Samudra Sengupta

Fantastic Voyage: Designing Self‐Powered Nanorobots

The use of swarms of nanobots to perform seemingly miraculous tasks is a common trope in the annals of science fiction.1 Although several of these remarkable feats are still very much in the realm of fiction, scientists have recently overcome many of the physical challenges associated with operating on the small scale and have generated the first generation of autonomous self-powered nanomotors and pumps. The motors can be directed by chemical and light gradients, pick up and deliver cargo, and exhibit collective behavior.

A Polymerization-Powered Motor†

Research into nanoand micromotors powered by catalytic reactions, or more broadly the study of autonomous motion at the microand nanoscale, has become an area of great current interest. Potential applications include the delivery of materials, self-assembly of superstructures, roving sensors, and other emerging applications. The motors described to date involve the catalytic conversion of small molecules, which typically results in a gradient of charged or neutral species that in turn drives the motor. Polymerization-powered motion has been reported in biological systems, for example, listeria has been observed to move by actin polymerization. However, there have been no reports of motion at the nanoand micrometer scale driven by polymerization. Given the large repertoire of known organometallic polymerization catalysts, the design of polymerization-driven motors would considerably increase the scope of catalytic reactions that could be employed to power autonomous motion. Furthermore, polymerization reactions offer the unique opportunity to both power motion and simultaneously allow the deposition of polymer along the motion track. Herein, we present the first motor to be powered by a polymerization reaction outside biological systems. The motor is powered by ringopening metathesis polymerization (ROMP) of norbornene. These motors show increased diffusion of up to 70 % when placed in solutions of the monomer. Furthermore, the motors were observed to display the phenomenon of chemotaxis when placed in a monomer gradient; an extremely rare example outside biology. Generating motion by polymerization has been previously suggested, although not demonstrated. We chose to employ a form of Grubbs ROMP catalyst for our initial study because of its relatively high stability and high polymerization activity with norbornene (Figure 1). The motors were fashioned by first synthesizing gold–silica Janus particles. This was performed using 0.96 mm silica particles. These particles were deposited as thin films using a published method. Then gold was deposited onto the monolayers creating the asymmetric Janus particles. The particles were then chemically modified with the Grubbs catalyst on the silica side utilizing previously published methods (Supporting Information, Figure S1). XPS confirmed that the catalyst did attach to the motor surface. Catalytic activity was then tested by adding the functionalized particles to norbornene solutions and monitoring monomer consumption by gas chromatography. The turnover frequency (TOF) was found to be proportional to monomer concentration and begins to saturate at 1m norbornene (Supporting Information, Figure S2). SEM images of these particles before and after exposure to a monomer solution shows the formation of polymer at the particle surface (Figure 2). As discussed in the

Enzyme Molecules as Nanomotors

Using fluorescence correlation spectroscopy, we show that the diffusive movements of catalase enzyme molecules increase in the presence of the substrate, hydrogen peroxide, in a concentration-dependent manner. Employing a microfluidic device to generate a substrate concentration gradient, we show that both catalase and urease enzyme molecules spread toward areas of higher substrate concentration, a form of chemotaxis at the molecular scale. Using glucose oxidase and glucose to generate a hydrogen peroxide gradient, we induce the migration of catalase toward glucose oxidase, thereby showing that chemically interconnected enzymes can be drawn together.

Self-powered enzyme micropumps

Non-mechanical nano- and microscale pumps that function without the aid of an external power source and provide precise control over the flow rate in response to specific signals are needed for the development of new autonomous nano- and microscale systems. Here we show that surface-immobilized enzymes that are independent of adenosine triphosphate function as self-powered micropumps in the presence of their respective substrates. In the four cases studied (catalase, lipase, urease and glucose oxidase), the flow is driven by a gradient in fluid density generated by the enzymatic reaction. The pumping velocity increases with increasing substrate concentration and reaction rate. These rechargeable pumps can be triggered by the presence of specific analytes, which enables the design of enzyme-based devices that act both as sensor and pump. Finally, we show proof-of-concept enzyme-powered devices that autonomously deliver small molecules and proteins in response to specific chemical stimuli, including the release of insulin in response to glucose.

Substrate Catalysis Enhances Single-Enzyme Diffusion

We show that diffusion of single urease enzyme molecules increases in the presence of urea in a concentration-dependent manner and calculate the force responsible for this increase. Urease diffusion measured using fluorescence correlation spectroscopy increased by 16-28% over buffer controls at urea concentrations ranging from 0.001 to 1 M. This increase was significantly attenuated when urease was inhibited with pyrocatechol, demonstrating that the increase in diffusion was the result of enzyme catalysis of urea. Local molecular pH changes as measured using the pH-dependent fluorescence lifetime of SNARF-1 conjugated to urease were not sufficient to explain the increase in diffusion. Thus, a force generated by self-electrophoresis remains the most plausible explanation. This force, evaluated using Brownian dynamics simulations, was 12 pN per reaction turnover. These measurements demonstrate force generation by a single enzyme molecule and lay the foundation for a further understanding of biological force generation and the development of enzyme-driven nanomotors.

Chemotactic separation of enzymes.

We demonstrate a procedure for the separation of enzymes based on their chemotactic response toward an imposed substrate concentration gradient. The separation is observed within a two-inlet, five-outlet microfluidic network, designed to allow mixtures of active (ones that catalyze substrate turnover) and inactive (ones that do not catalyze substrate turnover) enzymes, labeled with different fluorophores, to flow through one of the inlets. Substrate solution prepared in phosphate buffer was introduced through the other inlet of the device at the same flow rate. The steady-state concentration profiles of the enzymes were obtained at specific positions within the outlets of the microchannel using fluorescence microscopy. In the presence of a substrate concentration gradient, active enzyme molecules migrated preferentially toward the substrate channel. The excess migration of the active enzyme molecules was quantified in terms of an enrichment coefficient. Experiments were carried out with different pairs of enzymes. Coupling the physics of laminar flow of liquid and molecular diffusion, multiphysics simulations were carried out to estimate the extent of the chemotactic separation. Our results show that, with appropriate microfluidic arrangement, molecular chemotaxis leads to spontaneous separation of active enzyme molecules from their inactive counterparts of similar charge and size.

DNA Polymerase as a Molecular Motor and Pump

DNA polymerase is responsible for synthesizing DNA, a key component in the running of biological machinery. Using fluorescence correlation spectroscopy, we demonstrate that the diffusive movement of a molecular complex of DNA template and DNA polymerase enhances during nucleotide incorporation into the growing DNA template. The diffusion coefficient of the complex also shows a strong dependence on its inorganic cofactor, Mg2+ ions. When exposed to gradients of either nucleotide or cofactor concentrations, an ensemble of DNA polymerase complex molecules shows collective movement toward regions of higher concentrations. By immobilizing the molecular complex on a patterned gold surface, we demonstrate the fabrication of DNA polymerase-powered fluid pumps. These miniature pumps are capable of transporting fluid and tracer particles in a directional manner with the pumping speed increasing in the presence of the cofactor. The role of DNA polymerase as a micropump opens up avenues for designing miniature fluid pumps using enzymes as engines.