1. Neethu Xavier St. Alberts College Cochin, India Microcantilever Biosensors: Making Sensors Reliable

2. Cantilever Sensors Cantilever undergoes bending due to molecular adsorption Cantilever resonance frequency also changes due to molecular adsorption (not useful in liquid medium since vibration is highly damped) Cantilever undergoes bending due to molecular adsorption Cantilever resonance frequency also changes due to molecular adsorption (not useful in liquid medium since vibration is highly damped) Interactions Steric Electrostatic van der Waals  Resonance frequency decreases Due to mass loading Cantilevers can be made into a biosensor by immobilizing molecular probes

3. 3 Immobilizing Receptors on a Cantilever First the cantilever is coated with a thin layer of gold. Selective layers are then self assembled on the gold surface – Polymer – Self-assembled monolayers (SAM) – Nanoparticles – Bio-molecules Designing surface coupling chemistry – Linkers For Optimum Stress Transduction – Adhesion Layers – Nanostructures Application techniques – Self-Assembly – Directed self assembly – Ink-jet deposition

4. Example 1: Real-Time Detection of Biotoxins in Solution Cantilevers with immobilized ricin antibodies undergo bending when exposed to ricin under solution Response time can be reduced by using smaller liquid volume 40 parts-per-trillion sensitivity Bending/nm Bio-Receptors Are Highly Selective

5. 5 Example 2: C-Reactive Protein (CRP) Detection Bulk functionalization C-Reactive protein Sample introductio n Bulk chemical blocking/inactivation Y YY Spotting of CRP antibody YYY Reaction with CRP Y Y Y 100 mg/L 5 mg/L 0 mg/L CRP is an acute phase protein, which is found in the human blood at very low concentrations (<0.1 mg/L). Bacterial infection will induce a dramatically increase in concentration (up to 300 mg/L)

6. Example 3: Detection of Ca 2+ ions  Ca 2+ ions are complexed with two amide groups  Surface free energy decreases 2- (4-mercaptophenoxy)-N,N-diethyl- acetamide SAM

7. Research Opportunity Despite its advantages such as sensitivity, selectivity, miniature size, cantilevers are not used commercially At present cantilever bending signal due to biosensing (antibody-antigen interaction) vary from cantilever to cantilever This uncertainty is preventing the cantilever sensors from becoming a useful tool for detection Hypothesis: Signal variation in a cantilever sensor is most probably due to non-uniformity of the immobilized probe layer

8. Challenges in Probe Immobilization: Uniformity The gold coating is not single crystalline The selective layer is not uniform Non-uniformity of probe layer causes signal to vary form cantilever to cantilever Patchy immobilization of receptors results in large standard deviations in the results Godin et al., Langmuir, 2004 Morphology of gold deposited at two different temperatures Patchy immobilization

9. Research Proposal Develop a reliable technique for immobilization of receptors such as antibodies on a cantilever Develop a protocol for gold deposition on cantilevers Reduce the number of steps involved in Immobilizing antibodies on the gold surface Develop techniques for characterizing the quality of coatings (AFM and FTIR) Characterize the functional cantilever using resonance frequency (before and after functionalization) to estimate the number of probe molecules on the surface