Research Program


Our research program is comprised of closely allied applied and basic thrusts. The aim of our applied research is to harness the mechanisms evolution has invented to monitor molecules in vivo to the problem of performing continuous, real-time molecular measurements in complex environments, including in the living body. In related studies we are exploring the rich and diverse physics with which biomolecules interact with surfaces.


Continuous, real-time molecular measurements in the living body

The availability of technologies capable of tracking the levels of drugs, metabolites, and biomarkers in real time in the living body would revolutionize our understanding of health and our ability to detect and treat disease. Imagine, for example, a dosing regime that, rather than relying on your watch (“take two pills twice a day”), is instead guided by second-to-second measurements of plasma drug levels wirelessly communicated to your smartphone. Such a technology would likewise provide clinicians an unprecedented window into organ function and could even support ultra-high-precision personalized medicine in which drug dosing is optimized minute-by-minute using closed-loop feedback control. Towards this goal, we are developing a biomimetic, aptamer-based electrochemical sensor platform (Fig. 1) that supports the high frequency, real-time measurement of ­–and thus control over (Fig. 2)– specific molecules (irrespective of their chemical reactivity) in situ in the bodies of awake, freely moving subjects.

Figure 1: The first ever approach to the high frequency, real-time measurement of specific in situ in the living body via a platform that relies on binding, and not on the target molecule’s intrinsic chemical reactivity, to generate a signal that can be read within the body. Innovations enabling this include: (A) Reagentless, reversible, E-AB sensors, in which an aptamer re-engineered to undergo binding-induced folding is modified with a redox reporter and attached (via a self-assembled monolayer) to an electrode. The binding-induced conformational change causes a corresponding change in electron transfer rate that is easily measured using square wave voltammetry. (B) Bundled with its counter and reference electrodes, the resultant sensor is small enough and flexible enough to (C) be emplaced via a 22-gauge guide catheter. (D) We employ Kinetic Differential Measurements, which uses the difference between the signals generated at two different square wave frequencies (one at which the sensor signal increases upon target binding and a second at which the sensor signal decreases upon target binding) to correct for any drift seen in vivo. Critically, this approach employs a single aptamer on a single electrode, avoiding the problem of differential drift between a physically separated sensor/reference pair. (E) Using drift-corrected E-AB sensors we achieve excellent precision in the measurement of in-vivo drug levels over the course of many hours. Shown in red, for example, are 3 s resolved tobramycin measurements made in the jugular of a live rat (black line: a 30 s rolling average computed in real-time). (F) Expansion of the injection period further highlights the approach’s unprecedented, 3-second time resolution.


Figure 2: The high-precision maintenance of constant plasma drug levels. We have recently demonstrated closed-loop feedback control over plasma drug levels in live rats. (A) Here, for example, we present feedback-controlled pharmacokinetic profiles in which plasma concentrations of the antibiotic tobramycin were held at set points of 80, 50, or 20 µM for 40, 50, and 60 min, respectively, in three different rats. The precision afforded by actively optimizing the dosing rate every 7 s using real-time concentration measurements is reflected in variance of only ~ 5 µM around each set point. (B) Over longer runs feedback control can ensure that the drug’s concentration remains in the middle of its therapeutic window. (C) Once steady state is achieved the instantaneous dosing rate informs on the instantaneous rate of drug elimination (given the not unreasonable assumption that the animal’s volume distribution is fixed). These unprecedented, high resolution measurements (black curve: 3 min rolling average) of the drug’s elimination half life show that the drug’s metabolism varies by more than a factor of three within this subject over the course of a few hours, thus highlighting the need for improved methods of drug delivery.

Selected publications on this topic:

Arroyo-Currás, N., Somerson, J., Vieira, P., Ploense, K., Kippin, T. and Plaxco, K.W. (2017) “Real-time measurement of small molecules in awake, ambulatory animals.Proc. Natl. Acad. Sci. USA, 114, 645–650

Li, H., Dauphin-Ducharme, P., Arroyo-Currás, N., Tran, C., Vieira, P.A., Li, S., Shin, C., Somerson, J., Kippin, T.E. and Plaxco, K.W. (2017) “A biomimetic, phosphatidylcholine-terminated monolayer greatly improves the in-vivo performance of electrochemical aptamer-based sensors.Angewandte, 56, 7492-7495

Li, H., Dauphin-Ducharme, P., Ortega, G. and Plaxco, K.W. (2017) “Calibration-free electrochemical biosensors supporting accurate molecular measurements directly in undiluted whole blood.J. Am. Chem. Soc., 139, 11207–11213

Arroyo-Curras, N., Dauphin-Ducharme, P., Ortega, G., Ploense, K., Kippin, T., and Plaxco, K.W. (2018) “Sub-second-resolved molecular measurements in the body using chronoamperometrically interrogated aptamer-based sensors.ACS Sensors, 3, 360-366


Stealing nature’s tricks to build better biosensors

Recent years have seen the development of a broad class of optical and electrochemical sensors in which the binding of a specific molecular target is signaled via a large-scale conformational change in a protein- or nucleic-acid-based receptor.  The reagentless, rapidly reversible nature of this signaling mechanism supports continuous, real-time measurement of a wide variety of analytes, and, when coupled to electrochemical read-outs, its extraordinary selectivity allows this detection to be performed in even the most complex environments, including within the living body.  Like all processes reliant on single-site binding, however, these sensors still suffer from two potentially significant limitations: the useful dynamic range of single-site receptors is centered at a fixed target concentration (defined by the receptor’s dissociation constant) and spans a fixed width (defined by the hyperbolic shape of the Langmuir isotherm) (Fig. 3).  We have been exploiting the various mechanisms that evolution has invented in order to circumvent these very same limitations (e.g., allostery, cooperativity), and demonstrate their value in improving the utility of a wide range of artificial biosensors (Fig. 4)

Figure 3: The physics of single-site receptors (Top) are such that their useful dynamic range is fixed in width, position and shape. (Bottom right) Specifically, an 81-fold change in target concentration is required to transition a single-site receptor from 10% to 90% occupancy. Only over this “useful dynamic range” does a receptor respond sensitively to small changes in target concentration. (Bottom left) The range over which a receptor exhibits good specificity (the ability to discriminate between authentic target and chemically similar analogues) is likewise fixed. Shown, for example, is competition between binding the “proper” target and an analogue that interacts 10 kJ/mol less favorably. The “specificity window” over which the receptor robustly differentiates between these two is shown in red. Faced with these limitations evolution has invented a number of simple mechanisms by which this otherwise fixed dynamic range of single-site binding can be raised, lowered, extended, narrowed or otherwise optimized. In this account we discuss the adaptation of these same mechanisms to the biomolecular receptors employed in artificial biotechnologies, optimizing their input-output behavior for a variety of applications.


Figure 4: (Top) Nature uses cooperative binding in order to “steepen” binding curves, rendering systems more sensitive to small changes in the concentration of their target. We have developed a general method for engineering this into normally non-cooperative receptors. To do so we link a tandem repeat of one half of the receptor to a tandem repeat of the second half of the same receptor via a long, unstructured loop. The binding-induced association of the first pair of receptor “halves” must pay the entropic cost of loop closure, reducing the affinity of the first binding event relative to that of the second, thus producing a cooperative response. To demonstrate this approach we applied it a cocaine-binding aptamer, the complex, three-dimensional structure of which is not known beyond its predicted secondary structure. (Bottom left) As expected, the parent aptamer is not cooperative and exhibits a useful dynamic range within error of that expected for single-site binding. (Bottom right) In contrast, the cooperative, two-site receptor exhibits a dynamic range of just 13-fold; the resultant steepening of the binding curve leads to significantly greater sensitivity to small changes in target concentration.

Selected publications on this topic:

Vallée-Bélisle, A., Ricci, F. and Plaxco, K.W. (2009) “Thermodynamic basis for the optimization of binding-induced biomolecular switches and structure-switching biosensors. Proc. Natl. Acad. Sci. USA, 106, 13802-13807

Vallée-Bélisle, A., Ricci, F. and Plaxco, K.W. (2012) “Engineering biosensors with extended, narrowed, or arbitrarily edited dynamic range.J. Am. Chem. Soc., 134, 2876–2879

Ricci, F., Vallée-Bélisle, A., Porchetta, A. and Plaxco, K.W. (2012) “The rational design of allosteric inhibitors and activators using the population-shift model: in vitro validation and application to an artificial biosensor.J. Am. Chem. Soc., 134, 15177-15180

Porchetta, A., Vallée-Bélisle, A., Plaxco, K.W. and Ricci, F. (2012) “Using distal site mutations and allosteric inhibition to tune, extend and narrow the useful dynamic range of aptamer-based sensors.J. Am. Chem. Soc., 134, 20601-20604

Simon, A., Vallée-Bélisle, A., Ricci, F., Watkins, H.M. and Plaxco, K.W. (2014) “Using the population-shift mechanism to rationally introduce ‘Hill-type’ cooperativity into a biomolecular receptor. Angewandte, 53, 9471-9475

Simon, A., Vallée-Bélisle, A., Ricci, F., and Plaxco, K.W. (2014) “Intrinsic disorder as a generalizable strategy for the rational design of highly responsive, allosterically cooperative receptors.Proc. Nat. Acad. Sci. USA, 111, 1504815053


Proteins on surface

Proteins retain function when attached to some surfaces (e.g., the cell membrane) and yet often unfold and inactivate when attached to others (e.g., the artificial surfaces used in many technologies). Our understanding of why this is, however, has been hampered by a lack of quantitative experimental methods by which we can measure the thermodynamics of biomolecule-surface interactions. That is, despite a large body of qualitative literature describing how adsorption alters protein structure, and a large number of empirical studies searching for adsorption-resistant surfaces, quantitative, experimentally testable insights into how and why proteins unfold on some surfaces and not others have proven elusive. In response, we have developed a new experimental (electrochemical) approach for measuring the folding free energy of biomolecules site-specifically attached to well-defined, macroscopic surfaces (i.e., flat at the molecular length scale) (Fig. 5, left). Comparison with bulk-solution-phase folding free energies then informs on the thermodynamics of the biomolecule’s interactions with the surface and, in turn, the mechanisms that drive them. Using this novel approach we have, for the first time, accurately measured the free energy with which proteins and nucleic acids interact with a set of chemically distinct macroscopic surfaces (Fig. 5, right). In parallel, we have also developed a simple, first-principles theory that recovers our key experimental observations quantitatively, providing molecular details unavailable to experiment alone.

Figure 5: (Left) We can monitor the chemical denaturation of a surface-attached biomolecule by employing square-wave voltammetry to monitor electron transfer from an attached redox reporter. By placing the redox reporter on the protein such that its electron transfer rate changes upon unfolding, the observed current changes upon denaturation. Critically, this process is reversible upon removal of the denaturant (grey curve). (right) Comparison of the chemical denaturation of the surface-attached biomolecule and that of the same construct free in bulk solution then informs on the extent to which interactions with the surface alter the biomolecule’s stability. Shown are data for the FynSH3 domain, which is destabilized by 2 kJ/mol when attached to a hydroxyl-terminated surface.

Selected publications on this topic:

Watkins, H.M., Vallée-Bélisle, A., Ricci, F., Makarov, D.E. and Plaxco, K.W. (2012) “Entropic and electrostatic effects on the folding free energy of a surface-attached biomolecule: an experimental and theoretical study.J. Am. Chem. Soc., 134, 2120–2126

Watkins, H.W., Simon, A.J., Ricci, F. and Plaxco, K.W. (2014) “The effects of crowding on the stability of a surface-tethered biopolymer: an experimental study of folding in a highly crowded regime. J. Am. Chem. Soc., 136, 8923-8927

Kurnik, M., Ortega, G., Dauphin-Ducharme, P., Li, H., Caceres, A. and Plaxco, K.W. (2018) “Quantitative measurements of protein-surface interaction thermodynamics.Proc. Natl. Acad. Sci. USA, 115, 8352-8357

Watkins, H.M., Ricci, F. and Plaxco, K.W. (2018) “Experimental measurement of surface charge effects on the stability of a surface-bound biopolymer.Langmuir, DOI: 10.1021/acs.langmuir.8b01004