Creating Ultrafast Biosensors for Neuroscience
Doktorsavhandling, 2019

Neuronal communication is the basis for all our brain function and relies on regulated exocytosis, a cell function that involves release of quantal amounts of neurotransmitters into the gap space between interconnected neurons to serve as chemical signals. To study exocytosis, which is a fast process that occurs on the timescale of sub-milliseconds to milliseconds, a toolbox of analytical methods has been developed where the electrochemical based techniques offer quantitative and sufficient high temporal recording speed. However, neuronal activity involving non-electroactive neurotransmitters such as acetylcholine and glutamate have long time suffered from a limited detection speed about 3 orders of magnitude slow for capturing the rapid transients from exocytosis release by these non-electroactive compounds. In this work, we have focused on a new approach for the development of amperometric enzyme-nanoparticle-based sensors that significantly improve recording speed of important non-electroactive brain analytes and are suitable for ultrafast recording in neuroscience research.
    In Paper I, an acetylcholine sensor was designed and fabricated by modifying the sensor surface with gold nanoparticles (AuNPs) and two sequential enzymes, where the enzyme coating was limited to a monolayer in thickness to minimize enzyme product diffusion distance to be detected by the electrode surface. This novel sensor provided the first proof of concept to improving enzyme-based sensors speed by 2 orders of magnitude compared to existing technology and was fast enough to temporally resolve single millisecond vesicle release events of acetylcholine from an artificial cell model that mimics exocytosis.
    In Paper II, a new and analytical method was introduced that provided a significantly faster and a non-toxic way to quantify AuNP immobilized enzymes during sensor surface characterization in comparison to the previous method used in Paper I that involved using toxic cyanide solutions. This method was based on electrochemical stripping of AuNPs from the electrode surface after enzymes were attached, followed by quantifying the number of enzymes released, to determine the average number of enzymes attached to each single nanoparticle.
    In Paper III, an ultrafast glutamate sensor was developed by careful characterization of the conditions for controlling the enzyme coverage on a AuNP decorated electrode surface to a monolayer. By placing this novel sensor in the Nucleus Accumbens of rodent brain slice, recording of spontaneous glutamate activity and various isolated dynamic current transients from single exocytotic events on the sub-millisecond timescale were captured.
    In Paper IV, the conjugation of enzyme glucose oxidase (GOx) to AuNP surfaces was used to study how physical crowding affects enzyme stability and activity when immobilized at a highly curved nanoparticle surface. This work showed that by crowding a gold nanoparticle surface with its maximum number of enzymes that can theoretically fit, while maintaining a monolayer coverage, the retained enzymatic activity of immobilized enzyme improved 300% compared to GOx free in solution. Implementing these findings to a nanostructured electrochemical biosensor for glucose confirmed a recording speed for glucose on the millisecond timescale
    In Paper V, using our novel ultrafast glutamate sensor, a novel method was developed for quantification of the quantal glutamate content in single synaptic vesicles, and quantification of the quantal amount glutamate released from single exocytosis events in rodent brain tissue.

acetylcholine esterase

brain slice

exocytosis

amperometry

adsorption

Biosensor

quantification

gold nanoparticles

glucose oxidase

enzyme monolayer

enzymatic activity

artificial cell

acetylcholine

glutamate oxidase

glucose

choline oxidase

enzyme stability

glutamate

deformation

KA
Opponent: Martin Jönsson Niedziolka, Department of Physical Chemistry, The Polish Academy of Science, Warsaw, Poland

Författare

Yuanmo Wang

Chalmers, Kemi och kemiteknik, Kemi och biokemi

Amperometric Detection of Single Vesicle Acetylcholine Release Events from an Artificial Cell

ACS Chemical Neuroscience,; Vol. 6(2015)p. 181-188

Artikel i vetenskaplig tidskrift

Counting the number of enzymes immobilized onto a nanoparticle-coated electrode

Analytical and Bioanalytical Chemistry,; Vol. 410(2018)p. 1775-1783

Artikel i vetenskaplig tidskrift

Ultrafast Glutamate Biosensor Recordings in Brain Slices Reveal Complex Single Exocytosis Transients

ACS Chemical Neuroscience,; Vol. 10(2019)p. 1744-1752

Artikel i vetenskaplig tidskrift

Molecular Crowding and a Minimal Footprint at a Gold Nanoparticle Support Stabilize Glucose Oxidase and Boost its Activity. Yuanmo Wang, Rima Jonkute, Hampus Lindmark, Jacqueline D. Keighron and Ann-Sofie Cans

Counting the Number of Glutamate Molecules in Single Synaptic Vesicles. Yuanmo Wang, Hoda Fathali, Devesh Mishra, Thomas Olsson, Jacqueline D. Keighron, Karolina P. Skibicka, and Ann-Sofie Cans

All our human brain functions are based on neuronal activity, where neuronal cells communicating to one another through electrical and chemical signals. In the chemical transmission of signals, neurons release signaling compounds, called neurotransmitters, which are stored inside vesicles in the cell cytoplasm. Through the process of exocytosis, these vesicles fuse with the cell plasma membrane and release the vesicle cargo of signaling molecules into the extracellular space for neighboring neurons to pick up this chemical message. This exocytosis process is triggered by electrical nerve signals and occurs on the rapid timescale of sub-milliseconds to milliseconds. The electrochemical method amperometry allows studying exocytosis activity, the mechanism of this cell function and can perform quantitative measurement of the amount neurotransmitters released during neuronal activity. However, amperometry is limited to neurotransmitters that are electroactive and some of the key neurotransmitters in the brain such as glutamate and acetylcholine are non-electroactive and therefore need alternative detection schemes. This can be solved by using enzyme-based amperometric biosensors, however these biosensors have long suffered from limited detection speed and have been 2-3 orders of magnitude too slow for capturing rapid neurotransmitter events during exocytosis activity. To address this issues, we combined our skills in nanomaterial science, analytical chemistry, physical chemistry, surface chemistry and neuroscience to develop a new approach for designing and fabricating ultrafast enzyme-based electrochemical biosensors, which was accomplished by limiting the thickness of the enzyme coating at the sensor surface to a molecular monolayer, compared to conventional thicker enzyme layers used. Through careful studies of enzyme-nanoparticle interactions in bulk solution, we also optimized the conditions for boosting the retained enzyme activity of the immobilized enzymes. In this work, we show how this new technology is capable of temporally resolving single exocytosis activity of glutamate release in brain tissue of mouse and rat during spontaneous glutamate activity. We also used the new analytical tools for the development of a new method for analyzing the neurotransmitter content in single synaptic vesicles that are only 40 nm in diameter. Hence, this new technology developed in our lab introduces new means to rapidly detect and to quantify ultra-small amounts of non-electroactive neurotransmitters and that can be used to study synaptic vesicle physiology, pathogenesis and drug treatment for neuronal disorders where these neurotransmitters are involved.

Drivkrafter

Hållbar utveckling

Innovation och entreprenörskap

Styrkeområden

Nanovetenskap och nanoteknik (2010-2017)

Ämneskategorier

Analytisk kemi

Neurovetenskaper

Materialkemi

Annan kemi

Fundament

Grundläggande vetenskaper

Infrastruktur

Chalmers materialanalyslaboratorium

Lärande och undervisning

Pedagogiskt arbete

ISBN

978-91-7905-150-1

Doktorsavhandlingar vid Chalmers tekniska högskola. Ny serie: 4617

Utgivare

Chalmers tekniska högskola

KA

Opponent: Martin Jönsson Niedziolka, Department of Physical Chemistry, The Polish Academy of Science, Warsaw, Poland

Mer information

Senast uppdaterat

2019-08-09