Engineering improved measurement and actuation for nanoscale biophysics

Abstract

This commentary profiles the research interests of my recently established research group at The University of Chicago, as well as my own research trajectory and contributions to the field of nanoscale biophysics. I describe here certain open challenges of interest that drive my group’s current research efforts, along with my past efforts that have impacted these areas.

Introduction

I opened my lab at The University of Chicago in the Department of Molecular Engineering late last year (2019). My research group is broadly interested in pushing the limits of extracting information from nanoscale biological systems and in devising new ways to actuate components of these systems. These efforts will advance our collective capability to understand and experiment on biology from the bottom up.

Research interests

Nanoscale biophysics is maturing as a field. Our growing bottom-up understanding of cell biology has been driven by many advanced instruments for nanoscale observation, such as imaging (Betzig et al. 2006; Kim et al. 2007) and structural biology technologies (Drenth 2007; Bai et al. 2015), among many others. These observations have made it clear that the key features of life universally arise from sub-cellular spatial and temporal heterogeneities. Yet non-perturbative acquisition of information about nanoscale structure and events remains challenging; every technique in our single-molecule toolkit strikes a unique balance among trade-offs of spatial and temporal resolution, molecular specificity, the richness or complexity of information acquired, and limitations of the experimental conditions under which that data may be collected. There is unquestionably a need for continued investigation and innovation to optimize our access to information about nanoscale biological processes.

Actuation is the complementary capability to measurement; experimentation relies on repeated prediction, perturbation, and observation. But, nanoscale-targeted and on-demand manipulation of living systems remains challenging. Existing approaches to actuate or perturb the nanoscale biology of a cell are primarily macroscopic or bulk biochemical and physical techniques, such as genetic and chemical manipulation, or changing environmental conditions. Biochemically targeted (but spatiotemporally random) dilute nanoscale perturbations are possible (Mannix et al. 2008); or alternatively, micron-scale targeting is possible through specialized techniques such as electrophysiology and nanopipettes (Seger et al. 2012), optical tweezers (Ashkin et al. 1990), or optogenetics (Deisseroth 2011), among many others (Liu 2016; Nadappuram et al. 2019). New techniques and instruments are still needed to allow on-demand, nano-targeted, and direct manipulation and actuation of single objects in cells and tissue that will complement existing and developing capabilities for nanoscale observation.

Along with many others in the field, my research group is currently working to engineer novel experimental tools for precision measurement and control of single molecules. We will use these techniques to answer basic and applied biophysical, biochemical, and biomedical questions at the nanoscale.

Contributions to science

My own research to date has focused on developing and using novel strategies to confine, manipulate, and measure single biomolecules. As a Biomedical Engineering graduate student at Boston University in the lab of Prof. Amit Meller, I developed methods to improve control and readout of single DNA molecules pulled electrophoretically through a solid-state nanopore (Fig. 1a). At the time, solid-state nanopores were a relatively new technology for resistive sensing, and I was inspired by their potential versatility to pursue them as a means of directly sensing and actively probing DNA-protein interactions (Squires et al. 2017).

Fig. 1.

(a) Principle of resistive sensing by a solid-state nanopore. A single strand of DNA is drawn electrophoretically through the nanoscale aperture, temporarily reducing the measured ionic current. (b) Schematic of an Anti-Brownian ELectrokinetic trap. An excitation spot is quickly scanned over the trapping region at the center of a microfluidic cell to determine particle position, and electrodes are used to apply appropriate feedback voltages to electrophoretically move the particle constantly towards the center of the trap

My colleagues and I worked to improve the nanopore sensing platform, exploring modifications to the nanopore geometry, surface, and external volume in order to improve sensitivity and resolution: DNA translocation could be slowed via optical irradiation of the silicon nitride membrane (Di Fiori et al. 2013), or via interactions with a hydrophobic electrospun nanofiber mesh (Squires et al. 2013). We developed techniques to enable fluorescence readout of molecules transiting a nanopore to enable DNA barcoding (Assad et al. 2015) and parallelized detection from a nanopore array (Anderson et al. 2014). With a sufficiently thin and small nanopore, we were eventually able to resolve single zinc finger transcription factors bound to DNA (Squires et al. 2015a; Squires et al. 2017). I also conceived and demonstrated a simple, fast, and robust application for rapid pathogen typing by measuring DNA fragment lengths in a nanopore (Squires et al. 2015b).

Nanopores sense biomolecules by probing their geometrical shape, while fluorescence allows us to see them. As a postdoctoral fellow at Stanford University with Prof. W. E. Moerner, I became interested in measuring dynamic changes in the spectroscopic properties of photosynthetic biomolecules and complexes at the single-molecule level. To avoid the possible perturbative effects of surface immobilization or physical tethers, we employed a single-molecule technique previously developed in the Moerner Lab to cancel out the Brownian motion of a single particle in solution by fast electrokinetic feedback: the Anti-Brownian ELectrokinetic, or “ABEL,” trap (Cohen and Moerner 2006), shown schematically in Fig. 1b.

The dynamic organization and behavior of individual pigments in a photosynthetic light-harvesting complex are crucial to overall efficiency and adaptation. Even in a “simple” system consisting of a three-pigment monomeric phycobiliprotein (from a cyanobacterial light-harvesting antenna, the phycobilisome), we observed heterogeneous photodynamics and evidence that at least one of the embedded pigments can intermittently become a quencher (Squires and Moerner 2017).

The complex photophysical behavior we observed in the smallest building blocks of the phycobilisome directly influences larger scale emergent photodynamics in the full antennae complex. In a related study, we were able to trap a full phycobilisome complex and observe the effect of its interaction with a binding partner, the orange carotenoid protein, which binds and quenches the phycobilisome under high light as part of a unique photoadaptive mechanism found in certain cyanobacteria. Although it was previously believed that only one OCP effected quenching, we clearly identified two distinct quenched states and were able to computationally model and identify two rotationally symmetric binding sites for OCP (Squires et al. 2019a).

Most recently, we pioneered an update to the ABEL trap which employs interferometric scattering (rather than fluorescence) to track a trapped particle (Squires et al. 2019b). This will enable future investigations of a much broader class of single particles, including those that are intermittently or dimly fluorescent, or do not fluoresce.

Personal background

My interest in nanoscale biophysics has been both encouraged and aided by broad academic training and interests. I was raised in a family of scientists and engineers, including chemical and civil engineers, physicists, and computer scientists. I hold a B.S.E. in Mechanical and Aerospace Engineering from Princeton University with certificates in Engineering Physics, Mandarin Chinese, and Robotics. I earned my Ph.D. in Biomedical Engineering at Boston University as an NSF-GRFP and Clare Boothe Luce Fellow in the lab of Prof. Amit Meller. Most recently, I completed my postdoctoral training in Chemistry at Stanford University in the lab of Prof. W.E. Moerner. I have found this broad academic foundation to be extremely useful in approaching challenges in my research because biophysics is inherently so interdisciplinary, requiring integration of ideas and techniques from across the physical and biological sciences and engineering.

Conclusion

Together, my past and present research efforts have focused on advancing our capability to obtain and understand rich biophysical data describing details of the temporal, structural, and population-scale heterogeneities that arise at the molecular level and directly impact biological function. Despite the many groundbreaking advances that provide us windows and handles to measure and interact with biomolecules in vivo and in vitro, the complexity of cell biology and the technical challenges to accessing the nanoscale demand many future advances to enable optimal information and control over bottom-up biology.

Finally, on a personal note, I have been fortunate to study, train, and collaborate with biophysicists who are also outstanding role models and mentors, and to work with colleagues who are first-rate scientists and humans. I have always found the biophysics and single-molecule communities to be intellectually exciting and professionally welcoming; one particularly relevant exemplar of this is Laura Finzi, who selected my abstract for one of my first conference talks at a Biophysical Society annual meeting many years ago, and who put my name forward here. I am honored to be included among this cohort in recognizing the career of Michèle Auger, and I look forward to updating the biophysics community in future years about my lab’s progress towards the goals described here.

Funding

The author acknowledges current research funding from The University of Chicago and from the Neubauer Family Foundation.

Compliance with ethical standards

Conflict of interest

The author declares that she has no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by the author.