolecules such as viruses, DNA molecules, and proteins [1,2,3,4,5])
Transient changes in Iopen will occur when a particle slightly smaller than the pore traverses. This resistive-pulse can be analyzed to derive information regarding the particle size and even its morphology, concentration in the electrolyte, and affinity for the pore [6,7]: pulse amplitudes in the ionic current through the pore are proportional to the volume of the passing particles, the frequency of pulses is related to the concentration of particles in a sample flowing through the pore, and the residence time of a particle in the pore is related to its structure, affinity towards the pore, as well as its velocity
Under the impact of an outer electric field induced by the electrodes, an ionic current through the pore drags molecules to be analyzed through the pore which partially block the volume of the channel, resulting in an increased impedance which can be detected by a pico ammeter in a patch clamp setup
Summary
Resistive-pulse sensing (Coulter counting) is a powerful method for detection and characterization of micro- and nano-scale particles and biomolecules such as viruses, DNA molecules, and proteins [1,2,3,4,5]). Considerable effort has been recently devoted toward the development of artificially micropatterned apertures in engineered inorganic membranes These offer several advantages over their biological counterparts including size control over a wide range, increased chemical, electrical, mechanical, and thermal stability, tunable surface properties, and a potential for integration into different devices [13,14]. The most widely used methods employ ion or electron beams to create pores in silicon nitride or silicon oxide membranes [13,14,15] Such silicon-based membrane materials have been chosen for decades of fabrication experience (etching or sculpting processes) and general operations considerations (biocompatibility and mechanical, chemical and thermal stability). We give a concise overview over the EBL micropatterning and assembly sequence, complement EBL by self assembling a protein pore into the PMMA detector as a rapid prototyping approach to extend the area of applications by decreasing the pore size two orders of magnitude, electrically qualify and characterize the submicron pores, and report the first resistive-pulse sensing data on the transport of single stranded (SS) linear plasmid DNA molecules across a PMMA membrane of an all polymer electrophoretic flow detector system
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