UC Santa Cruz

Integrated optofluidics are one of the prominent choices for particle detection and manipulation which fuses optics and microfluidics in a single platform that enables sufficient sensitivity to probe individual particle, and especially single biomolecules. Among different optofluidic devices, anti-resonant reflective optical waveguides (ARROWs) form the basis of one of the most sensitive biosensors, also allowing integration of electrical single molecule sensing via solid state nanopores. Nanopores, which are small openings in a thin insulating membrane, are a fast-growing tool for label free electrical detection of single particles. Though young but nanopores have already proven their capabilities in sensing a variety of biomolecules with the greatest attention in nucleic acid sequencing which is being commercialized and used around the world. An innovative integration of two extremely powerful technologies like optofluidics and nanopores in a single ARROW platform enables particles to be probed with dual modalities, i.e. both optically and electrically. As the ARROW devices are primarily used for biosensing with facilities of further manipulation and trapping, there is huge room to add multiple functionalities with integrated nanopores including the use of nanopore as a smart gate to controllably deliver particles towards the optofluidic region with great precision. This work deals with the incorporation of nanopores with optofluidic devices to achieve new functionalities in the area of (chip-based) single molecule analysis. A first major breakthrough is the development and implementation of a feedback control system with the nanopore optofluidic device which is capable of detecting particle deliveries in real time and making further decision based on user’s instruction. With the feedback control nanopore gating, it is possible to turn off the electrical voltage across the nanopore after a single particle insertion which ensures isolation of a single particle and delivering that single particle into the fluidic micro-channel. This functionality is demonstrated by delivering single 70S ribosomes and DNA molecules into the optofluidic channel through feedback control nanopore gating. The feedback system is versatile for a wide range of biomolecules which have been justified by gating a variety of biomolecules including ribosomes, proteins, nucleic acids and NaCMC molecules. The feedback control gating offers reconfigurable settings thus, it is possible to adjust the gating functionalities based on user’s/experimental necessity. With the reconfigurable settings, deliberate delivery of two and three 70S ribosomes are demonstrated which can be set to any number if desired. Furthermore, automated delivery of 70S ribosomes and λ-DNAs is demonstrated with rates of several hundreds/min, which can be further boosted to near kHz range, illustrating the power and efficacy of the system for high throughput particle delivery and analysis. The feedback system is capable of analyzing translocation details (depth and duration) in real time and based on that it is possible to gate selective particles. This functionality has been demonstrated by selectively gating λ-DNAs from a mixture of 70S ribosomes, opening the door to selecting specific molecules for further study and producing purified subpopulations of particles when coupled with a microfluidic sorting system. The gated particles can be subjected to further analysis such as fluorescence detection and trapping for prolonged analysis. Fluorescence detection of voltage gated λ-DNAs are demonstrated which illustrates the feasibility of integration of feedback system with existing technologies. Furthermore, a sophisticated integration of the feedback system is shown with on chip anti-Brownian electrokinetic (ABEL) trapping. This functionality has been demonstrated by feedback gating and subsequent ABEL trapping a microbead.

ABEL trapping relies on fluorescence particle tracking and provides electrokinetic feedback force to adjust particle movement which is one of the supreme methods of particle trapping due to inherent advantages over optical trapping and other methods. In another part of the work, a novel ABEL trapping platform is developed which is capable of trapping particles in two dimensions (2D, full in-plane confinement) with better trap stiffness than previous 1D implementations. The trapping methodology, particle tracking algorithm is developed which is demonstrated by 2D trapping a microbead with 14x enhanced trapping stiffness compared to the old 1D ABEL trapping.

In the final part of the work, a novel and elegant method for dramatically increasing nanopore capture rates (event frequency) is demonstrated. Although the nanopore is a great tool for electrical detection of particles, most nanopore applications are limited due to the delivery of an insufficient number of analytes close enough to the pore to enable electrophoretic capture and detection. This severely limits the throughput (and extends the analysis time) and the limit of detection of the assay. An elegant solution to overcome the limitation is demonstrated which relies on preconcentration of targets on a micro-scale carrier bead followed by optical trapping the carrier beads at the vicinity of pore and thermally releasing them close to nanopore thus, increasing local analyte concentration. The practicality and efficacy of the methodology is experimentally demonstrated with ~80x enhanced capture rates by detecting DNAs corresponding to a melanoma cancer gene. As the method relies on accumulating targets and releasing them close to nanopore it should, in principle, be possible to detect targets at low concentrations. This functionality is demonstrated by Zika ns1 detection down to 2ng/mL which is a clinically relevant concentration. This demonstration illustrates the practicality and promising potential of the methodology which can be further developed towards diagnostics and possibly early stage disease detection.