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Quinoproteins, a novel grop of enzymes containing quinonoid cofactors by T4nRGwP

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									               Biosensors
A focus on peroxidase-modified electrodes and their practical
                       applications


                             by
                       Ivo Frébort
Biosensor
- an analytical device that exploits a biocatalytic reaction

  Consists of:   biocatalyst (enzyme, cells, tissue)

                 transducer (converts the biological or biochemical signal
                            into a quantifiable electrical or optical signal)
  First biosensor - Clark (1962):
  glucose sensor with glucose oxidase and oxygen electrode

  Glucose + O2                            Gluconic acid + H2O2




                                                       Oxygen electrode (1956)

                                                       working electrode: Pt cathode (-0.6 V)
                                                       reference electrode: Ag/AgCl

                                                       electrodes separated from measured
                                                       solution with a gas permeable mebrane



Leland C. Clark, Jr. with the first enzyme electrode
Construction of the biosensors
Sensing electrode: platinum, gold, various forms of carbon
Immobilization techniques: general method doesn’t exist
- enzyme physical entrapment
- covalent crosslinking

                                              O CH   (CH2)3 C H O
                                                Glutaraldehyde
                          BSA     E
                                                CH O     + H2N
electrode                               BSA
              o-ring                E
                          E   BSA                        Schiff base

 E E E                                               CH N
                        BSA             BSA               Reduction with NaBH4
 E   E      dialyzing         E
            membrane
                                                     C H2 NH
Covalent attachment to a support membrane or the electrode
        R
                        R       B        NH2
        N           O
                        N
                            +                           O
COOH + C        C                                   C
       N            O C                                 NH      B
                                    NH       R
                        NH
        R'                          C O
                        R'                                             N C N         DCC
Carbodiimide reaction               NH       R'
                                                                                            CMC
                                                                       N C N CH2CH2 N       O
                                                                                 H3C        tosyl-

                                                            C H3C H2   N C N   (CH2)3 N(C H3)2 EDC



        O CH    (C H2)3 C H O                                                       B      NH2
  NH2                                             N CH        (C H2 )3 CH O


                                                  NaBH4
     N CH    (CH2)3 CH N                 B                     NH C H      (CH2)3 CH NH          B

   Glutaraldehyde reaction
     Adsorption - glass, carbon, Au, Pt
     - often activation needed


     Adsorption of thiols to a gold electrode               Silanization of an oxidized metal electrode


                                       S    (C H2)2 NH2                                               O
           S   (C H2)2 NH2                                                   R Si (OC2H5)3       O Si R
       +                                                       --OH
           S   (C H2)2 NH2             S    (CH2)2 NH2
Au                              Au                                                                    O
                                                               --OH
                                                                                                 O Si R
                                                                                                      O
      S                  X       S
                                 S
                                                               (C 2H5)3S i   (C H2)3 NH2      APTES
      S                  X                          X
      S                  X       S
      S                  X       S                             (C H3)3Si     (C H2)3 O C H2   CH C H2
      S                  X       S                  X
      S                  X       S                                                              O
                                                                           CH3
                                                                                              GOPS
       Organized layer       Dilution with an inert thiol      C 2H5 O Si (CH2)3 NH2
                                                                           CH3                APDMES
Screen printing

                 Mobile wiper

                                        Matrix carrier
                                Paste

   Screen grid




     Screen print
   An enzyme electrode
                                      P1                P1    -


                S1                                             -

     Sample                                     E                       Electrode
                S2                                            -


                                      P2                P2    -
                        I                        P2*

                     Protective        Enzyme layer          Permselective
                     membrane                                membrane

1. Thin enzyme layer with high specific activity,
2. Good selection of membranes
   Response controlled by diffusion through the permselective membrane
   (not by enzyme kinetics)
   Enzyme activity low - thick membrane needed to achieve linear response, response slow
   Enzyme activity high - thin membrane OK, rapid response

3. Detection: Steady-state or flow injection analysis
Biosensor parameters
                                                1. Sensitivity
                   dS/dt                        2. Linear response
Analyte                     Signal              3. Detection limit
                                                4. Background noise
                             DS                 5. Baseline drift
                                                6. Selectivity
                                                7. Response
            Time         Background noise
                                                8. Operating stability
                                                9. Shelf life

      Detection                                               Analyte
S     limit
                                            N
                   DS/Dc Linear response                                 S=3N


                                                   Assay of the detection limit
                     c
Type of measurement
                                                     Analyte additions
                                              S




                                                             Time
Direct contact with the sample         Solution placed in a chamber

                                                               A

        Flow cell
                                          S


                                              Time             B
                IN               OUT
First generation biosensors - response to the substrates in solution

           Glucose + O2                Gluconic acid + H2O2
    1. Reduction of oxygen with a Clark type electrode at -0.6 V (vs SCE)
    2. Oxidation of hydrogen peroxide at a Pt electrode at +0.7 V
    3. Measuring of pH change

 Examples of hydrogen peroxide measurning biosensor
 Analyte       Enzyme             Reaction
 Alcohol      Alcohol oxidase     Ethanol + O2  Acetaldehyde + H2O2
 D-Glucose    Glucose oxidase     β-D-Glucose + O2  Gluconic acid + H2O2
 Lactose      Galactose oxidase   Lactose + O2  Galactose dialdehyde der. + H2O2
 L-Lactate    L-Lactate oxidase   L-Lactate + O2  Pyruvate + H2O2
 Starch       Amyloglucosidase    Starch + H2O  β-D-Glucose
              Glucose oxidase     β-D-Glucose + O2  Gluconic acid + H2O2
 Sucrose      Invertase           Sucrose + H2O  α-D-Glucose + β-D-Fructose
              Mutarotase          α-D-Glucose  β-D-Glucose
              Glucose oxidase     β-D-Glucose + O2  Gluconic acid + H2O2
     Types of transducers used in biosensors

Type                       Detectable species
Amperometric               O2, H2O2, I2, NADH
Ion-selective electrode    H+, Na+, Cl-
Field effect transistors   H+, Na+, Cl-
Gas sensing electrode      CO2, NH3
Photomultiplier            Light emission
                           ATP/Luciferase/Luciferin, H2O2/Peroxidase/Luminol, etc.

Thermistor                 Heat of reaction
Second generation biosensors
- mediated electron transfer between enzyme and electrode
- can be easily miniaturized
blood glucose measuring system in situ

Third generation biosensors
- direct electron transfer between enzyme and electrode

Cell-based based biosensors
- cheaper than purified enzymes,
Nocardia erythropolis cells immobilised in polyacrylamide or agar
(cholesterol oxidase)
Cholesterol + O2  Cholest-4-en-3-one + H2O2

Enzyme immunosensors
- many types, based on ELISA techniques
- often use chemiluminiscence or bioluminiscence
human chorionic gonadotropin - pregnancy
                   Examples of biosensors
Analyte      Biocatalyst           Transducer   Immobilization   Shelf life   Response

Alcohol     Alcohol oxidase             O2      Glutaraldehyde   2 weeks       1-2 min
Arginine    Streptococcus faecium       NH3     Entrapment       3 weeks       20 min
Cholesterol Nocardia erythropolis       O2      Entrapment       4 weeks       35-70 s
D-Glucose Glucose oxidase               O2      Covalent        3 weeks        1 min
Glutamate   Glutamate decarboxylase      CO2     Glutaraldehyde   1 week         10 min
NAD+        NADase + Escherichia coli    NH3      Membrane         1 week        5-10 min
Nitrate     Azotobacter vinelandii       NH3     Entrapment       2 weeks        7-8 min
Penicillin  Penicillinase                H+      Polyacrylamide   2 weeks        15-30 s
Urea        Urease                       NH4+    Polyacrylamide   3 weeks        20-40 s
Personal glucose meter for diabetics
(Medisense Britain, Ltd.)
Automated affinity systems
Biacore 2000 (Biacore)     KI 1 (BioTuL)
www.bioacore.com           www.biotul.com




IAsys (Affinity Sensors)   IBIS II (XanTec)
www.affinity-sensors.com   www.xantec.com
          Peroxidase-based electrodes


                                                              PEROXIDASE (EC 1.11.1.7)

                                                               Protein of 35-45 kDa, prosthetic
                                                               group - heme, Mn2+

                                                               Convenient sources:
                                                               horse radish root, soybean,
                                                               tobacco leaves, various fungi




Ruiz-Duenas, F. J., Martinez, M. J., Martinez, A. T.: Peroxidase from the ligninolytic fungus
Pleurotus eryngii. Mol Microbiol 31 pp. 223 (1999)
      The catalytic cycle of peroxidase

Native peroxidase + H2O2          Compound-I + H2O
(Fe3+)                            (Fe4+=O, Por+)

Compound-I + AH2             Compound-II + AH*
(Fe4+=O, Por+)               (Fe4+=O)

Compound-II + AH2          Native peroxidase+ AH* + H2O
(Fe4+=O)                   (Fe3+)
       Applications of peroxidase-based electrodes
1. Detection of hydrogen peroxide in aqueous solutions
  industry, environmental protection, clinical control
  photochemical smog, pathological processes in lungs, etc.

2. Detection of organic peroxides in water and organic solutions
  free radical injury, oxidative stress, autooxidation of unsaturated lipids

3. Detection of compounds based on peroxidase inhibition
  CN-, F-, hydroxylamine

4. Detection of aromatic amines and phenolic compounds
  environmental control: chlorophenols in water

5. Immunosensors based on peroxidase electrodes
  peroxidase conjugates with antibody, H2O2-producing enzyme conjugates

6. Detection of analytes based on peroxidase/oxidase-coupled
   reactions
  glucose, ethanol, lactate,choline, xanthine, cholesterol, bilirubin, glutamate
              Electrode designs

A. Surface modified electrodes
   Electrode material: graphite, glassy carbon, gold, SnO2
   Coupling: carbodiimide, glutaraldehyde, adsorption

B. Polymer-based electrodes
   Crosslinking with Os(bpy)23+/2+ redox polymer,
   electropolymerized polypyrrole, o-phenylethylamine

C. Bulk modified composite electrodes
   Graphite-silicone oil paste, paraffin oil paste,
   epoxy composite

D. Tissue-modified carbon paste electrodes
   Asparagus tissue, tobacco callus tissue,
   horseradish root, kohlrabi skin
   Mechanism of direct biocatalytic reduction of
hydrogen peroxide at peroxidase-modified electrodes


                     Compound-I
H2O                  (Fe 4+=O, Por+)
                                       e-
                                            Electrode

                                            Eappl< 0.6 V
                   Compound-II
                    (Fe 4+=O)
                                   e-       vs SCE
                  2H+
H2O2
                  H2O
                    Ferriperoxidase
                         (Fe 3+)
  Mechanism of mediated biocatalytic reduction of
hydrogen peroxide at peroxidase-modified electrodes

                        Compound-I
H2O                     (Fe 4+=O, P +)
                                          Mred

                                          Mox
                       Compound-II
                                                      Electrode
                        (Fe 4+=O)
                                          Mred
                      2H +
H2O2
                     H 2O                  Mox

                        Ferriperoxidase
                             (Fe 3+)

       Mediator: ferrocene, o-phenylenediamine, hydroquinone
                The mediators

                                         OH

                          NH2
            R
   +
  Fe
                          NH2
                                         OH

Ferrocene       o-Phenylenediamine   Hydroquinone
Detailed look at a practical example ...
 Copper amine oxidase-based electrodes for the assay
                of biogenic amines
Monitoring    the biomarkers of food freshness: histamine,
putrescine, cadaverine
Currently used methods: chromatographic techniques - they often
require sample pre-treatment steps and skilled operators; the
relatively long analysis time and high costs make these methods not
suitable for routine use
Aim of the work: design and construction of the amperometric
biosensors for monitoring of biomarkers
Two biosensor designs: monoenzymatic and bienzymatic, using
both the direct and mediated electron transfer pathways
Biological recognition element: copper amine oxidase (EC 1.4.3.6)
Mediator: poly(1-vinylimidazole) complexed with [Os(4,4'-
dimethylbipyridine)2Cl]+/2+ (PVI13-dmeOs)
Assay system: The biosensors were used in a flow-injection
analysis (FIA) line
The biogenic amines: histamine, putrescine and cadaverine
                  NH2
       N                                         H2N-(CH2)n-NH2

           N                            n=4: Putrescine; n=5: Cadaverine
           H
              Histamine
     Formed by the decarboxylation        Formed by the biodegradation
      of histidine biocatalysed by          of the aminoacids ornithine and
      various microorganisms                lysine by the action of
                                            putrefactive bacteria
     Stimulates smooth muscle
      contraction and relaxation,          Oversaturate the histamine-
      including heart motions               detoxifying enzymes, enhancing
                                            the toxicity of histamine
     Stimulates sensory and motory
      neurons
     Controls acid gastric secretion
     Copper amine oxidase (AO)                  Redox active polymer
                                                     (PVI13-dmeOs)
   Biological sources: bacteria, fungi,
    plants, animals                                         a            b

   Biological functions: involved in cell
                                                    N               N
    growth, proliferation and differentiation
   Cofactors:                                          N               N
           O
                      H
                      N
                                                                N            N
                                                                        Os2+/ 3+
                            &    Cu(II)
    HO                O                                         N       Cl N
           O
    Topa quinone (TPQ)          Copper

Catalyzed reaction: R-CH2-NH2 + H2O + O2  R-CHO + NH3 + H2O2
Flow-injection system used
Working mechanism for monoenzymatic electrodes

        H
        N
                           AOox                Electrode
    N          H2
            C C
            H2   NH2               NH3   2e-   Eappl.= +200 mV
        Histamine                              vs. Ag/AgCl

                                   H2O
            H
            N
                           AOred
        N
                C CHO
                H2
   Imidazoleacetaldehyde
    Working mechanism for bienzymatic electrodes


            H                                                                                                      Electrode
            N
                            AOox                  H2O2                  HRPred                     2 Os2+
                                                                                          2e-                2e-   Eappl.= -50 mV
    H2       N          (TOPA -native)                            (Fe3+- native)
                                                                                                                   vs. Ag/AgCl
    C C
       H2        H2O                                                                                2 Os3+
H2N                                               H2O                    HRPox
       Histamine                                              (Fe4+ =   O, P+ inactive)
                 NH3
            H
            N
                            AOred                 O2
                N       (TOPA -inactive)
  OHC C
      H2
Imidazoleacetaldehyde

                                           Electrode type C                                     No Os2+/3+
                                           Electrode type D
                  Biosensors characteristics


ELECTRODE    ANALYTE     Kmapp      Imax           S          DL     DR
  TYPE                   (µM)       (µA)       (mA/Mcm2)     (µM)   (µM)

 TYPE A     HISTAMINE    375±34   0.164±0.06    5.99±0.09    2.7    10-100

 TYPE B     HISTAMINE    730±33   0.360±0.08    6.76±0.05    2.2    10-200

 TYPE C     HISTAMINE    332±17   1.34±0.02    55.29±0.73    0.16   1-100
            PUTRESCINE   227±16   3.01±0.07    181.64±1.01   0.06   1-100
               H2O2       112±8   2.70±0.06    330.14±1.02          1-100
 TYPE D     HISTAMINE    901±85   4.85±0.41    73.74±1.73    0.33   1-150
            PUTRESCINE   512±40   7.26±0.53    194.11±1.37   0.17   1-400
               H2O2      977±92   22.8±1.68    319.59±1.63          1-250



   Native enzyme Km - putrescine 0.2 mM, histamine 0.35 mM
                               Monitoring real samples - turbot fish
                      30
                                   fish kept at -20°C
                                   fish kept at 25°C
                      25
g histamine/kg fish




                      20


                      15


                      10


                       5


                       0
                           0       2          4         6          8     10          12
                                                            Days
          •Amine content from fish kept in different conditions was extracted with 0.1M potassium
          phosphate buffer, pH 7.2, and analyzed by direct injection in the FIA system using the
          bienzymatic mediated electrode
Comparison of selectivity of the developed systems
                             350
                                                                        AO biosensor
                             300                                        AO-HRP biosensor
     Relative response (%)


                             250

                             200

                             150

                             100

                              50

                               0
                                   Hsm




                                                                                              TDAB
                                                                                       CDAB
                                         Csm

                                               Trm



                                                            EDA

                                                                  Agm
                                                      Spd




                                                                           Put

                                                                                 Cad
                                                     Amine substrate
  Further oxidation of the histamine reaction product
                                        H
                                        N
H2O   +   O2      AOred
                                    N        H2
                                          C C
                                          H2   NH2
                                     Histamine

                                        H
                                        N
NH3   + H2O2       AOox                                     Electrode
                                    N
                                            C CHO
                                            H2        2e-   Eappl.= +200 mV
                                                            vs. Ag/AgCl
                              Imidazoleacetaldehyde
                                        H
                                        N

                                    N
                                            C COOH
                                            H2

                               Imidazoleacetic acid
                     +
                  NH3                  AO           O
                          + O2 + H2O                       + H2O2 + NH4+
                  NH2                               NH2

            Putrescine
                                                    - H2O

Putrescine and cadaverine form
cyclic products - cannot be                        N
further oxidized !!!
                                            1-Pyrroline

                    +
                 NH3                   AO           O
                         + O2 + H2O                   + H2O2 + NH4+
                  NH2                               NH2

           Cadaverine
                                                    - H2O



                                                       N

                                            1-Piperideine
                       Conclusions
1. Combination of the monoenyzmatic (AO) and bienzymatic (AO-
HRP) electrode can be used for selective detection of histamine and
diamines (putrescine and cadaverine).

2. The biosensors were tested for detection of fish product poisoning
by putrefactive amines.

3. The monoenzymatic electrode (AO) is the first example of DET
with copper amine oxidase, which can proceed anaerobically.

4. With histamine as an analyte, both DET and further oxidation of
the product aldehyde contribute to the biosensor response.

								
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