Label-Free In-Flow Detection of Single DNA Molecules using Glass Nanopipettes

Abstract

With the view of enhancing the functionality of label-free single molecule nanopore-based detection, we have designed and developed a highly robust, mechanically stable, integrated nanopipette-microfluidic device which combines the recognized advantages of microfluidic systems and the unique properties/advantages of nanopipettes. Unlike more typical planar solid-state nanopores, which have inherent geometrical constraints, nanopipettes can be easily positioned at any point within a microfluidic channel. This is highly advantageous, especially when taking into account fluid flow properties. We show that we are able to detect and discriminate between DNA molecules of varying lengths when motivated through a microfluidic channel, upon the application of appropriate voltage bias across the nanopipette. The effects of applied voltage and volumetric flow rates have been studied to ascertain translocation event frequency and capture rate. Additionally, by exploiting the advantages associated with microfluidic systems (such as flow control and concomitant control over analyte concentration/presence), we show that the technology offers a new opportunity for single molecule detection and recognition in microfluidic devices.

On-Demand Delivery of Single DNA Molecules Using Nanopipets

“…26,27 The reproducibility of the molecular delivery between individual pulses is illustrated in Gershow and Golovchenko, have shown that the probability of recapture decreases dramatically with the time elapsed before the application of a reverse potential (V + here). 34 Indeed, recaptured DNA molecules were not observed in the V + current time traces as show in Supporting Fig.…”

“…In fact nanopipettes are predominantly used as an analytical platform for the detection of single molecules by transporting molecules from the outside reservoir to the inside of the nanopipette. 10,12,13,[24][25][26][27] . However, this does not take advantage of utilizing the nanopipette as a label-free single-molecule delivery vehicle.…”

Characterization of Nanopipettes

“…Typical SICM experiments are performed in moderate 19 to high ionic strengths (>100 mM), 3,31,32,45,67,68 as are many nanopipette measurements. 14,15,18,19 Under these conditions, the diffuse double layer is expected to be compressed to a sub-nanometer scale, and therefore undetectable, level according to: 50…”

“…14,15,18,19 Under these conditions, the diffuse double layer is expected to be compressed to a sub-nanometer scale, and therefore undetectable, level according to: 50…”

Resistive-pulse and rectification sensing with glass and carbon nanopipettes

“…They stressed several advantages of the nanopipette over the nanopore, including its mechanical strength and ease of fabrication. Another advantage-the small physical size and needle-like shape enabling precise positioning and experiments in small spaces-was utilized by Gong et al [68], who developed an integrated nanopipette-microfluidic device in which the pipette could be positioned at any point within a microfluidic channel. In this way, it was possible to detect and discriminate between DNA molecules of varying lengths travelling through the microfluidic channel.…”