Learning objectives
- Acquire knowledge of the principal techniques used for biomolecular analysis, with a focus on nucleic acids and proteins.
- Know how to interpret analytical data;
- Learn about the main applications in molecular diagnostics and chemical biology.
At the end of the course, the student is expected to:
- know the main biomolecular recognition processes underlying standard bioanalytical techniques and biosensors, including nucleic acid interactions, affinity binding processes and ligand-substrate interactions;
- have knowledge of the main biomolecular detection methods, including amplification techniques and immunoassays.
- be familiar with the main mass spectrometry-based instrumental techniques and the connected workflow used to analyze biomolecular species in complex samples.
- be able to read and process analytical data, also from a method validation perspective:
- recognize the potential, the limits and the drawbacks of the different techniques;
- develop critical thinking and creativity to address bioanalytical challenges
- build adequate communication skills and possess a good command of the terminology of the field.
- prove their ability to independently expand on the notions learned during the course, peruse the relevant literature and engage in the discussion of recent trends in bioanalytical chemistry.
Prerequisites
- - -
Course unit content
Principles of DNA nanotechnology and applications in bioanalytical chemistry: nucleic acid interactions and dynamic mechanisms, DNA-based reactions, bimolecular switches.
Nucleic acid amplification: enzymatic and non-enzymatic amplification techniques.
Immunoassays: ELISA and immunofluorescence assays, combined assays, lateral flow sticks.
Principles of gel electrophoresis: separation of nucleic acids and proteins.
Mass spectrometry in bioanalytical chemistry: general principles, ion sources, mass analyzers, qualitative and quantitative acquisition modalities, tandem mass spectrometry, fundamentals of proteomics, hyphenated liquid chromatography-mass spectrometry techniques.
Data interpretation: principles of chemometrics, analytical parameters, validation of analytical methods, interpretation of serology tests.
Laboratory: ELISA, electrochemical detection of DNA, strand displacement reactions.
Full programme
Principles of DNA nanotechnology and applications in bioanalytical chemistry: nucleic acid interactions and complex structures (duplex, triplex, G-quadruplex, i-motif, stem-loop hairpins, junctions, branched kissing loops); aptamers and their applications; artificial nucleic acids (PNA, LNA); dynamic DNA nanotechnology (strand displacement, toehold exchange, DNA switches, DNA walkers, effective molarity); enzyme-based amplification techniques (PCR, RT-PCR, LAMP, SDA, RCA, RPA, NASBA); enzyme-free amplification techniques (HCR, CHA).
Immunoassays: competitive/non-competitive; homogeneus/heterogenous; direct/indirect and sandwich ELISA; immunofluorescence; lateral flow assays; proximity ligation assays; agglutination assays.
Principles of gel electrophoresis: DNA separation on agarose and PAGE gels; protein separation on native and denaturing gels.
Mass spectrometry for biomolecular analysis: general principles; ESI and APCI ion sources; analyzers (quadrupole, TOF, ion trap, Orbitrap); tandem mass spectrometry MS/MS; qualitative and quantitative acquisition modalities; hyphenated LC-MS techniques; basic concepts of bottom-up proteomics; data dependent acquisition DDA; peptide fragmentation; MALDI; DESI.
Data interpretation: fundaments of chemometrics, analytical parameters and method validation (precision, trueness, dynamic range, LOD/LOQ, linear regression); calibration methods; matrix effect; analytical parameters in serological tests (specificity and selectivity, false negatives and false positives, Bayesian statistics, dissociation constants and binding affinity).
Laboratory: ELISA, electrochemical detection of DNA, strand displacement reactions.
Bibliography
Lecture notes.
Articles from the literature suggested by the instructor.
For mass spectrometry: “Mass Spectrometry: principles and applications”, E. de Hoffmann, V. Stroobant; Wiley, 2007.
Teaching methods
Frontal teaching
Laboratory: ELISA, electrochemical detection of DNA, strand displacement reactions.
Assessment methods and criteria
Oral exam
Other information
