Biosensors/ Aptasensors

Biosensors are devices used to detect the presence or concentration of a biological analyte, such as a biomolecule, a biological structure or a microorganism. They consist of three components: a recognition element that binds the analyte and produces a signal, a signal transducer and a reader device.

Aptamers are perfect tools for biosensor development due to their favourable properties, including

  • Versatility – Aptamers can be developed for virtually any kind of target under both, physiological or unnatural conditions.
  • Specificity – Aptamer specificity gives more reliable sensors with fewer false positives or background effects
  • Size – Aptamers are approximately 1/10 the size of an antibody, meaning that interactions occur closer to the sensor surface. This greatly increases the sensitivity of many sensor platforms.
  • Flexibility – most aptamers undergo a conformational change upon target binding. This is useful for redox reporter sensing as the distance between the sensor and reporter will change on target binding.
  • Adaptability – Common functional groups (biotin, thiol, amine etc.) are readily incorporated into aptamers during their synthesis. This allows straightforward immobilisation of the aptamer onto the sensor surface; converting a generic sensor platform into a target specific biosensor.
  • Reusability – In many cases, aptamer-target binding is regenerable enabling reuse of biosensor.
  • Displacement – our small molecule binding aptamers are released from surfaces on target binding. This means that interaction with a small molecule leads to a large ‘mass change’ on the surface (as the aptamer leaves). This amplifies the response seen from the small molecule.

These advantages make aptamer-based biosensor development significantly easier than with other affinity ligands.

Common Biosensor Formats

Aptamers are compatible with many well-established techniques for detection and monitoring biomolecular interactions; including Bio-Layer Interferometry (BLI), Surface Plasmon Resonance (SPR) , Isothermal Titration Calorimetry  (ITC) and Microscale Thermophoresis (MST). They are powerful tools in basic research, drug discovery and development, and downstream bioprocessing.

These methods are used to study molecular interactions, determine kinetic and affinity parameters (providing association rate constant ka, dissociation rate constant kd, dissociation constant KD); binding specificity profiling and ligand characterisation etc.

Small Molecule BLI (BioLayer Interferometry) utilising our Displacement Approach

  • Small molecule binding cannot be detected due to sensitivity limits of many biosensor instruments.
  • Conformation change / displacement of the aptamers upon small molecule binding, gives a ‘signal amplification’ effect.
  • This displacement can be used as a basis for small molecule detection in these platforms.
The displacement approach (used for small molecule aptamer selection) can be adapted to allow detection of small molecules on instruments that cannot normally detect these targets. Aptamer loading on the biosensor gives a clear positive signal (30-330 sec), followed by a reduction in signal during washes (330-510 sec). A clear difference is seen during the ‘Displacement phase’, when the sensor is incubated in the presence or absence of the aptamer target (red and blue traces respectively).
The response from displacement of surface bound aptamers is target concentration dependent. The level of response can be fitted using a Steady State Analysis to calculate an approximate affinity. (*Steady State Kinetics have been fit to aptamer displacement assay data. As the responses are not association curves, this is not a true KD measurement and is intended for comparative purposes only.)

Kinetic measurements of small molecules using aptamers and SPR (Surface Plasmon Resonance)

The enhanced sensitivity of the new generation of SPR biosensors (e.g. Biacore) allow rapid characterisation of kinetic and equilibrium binding properties of aptamers and their targets. These systems are generally sensitive enough to monitor small molecule binding interactions directly.

Direct measurements of aptamer interactions with small molecule targets are also possible with more sensitive instruments. SPR data shows clear target concentration dependent interactions between the immobilised aptamer and its small molecule chemotherapeutic target. A 1:1 Langmuir binding model was applied to this data to calculate a KD of 72 ± 16nM

Electrochemical Biosensors

The first scientifically proposed and successfully commercialised biosensors were based on electrochemical sensors for multiple analytes. In most electrochemical biosensors, a recognition element (in this case an aptamer) is conjugated to the sensor surface. The aptamer is also conjugated to a suitable reporting group (typically a redox reporter such as Methylene Blue). As with other sensor systems; target binding results in a conformational change in the aptamer, changing the distance between the reporter molecule and the sensor surface. This in turn leads to a quantifiable readout from the sensor.

Cartoon representation of an aptamer based biosensor for the detection of cocaine in a biological sample (left). Binding of the target induces a conformational change in the aptamer, moving the Methylene Blue (MB) redox reporter closer to the electrode. This conformational change results in a concentration dependent voltammetric response in the biosensor, which can be recorded and plotted against target concentration (right).
References:
J Am Chem Soc. 2006 Mar 15;128(10):3138-9. An electronic, aptamer-based small-molecule sensor for the rapid, label-free detection of cocaine in adulterated samples and biological fluids. Baker BR1, Lai RY, Wood MS, Doctor EH, Heeger AJ, Plaxco KW.