Physical Laboratory Automation in Synthetic Biology.

Synthetic Biology has overcome many of the early challenges facing the field and is entering a systems era characterized by adoption of Design-Build-Test-Learn (DBTL) approaches. The need for automation and standardization to enable reproducible, scalable, and translatable research has become increasingly accepted in recent years, and many of the hardware and software tools needed to address these challenges are now in place or under development. However, the lack of connectivity between DBTL modules and barriers to access and adoption remain significant challenges to realizing the full potential of lab automation. In this review, we characterize and classify the state of automation in synthetic biology with a focus on the physical automation of experimental workflows. Though fully autonomous scientific discovery is likely a long way off, impressive progress has been made toward automating critical elements of experimentation by combining intelligent hardware and software tools. It is worth questioning whether total automation that removes humans entirely from the loop should be the ultimate goal, and considerations for appropriate automation versus total automation are discussed in this light while emphasizing areas where further development is needed in both contexts.

Protein and DNA Yield Current Enhancements, Slow Translocations, and an Enhanced Signal-to-Noise Ratio under a Salt Imbalance.

Nanopores are a promising single-molecule sensing device class that captures molecular-level information through resistive or conductive pulse sensing (RPS and CPS). The latter has not been routinely utilized in the nanopore field despite the benefits it could provide, specifically in detecting subpopulations of a molecule. A systematic study was conducted here to study the CPS-based molecular discrimination and its voltage-dependent characteristics. CPS was observed when the cation movement along both electrical and chemical gradients was favored, which led to an ∼3× improvement in SNR (i.e., signal-to-noise ratio) and an ∼8× increase in translocation time. Interestingly, a reversal of the salt gradient reinstates the more conventional resistive pulses and may help elucidate RPS-CPS transitions. The asymmetric salt conditions greatly enhanced the discrimination of DNA configurations including linear, partially folded, and completely folded DNA states, which could help detect subpopulations in other molecular systems. These findings were then utilized for the detection of a Cas9 mutant, Cas9d10a─a protein with broad utilities in genetic engineering and immunology─bound to DNA target strands and the unbound Cas9d10a + sgRNA complexes, also showing significantly longer event durations (>1 ms) than typically observed for proteins.

On the origins of conductive pulse sensing inside a nanopore.

Nanopore sensing is nearly synonymous with resistive pulse sensing due to the characteristic occlusion of ions during pore occupancy, particularly at high salt concentrations. Contrarily, conductive pulses are observed under low salt conditions wherein electroosmotic flow is significant. Most literature reports counterions as the dominant mechanism of conductive events (a molecule-centric theory). However, the counterion theory does not fit well with conductive events occurring via net neutral-charged protein translocation, prompting further investigation into translocation mechanics. Herein, we demonstrate theory and experiments underpinning the translocation mechanism (i.e., electroosmosis or electrophoresis), pulse direction (i.e., conductive or resistive) and shape (e.g., monophasic or biphasic) through fine control of chemical, physical, and electronic parameters. Results from these studies predict strong electroosmosis plays a role in driving DNA events and generating conductive events due to polarization effects (i.e., a pore-centric theory).

DNA Coil Dynamics and Hydrodynamic Gating of Pressure-Biased Nanopores.

Nanopores are ideally suited for the analysis of long DNA fragments including chromosomal DNA and synthetic DNA with applications in genome sequencing and DNA data storage, respectively. Hydrodynamic fluid flow has been shown to slow down DNA transit time within the pore, however other influences of hydrodynamic forces have yet to be explored. In this report, a broad analysis of pressure-biased nanopores and the impact of hydrodynamics on DNA transit time, capture rate, current blockade depth, and DNA folding are conducted. Using a 10 nm pore, it is shown that hydrodynamic flow inhibits the early stages of linearization of DNA and produces predominately folded events which are initiated by folded DNA (2-strands) entering the pore. Furthermore, utilizing larger pores (30 nm) leads to unique DNA gating behavior in which DNA events can be switched on and off with the application of pressure. A computational model, based on combining electrophoretic drift velocities with fluid velocities, accurately predicts the pore size required to observe DNA gating. Hydrodynamic fluid flow generated by a pressure bias, or potentially more generally by other mechanisms like electroosmotic flow, is shown to have significant effects on DNA sensing and can be useful for DNA sensing technologies.

Nanodiagnostics: A review of the medical capabilities of nanopores.

Modern diagnostics strive to be accurate, fast, and inexpensive in addition to properly identifying the presence of a disease, infection, or illness. Early diagnosis is key; catching a disease in its early stages can be the difference between fatality and treatment. The challenge with many diseases is that detectability of the disease scales with disease progression. Since single molecule sensors, e.g., nanopores, can sense biomolecules at low concentrations, they have the potential to become clinically relevant in many of today's medical settings. With nanopore-based sensing, lower volumes and concentrations are required for detection, enabling it to be clinically beneficial. Other advantages to using nanopores include that they are tunable to an enormous variety of molecules and boast low costs, and fabrication is scalable for manufacturing. We discuss previous reports and the potential for incorporating nanopores into the medical field for early diagnostics, therapeutic monitoring, and identifying relapse/recurrence.

Measuring trapped DNA at the liquid-air interface for enhanced single molecule sensing.

Nanopore sensing is a promising tool with widespread application in single-molecule detection. Borosilicate glass nanopores are a viable alternative to other solid-state nanopores due to low noise and cost-efficient fabrication. For dielectric materials, including borosilicate glass, the capacitive noise is one of the major contributors to noise, which depends on the wall thickness and the surface area submerged in an ionic solution. Here, we investigated the root mean square (IRMS) noise and ionic conductance for borosilicate nanopores in different depths (i.e., tip submersion depth) ranging from the solution surface (assumed to be zero) to 5000 µm. Our findings demonstrate a decrease in IRMS noise as the pipette moves toward the surface. We further demonstrate that borosilicate nanopores can detect single lambda DNA (λ-DNA) molecules with a high signal-to noise ratio close to the liquid-air interface. Specifically, our results indicate a higher signal to noise ratio as the submersion depth is reduced owing to the reduced surface area and thus capacitive noise. Further, our experimental results show higher DNA capture frequency at the air-water interface due to a combined effect of evaporation and an evaporation-induced thermal gradient at the surface. Therefore, our findings demonstrate that borosilicate glass nanopores are suitable for studying interfacial concentration gradients of molecules, specifically DNA, with a higher signal to noise.