Biosensors for environmental applications

Human health...

Is tightly linked to the Health of the environment

Pollution
Pollution is the introduction into the environment of substances or energy liable to cause hazards to human health, harm to living resources and ecological damage, or interference with legitimate uses of the environment. The changes that are occurring in the environment caused by either natural processes or human activities, involve many large parameters and range from a slow increase in global temperature over years to the rapid accumulation of heavy metals and xenobiotics. Some of the changes may require careful monitoring over long time periods. Some of the elements and compounds that accumulate in the environment are present in concentrations so low that they are close to the limits of detection.

Causes?
The first major human influence on the environment was perhaps agriculture, and later the industrial revolution Many countries have a legacy of pollution and polluted sites.

Sources
The main groups of environmental pollutants which can contaminate land, water, and air are: Inorganic compounds such as metals Organic compounds such as sewage, petrochemicals Synthetic xenobiotic compounds such as phenol Micro-organisms such as pathogens and genetically engineered organisms (GMOs). The environmental pollutants can originate from contaminated sites of defunct industries such as gasworks, from existing domestic and industrial sources such as sewage and metals from industries like electroplating, from accidents, and illegal dumping.

What are dangerous levels?

The permissible levels and type of environmental contaminant are regulated by environmental legislation. UE legislation National laws are normally associated with a number of acts covering many types of pollutants, industries, and conditions (McEldowney and McEldowney, 1996).

The fate of pollutants

in the environment depends on the properties of the compound and the environmental conditions.

Monitoring
Monitoring must take into consideration the type or types of contaminants present (biological and chemical), their availability and the possibility of biomagnification and bioaccumulation. The environmental monitoring must be able to detect with accuracy and consistency pollutants present at very low levels.
A range of chemical analyses is used to determine the concentrations and type of pollutants- HPLC, GC, NMR, MS, IR etc.. This does not determine the real effect on the environment as the effects can be modified by availability, degradation, and transport of the pollutants. An alternative is to use a biological system to measure the pollutant..

Bioindicators, Biomarkers, Test organisms

The effects of pollutants on whole organisms representative of the environment, known as bioindicators. Bioindicator organisms are those that can be used to identify and quantify the effects of pollutants on the environment. Eg.: Mediterranean mussel Myttlus galloprovincialis ; frogs, fish eggs as a measure of water quality; Lichens; honey bees have been used as a bioindicator for the contamination of the atmosphere by heavy metals (cadmium, chromium, and lead). The effects of pollutants on physiological, biochemical, and molecular characteristics of organisms in the environment, known as biomarkers. Biomarkers can be defined as quantitative measures of changes in the biological system in response to pollutant exposure. Biomarkers can be placed into three groups: biochemical, immunochemical, and genetic. The effect of the pollutant on test organisms in the laboratory.

Bioindicators and biomarkers have the advantage that they measure the action of the pollutants in the real and complex environment where there may be many and complex interactions at sublethal levels.

Electrode types
Ion-selective electrodes

Enzymatic biosensors Whole cells based biosensors for heavy metals detection

Ion-selective electrodes. Metal speciation

Ion-selective electrodes
Notwithstanding, the ISE electroanalysis of complex environmental samples like lakes, rivers, seawater, soils, etc. is a challenging task due to a wide range of potential problems, as discussed elsewhere by De Marco et al. [11]: i) electrode fouling by the sample matrix components (e.g., chloride, hydroxide, organic ligands, other cation or anion interferences, etc.) causing erroneous response characteristics; ii) electrode drift leading to poor reproducibility as the sensor surface is altered on continuous exposure to real samples; iii) electrode dissolution causing a high surface excess of analyte that degrades the detection limit of the ISE beyond the realms of trace analyses in the environment; iv) electrode carry-over of the analyte which can cause cross-contamination of samples with the trace analyte; v) electrode instability as passivation of the ISE in real samples causes the response characteristics to become erratic and non-representative of the sample
assayed.

Ion-selective electrodes

Ion-selective electrodes

Hg2+ - detection
Hg,Hg Cl ,KCl satd //sample solution/PVCmembrane -ionophore/Pt electrode

Cu2+ -detection

Zn2+-detection
Ag | AgCl (saturated), KCl (3 M) | internal solution, Zn(NO3)2 (1.0 102 M) | PVC membrane | test solution| Hg2Cl2 (saturated), KCl (saturated) | Hg.

tetra(2-aminophenyl) porphyrin (TAPP)

Biosensors for environmental monitoring

The oxygen demand of wastewaters and effluents has been determined traditionally by measuring the BOD5 value -5 days are required to obtain a result. With a BOD biosensor a fast, online determination is possible. BOD biosensors can be either of the biofilm or respirometer type. In a biofilm BOD biosensor a microbial film is sandwiched between a porous membrane and the oxygen permeable membrane of a Clark oxygen electrode- A change in oxygen levels in the microbial culture will be proportional to the metabolizable content of the material to be measured. In some cases the electrical signal from the oxygen electrode has been replaced by optical signals using the luminous bacterium Vibrio phosphoreum.

Enzymatic biosensors

Enzymatic biosensors

Enzymatic biosensors

Enzymatic biosensors

Enzymatic biosensors

Biosensors for pesticide detection

Biosensors that can measure pesticide concentrations have been developed using the enzyme acetylcholinesterase combined with choline oxidase. Acetylcholinesterase converts acetylcholine to choline and choline oxidase converts the choline to betain and hydrogen peroxide. The H2O2 can be measured with an oxygen electrode. Pesticides inhibit the action of acetylcholinesterase and therefore produce a reduction in peroxide formation.

Biosensors for pesticide detection

In funcie de comportamentul lor fa de plante, pesticidele se clasific n: insecticide de contact - care rmn la suprafaa plantei, fr a intra n sistemul vascular (de ex.: parathion, fonofos dintre organofosforice; carbaryl dintre carbamai); insecticide sistemice - care ptrund n esutul vegetal i sunt transportate de sistemul vascular (de ex.: monocrotofos, fosfamidon dintre organofosforice; aldicarb, carbofuran dintre carbamai). Un alt criteriu de clasificare mparte pesticidele n funcie de utilizare: insecticide - pentru distrugerea insectelor. Ele pot fi subdivizate n insecticide pentru protejarea recoltelor, pentru domeniul veterinar, sntate public i conservare casnic; ierbicide - pentru distrugerea plantelor nedorite. Ele pot fi mprtiate direct la sol (adsorbite de rdcini) sau n aer (adsorbite de partea aerian a plantelor); fungicide - pentru distrugerea ciupercilor din culturi sau produse conservate; otrvuri - mpotriva roztoarelor, n special a obolanilor.

O(S) R1 R2 P X R1

O R2 C N R3

Biosensors for pesticide detection

P = O, familia fosfati P = S, familia tiofosfati (A)

(B)

Formula general a pesticidelor organofosforice (A) i carbamice (B).

Biosensors for pesticide detection

Biosensors for pesticide detection

Structura comparativ a AChE inhibate (A) i mbtrnite (B).

Structura enzimei inhibate (A) i a enzimei neinhibate (B)

Biosensors for pesticide detection

Biosensors for pesticide detection. AChE biosensor

Procentul de inhibare

unde: Io = curentul stationar nainte de contactul cu inhibitorul; I1 = curentul stationar dupa contactul cu inhibitorul.

Inhibitie relativa: RI = (dI/dt) / Io

unde: RI = inhibitia relativa, independenta de sensibilitatea biosenzorului pentru substrat; dI/dt = panta diminuarii raspunsului I n prezenta pesticidului, pentru un interval de timp foarte redus (dt) si constant pentru diferitele concentratii de pesticid; Io = raspunsul stationar al senzorului pentru o concentratie fixa de substrat.

Biosensors for pesticide detection. AChE

biosensor

Biosensors for pesticide detection. AChE biosensor

0.08

0.10

0.06

I / mA

0.00

I / mA

0.05

A1

A1
0.04

0.02

-0.05 -0.2 0.0

C1
0.2 0.4 0.6 0.8 1.0

0.00 -0.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4

/ V vs. Ag/AgCl,KClsat
0.10

/ V vs. Ag/AgCl, KCls a t

Rspunsul voltametric al tiocolinei pe electrod de platin (A) sau crbune past (B), n comparaie cu cel al soluiei tampon .

Ipa / mA

0.05

Randles-Sevcik equation: Ip,a = 2.99*105n(na)1/2ADSCh1/2[SCh]v1/2

0.00 0.0 0.1 v
1 /2

[A]

0.2 / (V/s)
1 /2

0.3

Biosensors for pesticide detection. AChE biosensor

150

400

r = 0.9958, n = 8 r = 0.9855, n = 8
100

50

300
0 0,00 0,35 0,70

200

100

Curba de calibrare a bioelectrodului Pt/AChE () i Pt/CoPC-AChE (). In detaliu este prezentat domeniul liniar al rspunsului bioelectrodului. Condiii experimentale: potenial aplicat, + 0.410 mV vs. Ag/AgCl, KClsat; [AChE]immobilizat = 0.48 U/mm2; pH 8; temperatura, 30 C
3 4

I (nA)

[ASChCl] (mM)

Biosensors for pesticide detection. AChE biosensor.

Calibration curves

Biosensors for pesticide detection. AChE biosensor

KM Imax

Biosensors for pesticide detection. AChE biosensor

Reprezentari secundare pentru calculul KI (A) si Ki (B) pentru paraoxon.

Biosensors for pesticide detection. AChE biosensor

Biosensors for pesticide detection. AChE biosensor

-0,3

-0,4

log(I/Io)

-0,5

Constanta aparenta de viteza ka = 2.6*103 M-1min-1

(r = 0.98 pentru electrodul Pt-AChE)

-0,6 0 2 4 6 8 10

[paraoxon] (M)

Biosensors for pesticide detection. AChE biosensor

Ec. Hill care descrie satisfacator legarea substratului la proteina alosterica.

Coeficientul x, denumit coeficient Hill, este o masura a cooperativitatii: daca x = 1, nu exista cooperativitate si cinetica este ideal hiperbolica, de tip Michaelis - Menten; daca x >1, cooperativitatea este pozitiva; daca x < 1, cooperativitatea este negativa. Pentru cazul concret al biosenzorilor pentru calculul coeficientul Hill, se poate extrapola tratamentul matematic elaborat pentru enzima n soluaie, trasnd urmatoarele reprezentari Hill: lg Y/(1 - Y) = f (lg I) unde: Y = I/Io; Io este semnalul obainut n absenaa inhibitorului ai I este variatia de curent datorata pesticidului ; I este proportional cu [S] conform ecuatiei 54. Hughes-Klotz: 1/Y = 1/x + K/(x [Inh]) Scatchard: Y / [Inh] = x / K - Y/K

0,0

Biosensors for pesticide detection. AChE biosensor

8
0,15

6
-0,5

lg [ Y / (1 - Y) ]

Y / [ paraoxon ] ( M

-1 0,10

1/Y

-1,0

)
0,05

-1,5 -0,4

-0,2

0,0

0,2

0,4

0,6

0,8

lg ( I)

0 0,0

0,5

1 / [ paraoxon ] ( M

1,0 -1

0,2

0,4

0,6

0,8

Hill

Hughes-Klotz

Scatchard

Biosensors for pesticide detection. Enzyme regeneration

Fosforilarea oximei permite restabilirea activitii enzimatice conform reaciei 7.14: (7.14) Procentul de reactivare se poate calcula utiliznd expresia:

% Reactivare
unde: aE, activitatea enzimei native; aEI, activitatea enzimei inhibate; aEIA, activitatea enzimei reactivate cu oxim. Gradul de reactivare spontan a AChE depinde de: pH, temperatur, structura chimic a radicalului fixat de atomul de fosfor.

Biosensor for phenol detection

Polifenol-oxidaza (EC. 1.14.18.1; PPO sau Ty), Lacaza (oxigen oxido-reductaza, EC. 1.10.3.2.),
2Cu 2 hidrochino na 2Cu p chinona 2H 1 2Cu O 2 2H 2Cu 2 H 2 O 2

Peroxidazele (H2O2 oxidoreductaz; EC. 1.11.1.7; HRP)

Sisteme bienzimatice PPO-lacaz PPO-HRP PPO-GDH

Biosensor for phenol detection

The oxidation of phenols to catechols and quinones by the enzyme tyrosinase requires oxygen. If the enzyme tryosinase is linked to an oxygen electrode levels down to about 50 parts per billion (ppb) could be detected.

Biosensor for phenol detection

Biosensors for heavy metals detection

Metals such as lead and cadmium can be detected by their inactivation of oxidases and dehydrogenases linked again to an oxygen electrode.

Biosensors for heavy metals detection

Biosensors for heavy metals detection

Biosensors for heavy metals detection

Amperometric recording obtained at a tyrosinase-based electrode under the optimum conditions, Eap = 0.4V and pH = 5. (1) Addition of catechol ([catechol] = 103 mol dm3), (2) addition of chromium(VI) giving an overall concentration of 105 mol dm3 and a consecutive injecting of aliquots of the chromium(III) solution into the cell to give an overall concentration of (3) 105 mol dm3, (4) 2 105 mol dm3.

DNA-based biosensor for heavy metals detection

Immunosensor

Principle steps of KinExA-based assay of metal ions. (D) Raw data curves from the KinExA instrument. Curve 1 corresponds to zero ligand concentration; curve 6 corresponds to a saturating ligand concentration. Curves 2 5 are concentrations of ligand between zero and saturation.

Whole cells based biosensors

Anatomy of a bioreporter organism

Whole cells based biosensors. Classification of light-emitting bacterial biosensors

Whole cells based biosensors

Whole cells based biosensors

Whole cells based biosensors

Conclusions
Thus biosensors can provide cheap, reliable, and accurate monitoring of the environment which will also give real-time analysis. Biotechnology will influence the monitoring of the environment in the following areas. Microbial diversity. The culture-independent study of microbial populations in situ. Biomarkers using microorganisms with the fluorescent protein (gfp) gene inserted will be used to monitor pollution in situ. Molecular biology techniques to follow the introduction of genetically modified micro-organisms released into the environment. Biosensors will be able to determine the levels of contaminants in the environment and perhaps provide real-time online monitoring.