Sensing Fast Processes in Biology

People: Chen He, Chen Wang, James Schwabacher, Jeremiah Kim

One thrust of our research is the creation of novel quantum dot (QD)-based sensors that enable “fast” (nanosecond to microsecond) sensing of biological processes. While the majority of cellular and microbiological processes occur on relatively fast-time scales (ns to μs), the traditional optical biological sensors that are used to study these processes operate at much slower conditions (ms to s)  as they rely on the slow diffusion of analytes or particles to initiate the sensing mechanism. This mismatch between the timescales of biological processes and current bio-sensors means that much our understanding of biology is in quasi-steady-state conditions, whereas biology is fundamentally a very dynamic non-equilibrium system. Our goal is to create QD-based sensors with rapid response times–capable of directly measuring cellular processes while they occur.

Slight changes in the extracellular and intracellular environment ( pH, ion concentrations, enzyme activities) can be directly correlated with biological processes and diseases. For example, the presence of cancer cells within a body often results in a decrease in the local extracellular pH.

General Schematic of a QD-based pH sensor

 

One goal of our research is to develop QD-based pH sensors where a change in the local environment of the QD (induced by the pH) results in a change in the optical spectra of the QDs. By utilizing molecules (or ligands) that are bound to the QD surface, we can avoid slow (μs-ms) diffusion-based processes that would limit our temporal resolution. Ultimately, we should be able to develop QD-sensors only limited by the migration time of protons in water (~ns).

Schematic showing how the protonation state of a phosphonate ligand on the surface of a QD results in a shift in the optical spectra of the QDs.

We have shown previously that the protonation state of phosphonate ligands on QDs affects the first exciton absorbance energy of the QD. By calibrating the degree of the first exciton absorbance energy shift to the exact number of protons proximate to the surface of the quantum dot, we can develop a fast QD-based pH sensor. The first exciton absorbance energy of the quantum dot can then serve as analytical probe of the local pH environment and provide insights into the timescales of pH changes in cellular environments.

 

Scheme of a charge-transfer based pH sensor

 

We are also exploring the use of charge-transfer (cT) as a probe for local pH by having a bound molecular acceptor proximate to the QD surface .We coat quantum dots using a mixed monolayer of dithiolate ligands with pH-responsive (yellow/grey) and electron-accepting (blue) moieties. Upon protonating/deprotonating the pH-responsive functional groups, we modulate the electrostatic potential at the surface of quantum dots and, therefore, the desorption/adsorption of electron-accepting groups, which determines the fluorescence intensity of the quantum dot ensemble. The response time of this process can potentially bypass the limit of diffusion (which is on the order of tens of nanosecond to microsecond), since the electron acceptors are covalently tethered to the surface of quantum dots.

 

We are also developing QDs that are sensitive to enzymatic processes such as the acetylation/de-acetylation of proteins, which has a considerable impact on  gene expression and cellular metabolism.

Schematic of a QD-based acetylation sensor: Depending upon the acetylation state of the QD ligands, the covalently attached methylviologen molecular is either tightly bound to the QD surface (quenching the photoluminescence of the QD) or repelled by the QD ligands (allowing the QDs to emit).

The above project works to create a fluorescent nanoparticle probe that utilizes electrostatic interactions between a tethered quencher molecule and its own surface coating to nondestructively monitor the balance of mitochondrial protein acetylation in real time.  This investigation will develop a novel method to study mitochondrial proteins and examine their relationship with mitochondrial disease.

In the figure, methylviologen (MV2+) is used as a positively charged quencher in the probe system that will have distance-dependent fluorescence. The distance is determined by the electrostatic environment caused by the amount of acetylation in the mitochondria. Acetylation keeps the peptide substrate neutral which allows the probe to interact with the surface of the QD thereby quenching fluorescence, while deacetylation adds a positive charge to the peptide substrate that repels the probe allowing fluorescence.