[nanoPost] Biosensor systems

 Currently under development at the University are biosensor systems that can be used at a macroscale (e.g. within multiwell plate technology), on the microscale (using existing chip based technology currently in development) or at the single-molecule level (where a nanoactuator is available). They are already developing a self-assembling biosensor to test for toxicity.

Biological material is not always first choice for use in a robust biosensor because of its inherent instability. However, it offers several advantages: biological materials (e.g. proteins and nucleic acids) naturally self-assemble and they can be disposed of easily (e.g. after contamination). In this system, the detected toxin “poisons” the sensor, but the sensor can be “washed away” and fresh sensor reassembled from original components.

These biosensors are generic in nature and can be readily adapted to almost any specific need. The components are modular and so only the reporter protein needs to be changed. This biosensor technology could be applied to detect any of the following: hydrophobic compounds (e.g. aromatic compounds); DNA interchelators (e.g. dioxins) and biological ‘poisons’ (e.g. heavy metals). The basic components within the sensors also have other applications including their use as nanoactuators and molecular motors. They provide a first step toward building bionanotechnology.

The enzymes they use for sensing are molecular motors. As they move toward single molecules and thus nanotechnology, with all the added benefits of parallelism and high throughput.

Biosensors have two components:

1. Molecular Recognition Elements (MREs)—to provide the selectivity of the sensor and the sensing unit.

2. A Transducer—to link the interaction of sensor and detected substance with a measurable output.

MREs are normally catalytic or affinity-based.  However, a third type of MRE may be dependent on the disruption of intermolecular forces within this component. This type of MRE would detect compounds through their ability to destabilise the biosensor. 

The biosensors under development  are based on a family of DNA-binding enzymes. By domain swapping the researchers can rapidly generate novel DNA specificity and have produced >36 different DNA-binding enzymes. The biosensor is designed for toxicity testing using an indicator reporter protein (Luciferase) that gives out light.  Loss of the light signal is an indication of the presence of a hydrophobic compound (most toxins fit this category).  Each DNA-binding protein interacts differently with each toxin producing different sub-assembly complexes that release different amounts of light from the reporter gene.

The researchers can readily locate these enzymes at known positions on a multiwell plate (or as single-molecules in a nanoscale device) using the “correct” DNA sequence within a purpose-designed oligoduplex. The oligoduplex DNA can be permanently fixed to various surfaces using existing DNA-array-type technology (see Figure 5 below). Each micro-well, or sensing position, can use one DNA oligoduplex and will thus only assemble one member of the 36 variants. This allows the creation of an array of different enzymes each with slightly different assembly properties and thus slightly different sensing capability for a toxin. This produces patterns of light output that vary from toxin to toxin and can be read in a luminometer.

The above biosensing depends upon disruption of the assembly pathway of a family of enzymes.  These enzymes are also molecular motors and motor activity can be lost under similar circumstances. Motor activity is also sensitive to interchelators (e.g. dioxins and drugs) that bind DNA. They can detect single-molecule motor activity through a moving magnet (attached to the DNA) allowing them to build a prototype biosensor/nanoactuator.

Researchers at NASA Ames Research Center have taken a different route. They cover the surface of a chip with millions of vertically mounted CNTs 30–50 nm in dia. When the DNA molecules attached to the ends of the nanotubes are placed in a liquid containing DNA molecules of interest, the DNA on the chip attaches to the target and increases its electrical conductivity. This technique, expected to reach the sensitivity of fluorescence-based detection systems, may find application in the development of a portable sensor.