Define biosensor.
A device that converts chemical information through a process of molecular recognition (usually through a biological molecule) which is transduced into an electric/optical signal which is fed into a computer for data processing.
What happens if you place a soft biomolecule on a hard surface? How can we overcome this?
- Hydrophobic protein-surface interactions will drive the denaturing of the molecule by unfolding and leading to a loss of function.
- This means they are incompatible – however smaller sensors (nanoscale) will have greater problems with this due to the lower number of molecules.
- Therefore micro and nano sensors need to be efficiently engineered to prevent loss of function.
We can make the surface more compatible by chemical modifications:
o Coating with organic molecules including silicon on glass surface will make them more organic which is more soft and biocompatible.
o On a gold surface, organic thiol compounds can be coated which will stick very well and is biocompatible.
o Both act as a buffer for the biomolecule.
– Also biotin-streptavidin chemistry is commonly used.
Describe the biotin-streptavidin chemistry.
- Its double ring structure binds extremely tightly to the protein streptavidin which is essentially irreversible.
- The streptavidin protein consists of four identical subunits (homotetramer) – which means they can be used on a sensor.
- Biotin has a LMW so it won’t affect function and streptavidin’s three other subunits can bind to biomolecules.
- This phenomenon is popular in biosensing.
Explain how the glucose test is an example of an electrochemical biosensor.
- It’s an enzyme sensor – the enzyme glucose oxidase oxidises glucose (it’s only substrate) – high specificity.
- The enzyme has a particular flavin cofactor – it oxidises glucose while reducing cofactor.
- The cofactor is then oxidised by oxygen, producing hydrogen peroxide.
- The hydrogen peroxide can then be re-oxidised at the electrode to oxygen, releasing two electrons and this generates a current.
- The electrode needs to have two properties –>
1. It needs to re-oxidise at the hydrogen peroxide at low voltage to avoid interference from naturally occurring reducing agents
2. It needs to make sure the electron transfer occur quickly to help turnover the enzyme and generate a larger current and signal.
- The electrode needs to have two properties –>
Why is graphene used as an electrode material? What is RGO?
- Graphene is a very popular electrode material, especially when combined with haemoglobin or Prussian blue – since hydrogen peroxide is rapidly oxidised.
- -This is due to graphene being a single sheet of carbon – the electrons can then easily be transferred.
- Pristine graphene is not suitable for nanobiosensors – reduce graphene oxide (RGO) is preferred which has more disorganised carbon sheets containing oxygen atoms because it is more hydrophilic and is more receptive to electron transfer.
Describe the method of surface plasmon resonance. What are its uses?
- Placing a thin layer of gold on a prism. A light source will be placed so that light will reflect on underside of gold layer which is reflected off and then detected.
- The gold layer needs to be treated so that antibodies will sit on without denaturing.
- For most angles of incidence, the light is reflected with 100% efficiency. However at the critical angle, the light is absorbed by the gold layer electrons.
- Here the free electrons in the gold which usually are moving at random, are stimulated to move as a collective/coherent wave – this collective wave is referred to as a surface plasmon.
- This phenomenon is important in biosensing – the critical angle is dependent on the refractive index of the layer – i.e. it’s determined by what’s bound to the gold layer.
- Binding of biomolecules will change the critical angle and can be seen on the graph.
(If the gold layer is too thick, the electron cloud won’t allow to become a collective wave and light is just reflected).
SPR can be used to plot time-dependent graphs to study the molecular kinetics of biomolecule binding. (youtube video)
Describe the process of nanocantilevers. What are its applications?
- Mechanical nanosensor –> a series of very thin individual silica cantilevers which are a few nm in thickness.
- The cantilevers are sensitive to surface stress – once they bind to the upper surface, they can bend due to a stress response since nothing is on the bottom surface (not due to the weight!).
- -The nanometre thickness is necessary for the bending to occur.
- Binding can be very sensitive to molecular interactions on the surface. Possible use as diagnostic tool.
- A laser beam (such as in AFM) is bounced off the cantilever – when something is bound and the cantilever bends, it is detected since the position of the light bouncing off changes.
- The binding of analytes can be detected in real time.
Application –> Detection of antibiotics – e.g. vancomycin – Vancomycin acts on bacteria by binding to peptides in bacterial cell wall.
o Vancomycin resistance is associated with a change in the bacterial cell wall – the resistant bacteria have a lactate instead of alanine (a loss of one hydrogen bond).
o Peptides were assembled onto gold coated cantilevers, the lactate and alanine onto adjacent cantilevers – and they were exposed to vancomycin.
o The cantilever with alanine would then bind vancomycin and bend downwards. The lactate (resistant) cantilevers did not deflect.
Describe the process of using nanowires.
- A voltage is applied to a silicon nanowire which is around 10nm in diameter and a current flows
- Nanowires can also be carbon nanotubes or organic polymers which are nanometres in diameter.
- The flow of electrons through the wire is dependent on the electrostatic charge of the surface molecules –> e.g. positive/negative charges will alter the flow of electrons.
- The net charge of the surface will alter resistance (and hence conductivity) to the flow of current through the nanowire.
Describe the chemiresistor.
- It is not a biological sensor but has several clinical applications.
- Based on gold nanoparticles —> they can be coated with ‘capping’ molecules. If they are in close proximity to each other we can measure conductivity.
- The electrons here will ‘hop’ from particle to particle (tunnelling) which determines the conductivity of the nanoparticle layer – this is determined on distance between the NPs and the capping molecules.
- If the NP layer is exposed to gas, they are adsorbed by the capping molecules – the level of adsorbing is determined by type of capping molecules.
- When molecules are adsorbed/desorbed, they alter distance of NPs and their conductivity.
- Therefore the conductivity of NPs is determined by what’s adsorbed onto capping layer – diff vapours will alter the conductivity in rapid fashion.
What is electronic noise in regards to the chemiresistor? Give an example of its application.
Electronic noise:
- the fact that an array of gold particles can be made, with each one coated with a capping molecule.
- Each capping molecule can adsorb several different vapours – such as in our nose, where our olfactory receptors can detect several different vapours.
- Therefore each capping molecule is differentially non-specific.
- Using statistical methods, we can train the software and the array to detect patterns of response characteristic of different volatile vapours.
- Therefore this is a generic method since different arrays can be calibrated in different ways.
E.g lung cancer diagnostics:-
- tests are done on healthy and cancerous exhalations to see their difference on the arrays and the arrays were then calibrated to detect differences between healthy and cancerous breaths by using these biomaerkers.
- With real samples, there was much more variation compared to the calibrated/simulated volatiles but is still sufficiently good to make a discrimination between volatiles from healthy and cancerous.
- Goal was to reduce it to a handheld device.
- These results show sensors based on gold nanoparticles could form the basis of an inexpensive and non-invasive diagnostic tool for lung cancer.
