Learning objectives
- Acquire knowledge of the principal techniques used for biomolecular analysis, with a focus on nucleic acids and proteins.
- Know how to interpret and process analytical data.
- Learn about the main applications in the field.
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.
- gain knowledge of the main biomolecular detection methods, including amplification techniques and binding assays.
- be familiar with the main mass spectrometry-based instrumental techniques and the connected workflow for the analysis of biomolecular species in complex samples.
- be able to read and process analytical data.
- be able to carry out validation of an analytical method.
- assess the potential and the limitations of different techniques.
- develop critical thinking and creativity to address bioanalytical challenges
- build adequate communication skills and demonstrate good command of the appropriate terminology.
- prove their ability to independently expand on the notions learned during the course by interfacing with the relevant scientific literature and being able to discuss recent trends in bioanalytical chemistry
Course unit content
Review of instrumental techniques for the generation of analytical signals: UV-vis absorption spectrophotometry, fluorescence emission spectrophotometry, voltammetry-based electroanalytical techniques.
Fundamentals of DNA nanotechnology and bioanalytical applications: reaction mechanisms between synthetic nucleic acids, static and dynamic structures, molecular architectures and sensors.
Nucleic acid amplification: enzymatic techniques, non-enzymatic techniques, techniques based on CRISPR-Cas technologies.
Immunoassays and binding assays: biochemical principles and Langmuir curves, enzymatic assays, fluorescence-based assays, lateral flow assays.
Gel electrophoresis: separation of nucleic acids and proteins.
Mass spectrometry for the analysis of biomolecules: general principles, ionization sources, analyzers, qualitative and quantitative acquisition methods, tandem mass spectrometry, general concepts in proteomics, hyphenated techniques based on liquid chromatography.
Data interpretation and measurement quality assessment: parameters for the validation of analytical methods and the interpretation of diagnostic tests.
Laboratory experiences: ELISA assays, electrochemical detection of DNA, mass spectrometry.
Full programme
Review of instrumental techniques for the generation of analytical signals: 1) UV-vis absorption spectrophotometry - interaction between electromagnetic radiation and molecules, electronic transitions, absorption bands, transmittance and absorbance, Lambert-Beer law, limitations and deviations, instrumentation ; 2) fluorescence emission spectrophotometry - origin of the phenomenon of fluorescence and of phosphorescence, Jablonsky diagram, Stokes shift, Kasha rule, mirror image rule, lifetime and quantum yield, quenching mechanisms, Stern-Volmer equation, instrumentation, quantitative measurements in fluorescence; 3) electroanalytical techniques in voltammetry – three electrode cell, current and reaction rate, mass transport and limiting current, quantitative measurements, faradic and non-faradic currents, pulsed voltammetry, electrochemical sensors, cyclic voltammetry.
Fundamentals of DNA nanotechnology and bioanalytical applications: interactions between nucleic acids, complex structures (duplex, triplex, stem-loop hairpin, junctions, origami); aptamers and SELEX process; artificial nucleic acids (PNA and LNA); dynamic reaction mechanisms in functional DNA nanotechnology (strand displacement, toehold-exchange), DNA and biomolecular switches.
Nucleic acid amplification: enzymatic amplification techniques (PCR, RT-PCR, LAMP, RCA, RPA, NASBA), non-enzymatic amplification techniques (HCR, CHA), techniques based on CRISPR-Cas technologies.
Immunoassays: antigen-antibody interactions, biochemical principles, Langmuir-type binding curves, direct, indirect and sandwich enzymatic assays (ELISA), competitive ELISA, immunofluorescence assays, lateral flow technologies.
Principles of gel electrophoresis: separation of large DNA fragments on agarose gels; separation of proteins and nucleic acids on native and denaturing polyacrylamide gels.
Mass spectrometry for the analysis of biomolecules: principles and general notions; ESI sources; quadrupole analyzers, TOF, ion trap, FT-ICR, Orbitrap; tandem mass spectrometry and related acquisition methods; combined liquid chromatography-LC-MS mass spectrometry techniques (RP-HPLC and SEC) and signal acquisition modalities (TIC, SIM, SRM, MRM), general outline of bottom-up proteomics and peptide fragmentation; MALDI mass spectrometry.
Data interpretation and measurement quality assessment: analytical validation parameters (precision, trueness, accuracy, linearity, dynamic range, LOD/LOQ, sensitivity, selectivity, specificity); interpretation of serological tests (specificity and selectivity, PPV, NPV, prevalence).
Laboratory experiences: ELISA assays, electrochemical detection of DNA, mass spectrometry.
Bibliography
Lecture notes.
Articles in scientific journals.
For the mass spectrometry course module: “Mass Spectrometry: principles and applications”, E. de Hoffmann, V. Stroobant; Wiley.
Teaching methods
In-person lectures.
Laboratory experiences.
