cod. 22985

Academic year 2022/23
1° year of course - Second semester
- Alessandro BERTUCCI - Simone FORTUNATI
Academic discipline
Chimica analitica (CHIM/01)
Attività formative affini o integrative
Type of training activity
55 hours
of face-to-face activities
6 credits
hub: PARMA
course unit

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


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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.


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.

Assessment methods and criteria

Oral exam

Other information