Carbon Nanotube Sensors for the Detection of Single Protein Molecules

By Benedette Cuffari, M.Sc.Feb 16 2017

As one of the most important constituents of life, proteins are polymers of amino acids, whose expression and secretion play a significant part in nearly all aspects of cellular metabolic and signaling pathways.

While inherent disorders can affect protein production, other aberrations in protein expression due to a variety of causes can also signify the presence of a disease state. Several methods exist in order to detect the presence, structure and function of proteins within a cell, allowing researcher to further understand the mechanisms in which the cell is able to respond to its environment.

Of these include enzyme-linked immunosorbent assays (ELISA), western blotting, radial immunodiffusion, or mass spectrometry¹. While useful in their application, a number of these analytical methods require several preliminary purification steps, fluorescent modification of the target protein of interest, and a strict requirement of maintaining the presence of the proteins to be restricted to the inside of the cell.

The addition of a fluorescent label to target proteins allows researchers the additional dimension for protein detection, as well as a deeper understanding of the various protein-to-protein interactions that can take place within the cell.

Despite its usefulness in the research setting, these approaches to protein detection and analysis are limited in their ability to detect proteins directly from their production source in an unpurified biological environment¹. Therefore, the need for a label-free detection method capable of sensitive protein quantification within a crude environment is of significant interest to researchers. In an effort to respond to this interest in advanced protein analysis techniques, the lab of Michael Strano, a Carbon P. Dubbs Professor of Chemical Engineering at MIT², have developed a label-free spatiotemporal optical protein detection parameter that is both sensitive and selective for crude analysis purposes.

Researchers in Strano’s lab used aptamers, which are nucleotide polymers derived from DNA, whose high affinity for alternating AT nucleotide sequences allow this molecule to be anchored for a particular target molecule, such as a protein.

An optical response to a target protein is elicited by the ability of the aptamer to anchor to the polymer surface of a semiconducting single-walled carbon nanotube (SWNT) ¹. Through this adherence, molecular recognition is captured, enabling for the selective fluorescence of the SWNT to occur only after coming into contact with the conjugate protein target.

SWNTs are found to be useful emitters, as they are the only fluorophores capable of maintaining an infinite lifetime without the typical susceptibility of fluorescent labels to cause “on-off blinking.” In fact, the ability of SWNTs to provide a long-term selective signal from its adherence to the aptamers allows for its usefulness for prolonged monitoring investigations of specific target proteins.

The immediate utility of this aptamer-SWNT platform was confirmed by the ability of the sensors to recognize and bind to RAP1, a signaling protein, and HIV1 integrase, a viral protein, in an E. coli bacterial cell line, the HEK 293 mammalian cell line, as well as in the P. pastoris yeast cell line¹.

Following the sensor application into these cell lines, the process of protein induction, synthesis and export were analyzed and measured. By utilization of the aptamer-SWNT sensors, both RAP1 and HIV1 integrase genes were successfully integrated into each of the applied cell lines. The experimental design by Landry et. al, successfully provided a molecular recognition element for the specific, yet reversible, detection of a target protein, while maintaining a stable linkage between the SWNT and the aptamer, ensuring its ability to be reproduced in future protein detection studies¹.

By eliminating the requirement of fluorescent labels to be added to proteins prior to analysis, unprecedented information regarding the molecular interactions is possible, as these observations are taken based on the crude biological characteristics of the proteins themselves, avoiding the possibility of false positives and conjugated labels to occur. This type of easily reproducible detection method allows for its future applications within the biopharmaceutical industry, for example, to be very possible. Future researchers utilizing these sensors have postulated its in testing and engineering cells to treat specific diseases, in which the patient’s own cells can be used to express a therapeutic protein that is then returned to the patient in need².

References

1. Landry, Markita Patricia, Hiroki Ando, Allen Y. Chen, Jicong Cao, Vishal Isaac Kottadiel, Linda Chio, Darwin Yang, Juyao Dong, Timothy K. Lu, and Michael S. Strano. "Single-molecule Detection of Protein Efflux from Microorganisms Using Fluorescent Single-walled Carbon Nanotube Sensor Arrays." Nature Nanotechnology (2017). Web.
2. Anne Trafton | MIT News Office. "New Sensors Can Detect Single Protein Molecules." MIT News. 23 Jan. 2017. Web. http://news.mit.edu/2017/new-sensors-detect-single-protein-molecules-0123.
3. Image Credit: shutterstock.com/maxuser

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