Immunochemical Methods and Biosensors for pollutants determination (General principles and application) 

antigen – antibody complex. Immunoassays (IAs) are techniques based on the formation of a thermodynamically stable antigen – antibody complex. 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 become important when: Fast measurement and evaluation are required  Highest possible detection strength is required  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.

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

Immunize with Antibodies formed DNP None BSA Anti-BSA DNP-BSA Anti-DNP Hapten - DNP Protein Carrier - Bovine Serum Albumin Immunize with Antibodies formed DNP None BSA Anti-BSA DNP-BSA Anti-DNP Anti-BSA Anti-DNP-BSA

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

Antibodies: production and labelling 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. LABELLING Radio-isotopes, Enzymes, Fluorescent, probes (Quantum dots), Chemi-luminescent probes, Metal tags

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

Antigen-antibody Interactions

Features of the Antigen-Antibody Interaction Reversibility Non-covalent Interactions Affinity Measure of the strength of the binding Ease of association or dissociation Avidity Increase in affinity due to multivalent binding The summation of multiple affinities

Non-covalent binding

Affinity and Avidity

Antibody-based assays

Enzyme-Linked Immunosorbent Assay ELISA Enzyme-Linked Immunosorbent Assay

immobilisation surface Direct competitive immunoassay (I) Specific Ab Ag E antigen- enzyme conjugate immobilisation surface Coating Incubation E E E S E Enzym. reaction Product measurement P Affinity reaction

Direct competitive immunoassay (II) I. No analyte - high detection signal E II. Analyte present - detection signal reduced E

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

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

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

SPECTROPHOTOMETER ADAPTED FOR ELISA PLATES ELISA PLATE WASHER ELISA PLATES SPECTROPHOTOMETER ADAPTED FOR ELISA PLATES

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

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

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

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

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

Test line Sample pad Analyte Ab-colloidal gold

Test line Sample pad Analyte Ab-colloidal gold

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

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

biosensor Recorder Biological component Signal transducer Analyte

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

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

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

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

ELISA Protein A MAb PAb LOD Sensitivity 0.6 ng/mL 7.6 ng/mL LOD (a – d) x c + 1 b + d MAb IgG 10 mg/mL MAb 1:10000 Ab2-AP 1:1000 PAb LOD Sensitivity 0.6 ng/mL 7.6 ng/mL LOD Sensitivity 0.07 ng/mL 0.6 ng/mL y = <x0> + 3s IgG 10 mg/mL   PAb 1:10000 Ab2-AP 1:1000 Sensitivity was calculated as tha amount of protein A needed to produce a 25% increase in the signal

ELISA S.aureus LOD Sensitivity 2 106 cell/mL 9 106 cell/mL LOD MAb PAb IgG 10 mg/mL IgG 10 mg/mL MAb 1:10000 PAb 1:10000 Ab2-AP 1:1000 Ab2-AP 1:1000 No cross-reactivity

AMPLI Q INT FORMAZAN NADPH Acetaldehyde NADH NAD+ Ethanol Alkaline phosphatase Pi Acetaldehyde INT NADH Alcohol deydrogenase Diaphorase NAD+ FORMAZAN Ethanol DAKO, Handbook for AmpliQ, 1997

ELISA S.aureus AMPLIQ Because a new lot of PAb showed lower affinity for Protein A/S. Aureus, next experiments were carried out using only Mab. MAb LOD LOD PAb 6 104 cell/mL 7 102 cell/mL IgG 10 mg/mL IgG 10 mg/mL Sensitivity Sensitivity MAb 1:10000 PAb 1:10000 2 105 cell/mL 6 103 cell/mL Ab2-AP 1:1000 Ab2-AP 1:1000

Magnetic Beads Good results in immunological field Ø 1-5 µm Magnetic particles are particles constituted from a dispersion of magnetic material (Fe2O3 and Fe3O4) 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. Good results in immunological field Ø 1-5 µm Measurements on real samples

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

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

Electrochemical measurement Addition of Enzymatic substrate for Electrochemical measurement Magnetic tube

ELIMC S.aureus ELIME S.aureus MAb LOD 1 104 cells/mL MAb LOD IgG 0.5 mg/mL Mab 1:50000 Ab2-AP 1:300 LOD Sensitivity 1 104 cells/mL 2 105 cells/mL MAb IgG 1.2 mg/mL MAb 1:1000 Ab2-AP 1:100 LOD Sensitivity 1 103 cells/mL 2 104 cells/mL

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 Sensitivity LOD 0.6 ng/mL 0.07 ng/mL 2 10 6 cell/mL 4 6 10 7 10 2 1 10 3 9 10 5 7.6 ng/mL 22 h 4 h Analysis Time

Air samples Sample 1 Sample 2 660 cell/m3 ± 15% 11700 cell/m3 ± 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 Sample 1 Sample 2 660 cell/m3 ± 15% 11700 cell/m3 ± 11%

Immunoassay test products validated by OSW

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

Acetilcholine Choline + Acetic ac. Choline + O2 + H2O Betaine + H2O2 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. Acetilcholinesterase Acetilcholine Choline + Acetic ac. Inhibited by pesticides Choline oxidase Choline + O2 + H2O Betaine + H2O2 Not Inibited Electrode H2O2 O2 + 2H+ + 2e-

Trasmission of the nervous impulse Acetilcholinesterase

Inhibition measurements I%=[( E0-Ei)/E0]•100 inhibited enzyme (E1) Non inhibited enzyme (E0) Time (min)

Inhibition of AChE with Paraoxon 1 2 3 4 TIME (minutes) I (nA) 6 Inhibition of AChE with Paraoxon Blank 2 ppb 6 ppb 10 ppb

HEAVY METALS DETERMINATION BASED ON THE USE OF INVERTASE ENZYME

REACTIONS E1= Invertase E2 = Glucose Oxidase Sucrose E1 + H2O D-Glucose + D-Fructose D-Glucose E2 + O2 Gluconic acid + H2O2 Electrode O2 + 2H++ 2e- H2O2

I1 I2 INHIBITION MEASUREMENTS Current Sucrose INV B Sucrose INV + Inhibitor I2 A Reaction Time Time

FIA Calibration with sucrose 10mM 20 40 60 80 [Hg2+] (ppb) Inibition % 100 FIA Calibration with sucrose 10mM

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

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