2. Immunochemical Methods and Biosensors for pollutants determination (General principles and application) Immunochemical Methods and Biosensors for pollutants determination (General principles and application) Danila Moscone Department of Chemical Science and Technology University "Tor Vergata" Rome, Italy danila.moscone@uniroma2.it

3. These methods already play an important role, especially in clinical chemistry, being used for the fast and safe detection of proteins, hormones, and pharmaceutical agents. Immunoassays (IAs) are techniques based on the formation of a thermodynamically stable antigen – antibody complex.

4.  Immunoassays become important when :  Fast measurement and evaluation are required  Fast measurement and evaluation are required  Highest possible detection strength is required  Highest possible detection strength is required  Large numbers of samples are to be expected  Large numbers of samples are to be expected  Only complex and expensive analytical methods are otherwise available. The greatest potential for the use of immunoassays in environmental analytical chemistry is in SCREENING i.e., for the selection of contaminated and uncontaminated samples for further validation analysis.  The greatest potential for the use of immunoassays in environmental analytical chemistry is in SCREENING i.e., for the selection of contaminated and uncontaminated samples for further validation analysis.

5. Terminology Immunogen: Substance able to generate an immune response Antigen:Original - Substance able to generate antibody. More general - Substance that can be recognized by antibody or T cells Hapten:Non-immunogenic substance. Usually low molecular weight. Induces antibody formation when coupled to a larger “carrier” molecule. Can bind antibody

6. Immunize withAntibodies formed DNP None BSA Anti-BSA DNP-BSA Anti-DNP Anti-BSA Anti-DNP-BSA Protein Carrier - Bovine Serum Albumin Hapten - DNP

7. Antibody structure Antigen binding sites Light Chain Heavy Chain. ANTIBODY (immunoglobulin) A biological molecule (protein) that specifically recognizes a foreign substance (antigen) as a means of natural defence

8. Antibodies: production and labelling LABELLING Radio-isotopes, Enzymes, Fluorescent, probes (Quantum dots), Chemi- luminescent probes, Metal tags PRODUCTION Animals have a large number of antibody producing cells, all producing a different antibody. When an animal (rabbit) is injected with antigen, proliferation of the corresponding antibody producing cell is promoted. Blood from the rabbit contains antibodies, originating from different cells with slight variations.

9. Antibodies Polyclonal Monoclonal Antibodies that are collected from sera of exposed animal recognize multiple antigenic sites of injected biochemical. Individual B lymphocyte hybridoma is cloned and cultured. Secreted antibodies are collected from culture media recognize ONE antigenic site of injected biochemical

10. Antigen-antibody Interactions

11. Features of the Antigen- Antibody Interaction ReversibilityReversibility Non-covalent Interactions AffinityAffinity Measure of the strength of the binding Ease of association or dissociation AvidityAvidity Increase in affinity due to multivalent binding The summation of multiple affinities

12. Non-covalent binding

13. Affinity and Avidity

14. Antibody-based assays

15. Enzyme-Linked Immunosorbent Assay ELISA

16. Specific Ab Ag E antigen- enzyme conjugate immobilisation surface Affinity reaction E E S P Enzym. reaction Product measurement E Incubation E E Coating

17. I. No analyte - high detection signal E E E E E E II. Analyte present - detection signal reduced E E

19. The enzymatic product concentration is inversely proportional to the analyte (standard or sample) amount ANTIGEN COATING ENZYMATIC REACTION S P Indirect competitive ELISA format Indirect competitive ELISA format BLOCKING FREE Ag and SPECIFIC Ab ADDITION SECONDARY LABELLED Ab

20. ELISA SANDWICH FORMAT Y Y Y Y Y Y 2nd antibody with enzyme Antibody/Antigen Antibody Y Y Y enzyme produces colour

21. signal/concentration curve Signal (enzyme activity) Antigen concentration Functional concentration range

22. ELISA PLATE WASHER ELISA PLATES SPECTROPHOTOMETER ADAPTED FOR ELISA PLATES

23. Lateral Flow Strips (Dipsticks) Immunochromatography (Lateral Flow) Biochemical components are separated across an absorbent membrane into discrete distinct regions. Apply sample solution, upon application of sample biochemicals dissolve Immobilised Antibody area Control area Positive: no antigen Negative: antigen present

24. analyte Test line Ab-colloidal gold Predator support Sample pad QUALITATIVE TEST

25. Test line Sample pad QUALITATIVE TEST:Analyte absent in the sample QUALITATIVE TEST: Analyte absent in the sample Analyte Ab-colloidal gold

26. If the analyte is ABSENT in the sample the line will be colored Test line Sample pad Analyte Ab-colloidal gold

27. Test line Sample pad Analyte PRESENT in the sample Analyte Ab-colloidal gold

28. Test line Sample pad Analyte Ab-colloidal gold

29. Test line Sample pad Analyte Ab-colloidal gold

30. Sample pad Test line If the analyte is present in the sample the line will be not colored Analyte Ab-colloidal gold

32.  We can use these immunochemical elements to assemble a special kind of biosensors called Immunosensors

33. Analyte Biological component Signal transducer Recorder

34. What do they have in common? Analyte / bioreceptor / transducer / processor Biosensor Small molecules / olfactory membrane / nerve cells / brain Visible light / rods and cones / nerve cells / brainNoseEye

37. Staphylococcus aureus Enterotoxins: A, B, C, D, E (thermostable);  Coagulase;  Thermonuclease. 100-200 ng of enterotoxins are sufficient to cause toxinfection in immuno-compromised subjects. gram-positive, non spore-forming bacterium able to synthetise:

39. Conventional ELISA Proteina A Conventional ELISA S. aureus ELISA/AMPLI S. aureus ELIMC S. aureus ELIME S. aureus DEVELOPED TEST:

40. Spectrophotometric ELISA Protein A/S.aureus Human IgG Specific antibody (MAb o PAb) Secondary antibody-AP AP p-NPPp-NITROPHENOL

41. MAb IgG 10 mg/mL MAb 1:10000 Ab 2 -AP 1:1000 PAb IgG 10 mg/mL PAb 1:10000 Ab 2 -AP 1:1000 LOD Sensitivity 0.6 ng/mL 7.6 ng/mL LOD Sensitivity 0.07 ng/mL 0.6 ng/mL y = (a – d) x c +1 b + d y = + 3s ELISA Protein A Sensitivity was calculated as tha amount of protein A needed to produce a 25% increase in the signal

42. MAb IgG 10 mg/mL MAb 1:10000 Ab 2 -AP 1:1000 PAb IgG 10 mg/mL PAb 1:10000 Ab 2 -AP 1:1000 LOD Sensitivity 2 10 6 cell/mL 9 10 6 cell/mL LOD Sensitivity 2 10 4 cell/mL 2 10 5 cell/mL ELISA S.aureus No cross- reactivity

43. Acetaldehyde NADPH NADH NAD + INT FORMAZAN Ethanol Alcohol deydrogenaseDiaphorase Alkaline phosphatase PiPi DAKO, Handbook for AmpliQ, 1997 AMPLI Q

44. ELISA S.aureus AMPLIQ MAb IgG 10 mg/mL MAb 1:10000 Ab 2 -AP 1:1000 PAb IgG 10 mg/mL PAb 1:10000 Ab 2 -AP 1:1000 LOD Sensitivity 6 10 4 cell/mL 2 10 5 cell/mL LOD Sensitivity 7 10 2 cell/mL 6 10 3 cell/mL

45. Good results in immunological field Ø 1-5 µm Measurements on real samples Magnetic Beads Magnetic particles are particles constituted from a dispersion of magnetic material (Fe 2 O 3 and Fe 3 O 4 ) and then covered with a thin shell of polymer which contains the magnetic material and also serves to define a surface area for the absorption or coupling of a large variety of other molecules.

46. ELIMC (Enzyme Linked ImmunoMagnetic Colorimetry) All reactions were carried out in eppendorf tubes No intermediate washings AP p-NPP p-NITROPHENOL Microtitre ELISA

47. ELIME (Enzyme Linked ImmunoMagnetic Electrochemistry) a-naphthyl phosphate a-naphthol AP + + NaH 2 PO 3 Selectivity Ag-Ab; Sensibility of electrochemical detection; Possibility of concentrating magnetic particles on the electrode surface. DPV Potential range 0-600 mV Scan speed 100 mV/s Pulse width 50 ms Modulation time 60 ms Interval time 0.16 s

48. Magnetic tube Addition of Enzymatic substrate for Electrochemical measurement

49. ELIMC S.aureus ELIME S.aureus MAb IgG 1.2 mg/mL MAb 1:1000 Ab 2 -AP 1:100 LOD Sensitivity 1 10 3 cells/mL 2 10 4 cells/mL MAb IgG 0.5 mg/mL Mab 1:50000 Ab 2 -AP 1:300 LOD Sensitivity 1 10 4 cells/mL 2 10 5 cells/mL

50. Mab ELIME Mab ELIMC Pab ELISA AmpliQ Mab ELISA AmpliQ Pab ELISA S.a Mab ELISA S.a Pab ELISA prot A Mab ELISA prot. A SensitivityLOD 0.6 ng/mL 0.07 ng/mL 2 10 6 cell/mL 2 10 4 cell/mL 6 10 4 cell/mL 7 10 2 cell/mL 1 10 3 cell/mL 1 10 4 cell/mL 9 10 6 cell/mL 2 10 5 cell/mL 2 10 5 cell/mL 6 10 3 cell/mL 2 10 4 cell/mL 2 10 5 cell/mL 0.6 ng/mL 7.6 ng/mL 22 h 4 h Analysis Time

51. Air samples Sample 1 Sample 2 660 cell/m 3 ± 15% 11700 cell/m 3 ± 11% Two air samples from hospital rooms Sampling carried out by a SAS air-sampler. Flow rate 35 litri/min, for 30 minuts, collin 30 ml of buffer

52. Immunoassay test products validated by OSW

53. Anticholinesterase activity measurement by an enzyme biosensor: application in water analysis

54. This method is a fast, cheap, and good analytical choice to measure the total anti-ChE charge in the sample, an important toxicological index defined as the amount of compounds which causes a % of ChE inhibition equivalent to that produced by a known amount of a pesticide (e.g. Paraoxon) taken as reference compound. Acetilcholine Choline + Acetic ac. Acetilcholinesterase Inhibited by pesticides Choline + O 2 + H 2 O Betaine + H 2 O 2 Choline oxidase Not Inibited H2O2H2O2 O 2 + 2H + + 2e - Electrode

55. Trasmission of the nervous impulse Acetilcholinesterase

56. Time (min) I%=[( E 0 -E i )/E 0 ]100 Non inhibited enzyme (E 0 ) inhibited enzyme (E 1 ) Inhibition measurements

57. 0123 4 TIME (minutes) I (nA) 0 2 4 6 Inhibition of AChE with Paraoxon Blank 2 ppb 6 ppb 10 ppb

58. HEAVY METALS DETERMINATION BASED ON THE USE OF INVERTASE ENZYME ON THE USE OF INVERTASE ENZYME

59. REACTIONS Sucrose E1E1 D-Glucose D-Glucose + D-Fructose D-Glucose E2E2 + O 2 H 2 O 2 Gluconic acid + H 2 O 2 H2O2H2O2H2O2H2O2 Electrode 2e - O 2 + 2H + + 2e - E 1 = Invertase E 2 = Glucose Oxidase + H 2 O

60. INV INV + Inhibitor Sucrose I1I1 I2I2 Reaction Time A B Time Current INHIBITION MEASUREMENTS

61. 0204060 80 [Hg 2+ ] (ppb) Inibition % 0 20 40 60 100 FIA Calibration with sucrose 10mM 80

62. Biosensors applied to the determination of pollutants in real samples From: S. Rodriguez-Mozaza, M. J. L´opez de Aldaa, M.-P. Marcob, D. Barcel´oa,, Talanta 65 (2005) 291–297