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Significance Statement
Label free assays have wide application in the medical sector. Field effect transistors, nanotubes, micro cantilever arrays and surface plasmon resonance are some of the label free techniques which depend on surface reaction for the detection of analytes. However, when the analyte concentration is low, the corresponding surface reaction is a slow process as diffusion of analytes to the surface is limited due to the presence of a large depletion layer. It is thus difficult to detect low concentration analytes using these label free techniques, within a short time.
In principle, this issue could be addressed if the substrates and the analytes are confined in nanoscale spaces. In such cases, analytes only need to travel a short distance to reach the surface and interact with the substrates. Assays based on this concept have been developed already and have their applications in bio-sensing and enzyme kinetic characterization. Yet, separate signal transducers are required for these assays for the conversion of reaction progress into readable signals. This leads to system complexity and increases the operation cost. Furthermore, in these nanoconfinement based assays, the analytes still need to be continuously supplied from the reservoir(s), which is still a very time-consuming process.
Dr. Chuanhua Duan, Dr. Arun Majumdar and colleagues developed a label-free nanochannel based sensor, in which the nanochannel itself is the signal transducer, to detect enzymes using surface enzymatic reactions. In these reactions, the target analytes, the enzymes, are not consumed as they are catalysts. Although it is still a long distance for the enzymes to diffuse from one end to the other end of the nanochannel, repeatability of the enzymes to interact with the immobilized substrates leads to short response time. The research work is published in peer-reviewed journal, ACS Nano.
Enzymatic reactions are very important for many biological processes. To quantify the activity of enzymes, fluorogenic or chromogenic labeled substrates are among the current approaches generally being used. These approaches consume more time and are expensive. Using nanochannel devices to detect the enzyme activity brings a low cost, highly sensitive approach. In this paper, the authors demonstrate the enzymatic reactions (on immobilized substrates) in nanochannel devices by electrical detection. They also discover that nanoscale confinements would lead to significant changes in reaction kinetics.
The nanochannel devices, containing ten nanochannels, four reservoirs and two microchannels are fabricated by the classical “etching and bonding scheme”. The enzymatic reaction that they investigate is the trypsin-poly-L-lysine reaction and trypsin is their target enzyme. In a typical experiment, the authors first introduce poly-L-lysine into the silica nanochannel. The electrostatic interactions cause the ploy-L-lysines, which are positively charged, to bind to the negatively charged silica nanochannel surface. Trypsin is then introduced to digest the poly-L-lysines into di- and tri-lysines. By measure ionic conductance of the nanochannel device before and after the introduction of trypsin, the authors can correlate the conductance change, which is a function of surface charge and reflects the progress of the surface enzymatic reaction, to the trypsin concentration. They demonstrate detection of trypsin with concentrations ranging from 5 ng/ml to 50 mg/ml within an hour. They also demonstrate the specificity of this nanochannel sensor by comparing the activity of trypsin and the activity of two other proteases, i.e. thrombin and chymotrypsin.
In addition to demonstrating the excellent performance of the nanochannel enzyme sensor, the authors also use it to quantitatively study enzyme kinetics in nanoscale confinements. They find that the reaction progress reveals a linear time dependence at low enzyme concentrations and a square-root time dependence at high enzyme concentrations. These dependences are not observed for enzymatic reactions in solutions or on plain surfaces, which result from the catalytic nature of enzymes and non-specific enzyme-surface interactions in nanoscale confined spaces, respectively.
In summary, this study demonstrated detection of enzymes using 2D nanochannel devices as fast label-free electrical bio-sensor. The designed nanochannel sensor can also be considered as a suitable platform for measuring enzyme kinetics in confined spaces.
Reference
Chuanhua Duan1, Mohammad Amin Alibakhshi1, Dong-Kwon Kim2, Christopher M. Brown3, Charles S. Craik3, Arun Majumdar4, Label-Free Electrical Detection of Enzymatic Reactions in Nanochannels, ACS Nano, Volume 10, 2016, Pages 7476-7484.
[expand title=”Show Affiliations”]- Department of Mechanical Engineering, Boston University, Boston, Massachusetts 02215, United States
- Department of Mechanical Engineering, Ajou University, Suwon 443-749, South Korea
- Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94158, United States
- Department of Mechanical Engineering, Stanford University, Stanford, California 94305, United States
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