Research Article Toxicity Biosensor for Sodium Dodecyl...

Research ArticleToxicity Biosensor for Sodium DodecylSulfate Using Immobilized Green FluorescentProtein Expressing Escherichia coli

Lia Ooi1 Lee Yook Heng12 and Asmat Ahmad2

1Southeast Asia Disaster Prevention Research Initiative (SEADPRI-UKM) LESTARI National University of Malaysia 43600 BangiSelangor Malaysia2Faculty of Science and Technology National University of Malaysia 43600 Bangi Selangor Malaysia

Correspondence should be addressed to Lee Yook Heng leeyookhengyahoocouk

Received 17 September 2014 Revised 17 December 2014 Accepted 18 December 2014

Academic Editor Qingjun Liu

Copyright copy 2015 Lia Ooi et al This is an open access article distributed under the Creative Commons Attribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Green fluorescent protein (GFP) is suitable as a toxicity sensor due to its ability to work alone without cofactors or substrates Itsreaction with toxicants can be determined with fluorometric approaches GFP mutant gene (C48SS147CQ204CS65TQ80R) isused because it has higher sensitivity compared to others GFP variants A novel sodium dodecyl sulfate (SDS) toxicity detectionbiosensor was built by immobilizing GFP expressing Escherichia coli in k-Carrageenan matrix Cytotoxicity effect took place in thetoxicity biosensor which leads to the decrease in the fluorescence intensity The fabricated E coliGFP toxicity biosensor has a widedynamic range of 4ndash100 ppm with LOD of 17 ppm Besides it possesses short response time (lt1min) high reproducibility (076RSD) and repeatability (072 RSD 1198772 gt 098) and long-term stability (46 days) E coliGFP toxicity biosensor has been applied todetect toxicity induced by SDS in tap water river water and drinking water High recovery levels of SDS indicated the applicabilityof E coli GFP toxicity biosensor in real water samples toxicity evaluation

1 Introduction

Green fluorescent protein (GFP) was first detected inAequorea victoria a type of jellyfish in 1961 [1] but thecloning of the GFP gene only took place 29 years later[2] GFP possesses several characteristics which make itexceptional among the characteristics it does not requirecofactors or substrates is stably expressed as a fusion pro-tein is relatively nontoxic and can be readily detected byfluorescence microscopy and other fluorometric techniques[3] The wild type chromophore is excited with blue lightor UV at 396 nm or 475 nm and emits green fluorescenceat 508 nm [4] There are many GFP variants that havebeen created with shifted absorbance and emission spectraimproved folding and expression properties The creation ofblue cyan yellow and red GFP variants coupled with newfluorescence imaging approaches has created more potentialof GFP in proteins and biosensor studies [3] Escherichia coli

GFP is one of the examples where GFP mutant gene (C48SS147CQ204CS65TQ80R) was introduced and expressed inE coli bacteria

Water resources are reported to be polluted by pollu-tants (nanoparticles pesticides pharmaceutical industrialwaste by-products from water treatment plants etc) veryfrequently in these few decades Surfactants are one ofthe pollution agents [5] and the presence of surfactants aspollutants is also reported in filtered and treated tap waterand drinking water Surfactants play an important role indecreasing the surface tension of water and allow removaland breakdown of stain or grease particles to take placeSodium dodecyl sulfate (SDS) also known as sodium laurylsulfate (SLS) a primary alkyl sulphate with chemical formulaCH3(CH2)11OSO3Na is a member of alcohol sulfate family

SDS is one of the anionic surfactants and it takes up a largepart in human life from appearing in the household [6] SDSwill get into the water resources through outfalls of waste

Hindawi Publishing CorporationJournal of SensorsVolume 2015 Article ID 809065 9 pageshttpdxdoiorg1011552015809065

2 Journal of Sensors

water or through direct application such as agrochemicalsprays [7 8] dispersants and pesticides [6] SDS is widelyused in biochemical research in cell lysis in DNA extractionvia SDS-PAGE and as viral biocide [9]

SDS has been reported toxic to aquatic organisms Infish species it enhances organ morphologies such as kidneyand spleen alteration of metabolism rate and swimmingability and changes in growth and death rates [10ndash12]SDS shows acute effect to fertilization of ova and spermdecreasing probability of fertilization in fish [12] SDS is alsoreported to be toxic to mammals such as rodents and human[13] Exposure of SDS to mammals results in physical andbiochemical effects skin irritation hyperplasia alterationof serum lipid composition damage of cells and decreasein cell proliferation [14ndash16] Detection of SDS via chemicalanalytical methods through capillary electrophoresis gaschromatography HPLC and UV-V has drawbacks such asdifficulty to determine LOD with large sample volume lowdetection and volatilization rates production of toxic wasteduring operational analysis high cost and causing damageto sample during detection process [17ndash19] The use of dyeto bind with SDS in water has been suggested for thequantification of SDS concentration but there is challenge toproduce specific binding of the dye with SDS [20 21] Notmany SDS-detection biosensors have been reported Whileseveral bacterial biosensors were produced to assist SDSdetection most of them were reported to have long responsetime (minutes to hours) short dynamic linear range lowreproducibility and short term of stability [22ndash24] In thisstudy we report a sensitive SDS-detection toxicity biosensorfabricated with immobilization of E coli GFP which wasfound to have improved properties

2 Materials and Methods

21 Materials Ampicillin 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES) sodium dodecyl sulfate(SDS) and sodium chloride (NaCl) were purchased fromSigma-Aldrich Nutrient agar and glycerol were purchasedfrom MERCK and HMBC Chemicals respectively Yeastextract and tryptone were purchased from Becton Dickinsonand Company All materials used in this study are of thehighest purity available Solutionswere prepared in deionizedwater (Barnstead ROThermo Scientific)

22 Cultivation of Recombinant Bacteria E coli GFP E coliGFP colonies were produced by spreading the bacterial stockon nutrient agar and incubated under 37∘C for 18 hoursSingle colony of bacteria has been inoculated in 4mL Luria-Bertani medium (LB 10 gL tryptone 5 gL yeast extract10 gL of NaCl pH 70) with 100 120583gmL ampicillin addedPreculturing of E coli GFP bacteria has been carried outin a rotary thermoshaker and has been set at 250 rpm37∘C for 18 hours (until OD

600reached 13 ABS) 500 120583L of

yielded recombinant bacteria from the preculture was furthercultivated in 50mL LB medium for 4 hours (optical density600 nm reached 08ndash084 ABS) [25] Bacteria E coliGFP thathas been cultured was centrifuged under 3000 rpm at 25∘Cfor 10min the supernatant was discharged It was then

Container

E coli GFP bacterial cell

Matrix of k-Carrageenan

Figure 1 The design of E coli GFP toxicity biosensor

washed twice with 5mM 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES) bufferwith 171mMNaCl pH70 under the same condition [26] Bacterial cells in pelletformwere suspended in 10mLHEPES buffer and kept in 4∘CThe bacteria culture is ready to be used in the following steps

E coliGFP bacteria strain was selected because it is moresensitive and fast in giving a response compared with therest of the GFP mutants Wild type GFP was not consideredbecause of its weak fluorescence signal Single colonies wereobtained from the nutrient agar after 20 120583L of E coli GFPfrom glycerol stock was spread equally and allowed to growon the agar Ampicillin was added into the nutrient agar aswell as the LBmedium to ensure plasmidmaintenance of thebacteria E coli GFP The bacteria cultures were washed twicewith HEPES buffer to eliminate excessive LB medium andalso organic wastes that were produced during the growingand duplication process of the bacteria

23 Fabrication of E coli GFP Biosensor Toxicity biosensoris fabricated by immobilizing E coli GFP bacterial cells ink-Carrageenan matrix Immobilization process was carriedout on a hot plate with temperature set to 40ndash45∘C Around container (119889 = 8mm) was used as a mould for theimmobilization to take place Figure 1 illustrates the design ofE coli GFP toxicity biosensor Optimized amount of E coliGFP cells (warmed in 35ndash40∘C water bath) was mixed withoptimized concentration of k-Carrageenan agar (kept in liq-uid form under 40ndash45∘C heating) Temperature for biosensorpreparation was set in between 35 and 45∘C which is asuitable temperature range for active survival of bacteriaMixture was stirred well to make sure there was no formationof gas bubbles in thematrix and to produce a smooth surfaceHomogenous mixture was kept in 4∘C overnight to allowthorough solidification to take place

231 Immobilization Matrix Optimization Six concentra-tions of k-Carrageenan matrix were prepared 10 1214 16 18 and 20 All of themwere prepared bymix-ing the specific amount of k-Carrageenan powderwith deion-ized water in which the temperature was fixed at 75∘C whichis the temperature needed for k-Carrageenan powder to dis-solve homogeneously in the solvent higher temperature willdenature the k-Carrageenan structure Continuous stirringwas set to allow homogenous mixture to form A constantamount of E coli GFP was then immobilized in 100 120583L of k-Carrageenanmatrix for all six concentrationmeasures Eachconcentration was prepared in 3 repetition setsThemixtureswere kept in 4∘C overnight Toxicity biosensors with different

Journal of Sensors 3

concentrations of k-Carrageenan matrix were then inter-faced with fluorescence spectrometer (discussed in detail inSection 24) The parameter of biosensor which gives thehighest fluorescence reading will be chosen as the optimumone and that parameterwill be set as constant in the followingoptimization steps

232 E coli GFP Cell Density Optimization Five differentdensities of E coli GFP bacterial cells were prepared 5841168 146 1752 2336 and 292120583gmL All of them wereobtained by dilution and concentration of bacterial culture inLB medium (obtained via process mentioned in Section 21)The specific amount of E coli GFP was then immobilizedin 100 120583L of k-Carrageenan matrix with the optimizedconcentration measures in Section 231 Each amount of Ecoli GFP was immobilized in four similar repetition sets Themixtures were kept in 4∘C freezer overnightThe as-preparedbiosensors were then interfaced with fluorescence spectrom-eter (details discussed in Section 23) the optimized celldensity was determined from set of biosensors which gave thehighest fluorescence reading

24 Fluorescence Response Measurement Toxicity biosensorswhich consist of E coli GFP cells immobilized in the k-Carrageenan matrix were tested with fluorescence opticalfibre probe which acted as a transducer Fluorescence signalsof each toxicity biosensor have beenmeasuredwith an opticalfibre probe which was placed 10mm above the biosensorwhere the probe was connected to a Perkin Elmer fluores-cence spectrometer The excitation wavelength was fixed at395 nm and emission wavelength was scanned from 400 to500 nm Bandwidths of excitation and emission were bothset at 5 nm The reading of fluorescence signals has beenmeasured and read at 436 plusmn 2 nm Optical fibre probe was set10mm above the biosensor samples each time to ensure thatno additionalmanipulated variable was added into the exper-iment Excitation and emission bandwidths were fixed dueto the same reason 5 nm wide was the best gap to keep awaythe noises created during the experiment and at the sametime was sufficient enough to capture returning fluorescencesignals

25 Performance Evaluation of Toxicity Biosensor E coli GFP

251 Stability of E coli GFP Toxicity Biosensor The opera-tional definition of stability of E coli GFP toxicity biosensoris the time frame when the biosensor gives out persistentfluorescence signal not less than 75 as compared to ini-tial measurement Fluorescence signal is the referral unitfor biosensor stability 10 sets of biosensor with optimizedimmobilization parameters have been prepared sealed withparafilm and stored in 4∘C A drop of 10 120583L of pH 70HEPES buffer was dropped onto the biosensor each timeafter measurement to maintain the moisture of the matrixbefore it was resealed with parafilm and returned into 4∘Cfreezer Fluorescence emissions of the toxicity biosensor weremeasured daily or within interval until a sharp drop in thefluorescence signal of toxicity biosensor observed

252 Repeatability and Reproducibility Studies The repeata-bility value of the E coli GFP toxicity biosensor was deter-mined by calculating the percentage of relative standard devi-ation (RSD) of the fluorescence intensity of ten sets of toxic-ity biosensor which were all prepared with optimized param-eters and exactly the same technique while the reproducibil-ity value of the E coliGFP toxicity biosensor was determinedby calculating the RSD of the response of the toxicitybiosensors whichwere prepared from three different batcheswith five replicates each towards a series of SDS concentra-tions (20 40 60 and 100 ppm)The exposure time was 5min

253 Determination of IC50 of SDS towards E coli GFPToxicity Biosensor Half maximal inhibitory concentration(IC50) is a measure to determine the toxicity level of achemical towards living organisms The IC50 value of SDStowards the E coli GFP cells immobilized onto the toxicitybiosensor is determined by exposing the biosensor to 12concentration parameters of SDS in the range of 1ndash100 ppm50 120583L of SDS from each concentration was exposed to thetoxicity biosensor Approximately three replicates were pre-pared for each parameter Toxicity biosensors were interfacedwith fluorescence optical fibre probe and readings wererecorded before the exposure to surfactant SDS (119865

119900) and

5min after exposure (119865119905) Percentage of relative fluorescence

unit (RFU) for each SDS concentration was determinedusing the formula as follows

RFU =119865119900minus 119865119905

119865119900

times 100 (1)

The value of RFU indicates the inhibitory level of SDStowards E coli GFP toxicity biosensor A graph of inhibitorypercentage versus SDS concentration is plotted The IC50value is determined from the graph

254 Evaluation of E coli GFP Toxicity Biosensor towardsSDS Detection The calibration curve of E coli GFP toxicitybiosensor was determined by exposing the biosensor towards50 120583L SDS with 29 concentrations ranging from 01 to1000 ppmThe changes in fluorescence before and 5min afterexposure of the toxicity biosensor were determined Fivereplicates were prepared for each concentration RFU foreach measure was calculatedThe linear range and the lowestdetection limit of the toxicity biosensorwere established fromthe calibration curve

255 Real Sample Exposure and Recovery Studies Watersamples were collected from three different sources fromareas around the National University of Malaysia (UKM) (a)tap water collected from the Chemical Sensor and BiosensorLab UKM (b) river water collected from Langat RiverCheras and (c) filtered drinking water collected from Kajangtown Water sample (b) was filtered with a Whatman quali-tative paper number 6 and number 2 to remove suspendedparticles that will distort the readings of fluorescence inten-sity SDS solutions with five concentration measures (10 2050 80 and 100 ppm) were prepared in the real water samplesE coliGFP toxicity biosensors were exposed to SDS in differ-ent water backgrounds Induction response with the addition


320

340

360

380

400

420

440

460

480

500

08 1 12 14 16 18 2 22

Fluo

resc

ence

inte

nsity

(au

)

Concentration of k-Carrageenan ()

Figure 2 The fluorescence emission spectra of the bacteria E coliGFP cells immobilized in k-Carrageenan matrix (119899 = 4)

of surfactant SDS has been carried out The experiment wasdone with five-sample repetition The ability of E coli GFPtoxicity biosensor to work under real water background hasbeen studied

3 Results and Discussion

31 The Optimization of E coli GFP Toxicity Biosensor Inthe fabrication of E coli GFP toxicity biosensor two majorparameters have been studied the concentration of theimmobilization matrix and the E coli GFP cell density to beimmobilizedMatrix k-Carrageenan has been selected for theease of its preparation procedure its gelling temperature fallsin the range that is tolerable by E coli GFP cells it is nottoxic to the bacterial cells and its clear nature allows opticalmeasurement to be done k-Carrageenan is hydrophilic andwill be surrounded by water molecules which brought to theprocess of gelation [28]The presence of Na+ cations from thecell suspension buffer enhances the formation of double helixdomain of k-Carrageenan as proposed by the DomainModelwhich gives a more rigid structure to the toxicity biosensor[29ndash31]

Figure 2 shows the effect of the concentration of immo-bilization matrix to the fluorescence intensity of the toxicitybiosensor It can be understood that the gel viscosity andrigidity increasewith the gel concentration but the optimizedgel concentration appeared to be 18 instead of 20 Con-tinuous increment in the fluorescence readings was observedfor E coli GFP immobilized in 10ndash18 k-Carrageenanmatrix 10 of k-Carrageenan appeared to be too watery andwas unable to fix the bacteria stationarily causing bacteriacells to sediment overnight due to gravitation force Thestacking of bacteria cells at the bottomof the biosensormouldblocked the emission of fluorescence from being detectedaccordingly by the fluorescence spectrometer giving out lowreadings Same explanations applied to biosensors fabricatedin 12ndash16 of k-Carrageenan which showed improvingimmobilization ability while for k-Carrageenan 20 it wastoo thick to be manipulated in the immobilization approachThe gelation took place within a few seconds when gel-cell

380

400

420

440

460

480

500

520

4 9 14 19 24 29

Fluo

resc

ence

inte

nsity

(au

)

E coli GFP density (120583gmL)

Figure 3 The fluorescence emission spectra of the bacteria E coliGFP cells with varying cell densities (584 1168 146 1752 2336and 292 120583gmL) immobilized in 18 k-Carrageenanmatrix (119899 = 4)

050

100150200250300350400450500

0 10 20 30 40 50

Fluo

resc

ence

inte

nsity

(au

)

Period (day)

Figure 4 Long-term stability of E coli GFP toxicity biosensor

mixture was prepared Even distribution of the bacterial cellscould not be achieved before the gel solidified leading tostacking of bacteria 18 is the optimum concentration ofk-Carrageenan which fixes the bacteria cells firmly whileallowing thorough stirring of mixture

Figure 3 shows the effects of immobilized E coli GFP celldensity on the fluorescence emission of the toxicity biosensorSimilar stacking theory applied in the optimization of celldensity of E coli GFP graph where the decrease in the fluo-rescence readings after 1168 120583gmL was due to overloadingof bacteria Increasing bacteria quantity beyond the immobi-lization limit of the k-Carrageenan matrix will lead to over-crowded bacteria cells in a constant amount of matrix Thissituation ends with stacking of bacteria cells Fluorescencelight emitted by the bacterial cells at the bottom of the con-tainer will not be able to be detected by the fluorescence spec-trometerThis situation brought the reverse results where themore bacteria were being immobilized the less fluorescencereadings were being recorded

32 E coli GFP Toxicity Biosensor Performance The E coliGFP toxicity biosensor is stable for a period of 46 days(Figure 4) The biosensor signal remained in the range of


Table 1 Response of E coli GFP toxicity biosensors prepared fromthree different culture batches towards SDS (20ndash100 ppm) exposure119899 = 5

Set biosensor Sensitivity (ppm) 1198772 value

1 6139 plusmn 032 098772 6219 plusmn 076 098753 6212 plusmn 062 09833

0

100

200

300

400

500

600

700

1 2 3 4 5 6 7 8 9 10

Fluo

resc

ence

inte

nsity

(au

)

E coli GFP toxicity biosensor ( )

Figure 5 Stability of fluorescence signal of E coli GFP toxicitybiosensors which were prepared under constant conditions

44204 plusmn 1394 for the first 43 days The fluorescence signaldropped 7936 from Day 1 and decreased to 280 startingDay 47 The decrement of fluorescence signal is caused byincreased cell death due to depletion of nutrient and toxicityof respiratory residue

A series of ten E coli GFP toxicity biosensors have beenfabricated and the fluorescence intensity of each biosensorwas detected and compared (Figure 5) Fluorescence signalsobtained fall in the range of 58250 plusmn 445 giving a value of076 for RSD

Repetition of SDS exposure (20ndash100 ppm 119899 = 5) forE coli GFP toxicity biosensor prepared from three differentbatches of bacteria culture (labeled sets 1ndash3) shows similarlevel of sensitivity (Table 1) All three sets of E coli GFPtoxicity biosensor gave an average sensitivity of 6190 plusmn044ppm with 1198772 value gt 098 The toxicity biosensorresponse towards SDS gave RSD of 072

33 Response of E coli GFP Toxicity Biosensor towards SDSDetection The fluorescence intensity of the E coli GFPtoxicity biosensor decreased when it was exposed to SDSDifferent concentrations of SDS induced different levels ofdecrement in the biosensor response SDS causes cell toxicityto E coli GFP which led to decline of fluorescence signalFigure 6 shows the overall responses of theE coliGFP toxicitybiosensor to 10 ppm SDS exposure Before exposure to SDSsolution the biosensor gave out a total fluorescence intensityof 97656 We can understand that at this stage the ldquoalwaysonrdquo E coli GFP biosensor expressed the GFP proteins to itsfullest while after a few seconds when 10 ppm SDS solutionwas added the fluorescence intensity dropped to 88258 TheSDS toxicity started to take place and that gave effect on

0100200300400500600700800900

1000

425 450 475 500 525 550 575 600

Without SDS

Emission wavelength (nm)

0min after exposed to SDS5min after exposed to SDS

Fluo

resc

ence

inte

nsity

(au

)

Figure 6 Fluorescence emission spectra of the bacteria E coli GFPtoxicity biosensor before and after the exposure to 10 ppm SDS

the fluorescence signals Within 5min after the exposuremore inhibitory reactions between the surfactant and GFPhave taken place which results in further reduction in thefluorescence intensity to 83035

Many derivatives of GFPs have been used in the fabri-cation of whole-cell biosensors for environmental pollutionmonitoring purposes [32ndash36] In bacteria E coli GFP theactive site that was used in toxic detection and binding isthe cysteine groups that were introduced into the beta barrelstructure The redox state of the cysteine determines thefluorescent properties of E coliGFP toxicity biosensorThereare three modes of action that took place which contribute tothe observed response (i) redox reaction of thiol group of theGFP (ii) denaturation of GFP protein which leads to loss ofprotein function and (iii) disturbance of cell capsulersquos surfaceprotein which leads to cell death [26 37 38]

Binding of SDS to the thiol group of the GFP enhancesredox reaction that gave effect on the fluorescence signalsThe oxidation state of cysteine plays an important role inprotein structure and formation In its thiol form cysteineis the most reactive amino acid and is often used foradding fluorescent groups In oxidized forms cysteine formsdisulfide bonds which are the primary covalent cross-linksfound in proteins that stabilize the native conformation ofa protein Cysteine is uniquely suited to sensing a range ofredox signals as the thiol side-chain (ndashSH) can be oxidizedto several different reversible redox states such as disulphide(RndashSndashSndash1198771015840) sulphenic acid (RndashSOH) and S-nitrosothiol (RndashSNO) [39]

SDS is known to cause denaturation of protein by bind-ing to folded protein its charged counterion will disturbthe balance of the intrinsic charges of the protein andeventually unfold the protein with its negative charge [37]Dilution of protein takes place when GFP consisting of4-hydroxybenzylidene imidazolinone which is commonlyknown as Y66 chromophore is exposed to SDS Y66 chro-mophore is responsible for the fluorescence emission of the


0102030405060708090

100

0 20 40 60 80 100SDS concentration (ppm)

Inhi

bito

ry (

)

Figure 7 Dose-response curve of E coli GFP toxicity biosensortowards SDS exposure Red arrow with dotted line indicates theposition of IC50 value of SDS (119899 = 3)

10

30

50

70

90

110

0 1 2 3

020406080

100

0 05 1 15 2 25

y = 63145x minus 34277

R2 = 09925

minus1

Log[SDS] (ppm)

Inhi

bito

ry (

)

Figure 8 The calibration curve of E coli GFP toxicity biosensortowards SDS exposure Exposure time was set at 5min 119899 = 5 Insertshows the dynamic linear range

E coli GFP toxicity biosensor [40] Changing of proteinconfirmation inhibits the emission of the fluorescence

Besides the unfolding of GFP SDS is also believed to bindwith proteins embedded on the phospholipid bilayer of the Ecoli GFP cell capsule During the exposure of SDS to the Ecoli GFP toxicity biosensor the surfactant decreases surfacetension of the phospholipid bilayer Binding of SDS changesthe confirmation of embedded capsule proteins which leadsto the breakdown of phospholipid bilayer Dispersion ofphospholipid bilayers in cell membrane by SDS is also possi-ble Cellmembranes play role in holding the bacteria contents(nucleus proteins cytoplasm etc) together When the SDSworks in decreasing the surface tension of the membranethe cell membrane will deteriorate and dissemble [38] Ecoli GFP bacterial cells decompose when the cell membraneswere gone and hence no more fluorescence signal is beingproduced

Table 2 Recovery data of E coli GFP toxicity biosensor for thedetection of SDS in tap water 119899 = 5

Real SDSconcentration (ppm)

Detected SDSconcentration (ppm)

Percentage ofrecovery ()

10 958 plusmn 008 958320 2070 plusmn 028 1035250 5581 plusmn 090 1116380 8123 plusmn 101 10154100 9136 plusmn 124 9136

Table 3 Recovery data of E coli GFP toxicity biosensor for thedetection of SDS in Langat River water 119899 = 5





Table 4 Recovery data of E coli GFP toxicity biosensor for thedetection of SDS in filtered drinking water 119899 = 5





34 Median Inhibitory Concentration of SDS The responseof E coli GFP toxicity biosensor towards SDS exposureshows a sigmoid dose-response curve (Figure 7) Percentageof SDS toxicity inhibition increases when the concentrationof SDS increases The decrease in fluorescence signal of Ecoli GFP toxicity biosensor after SDS exposure is an effectof cytotoxicity IC50 of SDS towards E coli GFP toxicitybiosensor is determined as 2275 ppm This indicates thathalf of the total responsive activity of the toxicity biosensorwill be induced when it is exposed to that dosage of SDSSDS induces activity of E coli GFP toxicity biosensor at anexponential rate at lower concentrations while the responseis close to plateau at higher levels due to maximum toxicityeffect

35 E coli GFP Toxicity Biosensor towards SDS DetectionE coli GFP toxicity biosensor has been applied to detectSDS in a wide range of 01ndash1000 ppm The SDS inhibitionresponse has been studied (Figure 8) Inhibitory effect of SDStowards E coliGFP toxicity biosensor can be observed clearlyfrom concentration of 20 ppm and onwards The sigmoidalcalibration curve indicates that E coliGFP toxicity biosensorhas a wide detection range with dynamic linear range falling


Table 5 Comparison between E coli GFP toxicity biosensor and reported SDS immobilized whole-cell bacteria biosensors

Parameter This study [22] [23] [27]Bacteria E coli GFP Pseudomonas rathonis Pseudomonas sp Comamonas testosteroni TIImmobilization matrix k-Carrageenan Gel agar Gel agar Gel agarDynamic linear range (ppm) 40ndash100 10ndash200 004ndash090 mdashLowest detection limit (ppm) 17 sim025ndash075 248 times 10minus4 025ndash05IC50 (ppm) 2275 mdash 016 plusmn 002 mdashResponse time (min) lt10 17ndash25 17ndash25 12ndash15Reproducibility () 076 313 mdash 450Repeatability () 072 mdash mdash mdashBiosensor stability (days) 46 2-3 3ndash5 10

at 4ndash100 ppm SDS with concentrations of 300ndash1000 ppminduces high level of inhibition which leads to a plateau atsim100 SDS with concentration falling in that range giveshigh toxicity effect to E coli GFP cells resulting in massivecell death which gives rise to a quick drop in the fluorescencesignal LOD of E coli GFP toxicity biosensor for SDSdetection has been determined to be 17 ppm SDS at lowerconcentration might take longer (gt5min) to cause toxicityor the toxicity level is too low and the damage is resistible byE coli GFP cells

36 Recovery Performance of E coli GFP Toxicity BiosensorE coli GFP toxicity biosensor was exposed to a series of SDS(concentration 10ndash100 ppm) prepared in three different realwater sample backgrounds Recovery data for the biosensorperformance in each water background were shown inTables 2ndash4 E coli GFP toxicity biosensor showed a recoverypercentage of 9136ndash11163 when working under tap waterbackground (Table 2) For E coli GFP toxicity biosensorwhich worked under the real river water background gavea recovery level of 9142ndash10725 (Table 3) while for waterbackground of drinking water E coli GFP toxicity biosensoris able to detect SDS for a recovery percentage of 9125ndash11101 (Table 4) The biosensor response slightly variesfrom a controlled experiment conducted in deionized waterbackground (data not shown) due to interaction of SDS andEcoliGFP with free nontarget radicles present in the real watersamples Nevertheless high level of recovery performanceenables E coli GFP toxicity biosensor to be applied in realenvironment

Optimized E coli GFP toxicity biosensor performanceis compared with reported immobilized whole-cell bacteriabiosensors that were fabricated to apply in SDS detection(Table 5) In our knowledge there was no SDS-detectionbacterial biosensor fabricated using k-Carrageenan as amatrix for cell immobilization being reported Our toxicitybiosensor has a comparatively wide dynamic linear rangeAlthough the LOD of E coli GFP toxicity biosensor is notas low as others it has an IC50 value which is higher ascompared to the one reported by Taranova et al [23] E coliGFP toxicity biosensor is able to respond to SDS detection inseconds while the reported biosensors would need minutesThe fabricated E coli GFP toxicity biosensor possesses highreproducibility and repeatability as compared to the rest

Furthermore our biosensor is stable for a longer time amongcomparison

4 Conclusions

A sensitive and optimized E coli GFP toxicity biosensor hasbeen fabricated with minimum response time as short as5ndash10 s Sodium dodecyl sulfate shows an inhibition reactionon the fluorescent ability of the bacteria which was dueto alteration of GFP protein and capsule surface embeddedfunctional protein upon SDS binding and lipid dispersioncriteria of SDS surfactant towards the phospholipid bilayerbacterial cell membrane E coliGFP toxicity biosensor is ableto work in real water backgrounds to detect toxicity inducedby SDS in tap water river water and drinking water Thisstudy gives an alternative for SDS toxicity detection in waterresources which is comparatively economic portable andeasy to prepare

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

This work is funded by the National University of Malaysia(UKM) via Research Grants DPP-2014-060 and NNDND2TD11-009

References

[1] O Shimomura ldquoThe discovery of aequorin and green fluores-cent proteinrdquo Journal of Microscopy vol 217 no 1 pp 3ndash152005

[2] D C Prasher V K Eckenrode W W Ward F G Prendergastand M J Cormier ldquoPrimary structure of the Aequorea victoriagreen-fluorescent proteinrdquo Gene vol 111 no 2 pp 229ndash2331992

[3] J Lippincott-Schwartz and G H Patterson ldquoDevelopment anduse of fluorescent protein markers in living cellsrdquo Science vol300 no 5616 pp 87ndash91 2003

[4] R Heim D C Prasher and R Y Tsien ldquoWavelength mutationsand posttranslational autoxidation of green fluorescent proteinrdquo


Proceedings of the National Academy of Sciences of the UnitedStates of America vol 91 no 26 pp 12501ndash12504 1994

[5] M Stuart D Lapworth E Crane and A Hart ldquoReview of riskfrom potential emerging contaminants in UK groundwaterrdquoScience of the Total Environment vol 416 pp 1ndash21 2012

[6] V Chaturvedi and A Kumar ldquoToxicity of sodium dodecylsulfate in fishes and animals A reviewrdquo International Journal ofApplied Biology and Pharmaceutical Technology vol 1 no 2 pp630ndash633 2008

[7] J R Marchesi S A Owen G F White W A House and NJ Russell ldquoSDS-degrading bacteria attach to riverine sedimentin response to the surfactant or its primary biodegradationproduct dodecan-1-olrdquo Microbiology vol 140 no 11 part 1 pp2999ndash3006 1994

[8] C G van Ginkel ldquoComplete degradation of xenobiotic surfac-tants by consortia of aerobic microorganismsrdquo Biodegradationvol 7 no 2 pp 151ndash164 1996

[9] J Piret A Desormeaux and M G Bergeron ldquoSodium laurylsulfate a microbicide effective against enveloped and nonen-veloped virusesrdquo Current Drug Targets vol 3 no 1 pp 17ndash302002

[10] E Barbieri P V Ngan and V Gomes ldquoThe effect of SDSsodium dodecyl sulfate on the metabolism and swimmingcapacity of Cyprinus carpiordquo Revista Brasileira de Biologia vol58 no 2 pp 263ndash271 1998

[11] A J S Rocha V Gomes P V Ngan M J A C R Passos andR R Furia ldquoEffects of anionic surfactant and salinity on thebioenergetics of juveniles of Centropomus parallelus (Poey)rdquoEcotoxicology and Environmental Safety vol 68 no 3 pp 397ndash404 2007

[12] M Rosety F J Ordonez M Rosety-Rodrıguez et al ldquoCompar-ative study of the acute toxicity of anionic surfactans alkyl ben-zene sulphonate (ABS) and sodium dodecyl sulphate (SDS) ongilthead Sparus aurata L eggsrdquo Histology and Histopathologyvol 16 no 4 pp 1091ndash1095 2001

[13] N J Fendinger D J Versteeg E Weeg S Dyer and R AR Rapaport ldquoEnvironmental behavior and fate of anionicsurfactants partnersrdquo in Environmental Chemistry of Lakes andReservoirs pp 527ndash557 American Chemical Society Washing-ton DC USA 1994

[14] M Lindberg B Forslind S Sagstrom and G M RoomansldquoElemental changes in guinea pig epidermis at repeated expo-sure to sodium lauryl sulfaterdquo Acta Dermato-Venereologica vol72 no 6 pp 428ndash431 1992

[15] Y Miura H Hisaki B Fukushima T Nagai and T IkedaldquoDetergent induced changes in serum lipid composition in ratsrdquoLipids vol 24 no 11 pp 915ndash918 1989

[16] J J M van de Sandt T A Bos and A A J J L Rutten ldquoEpider-mal cell proliferation and terminal differentiation in skin organculture after topical exposure to sodium dodecyl sulphaterdquo InVitro Cellular and Developmental BiologymdashAnimal vol 31 no10 pp 761ndash766 1995

[17] E Olkowska Z Polkowska and J Namiesnik ldquoAnalytics ofsurfactants in the environment problems and challengesrdquoChemical Reviews vol 111 no 9 pp 5667ndash5700 2011

[18] R Alzaga A Pena L Ortiz and J M Bayona ldquoDeterminationof linear alkylbenzensulfonates in aqueous matrices by ion-pair solid-phase microextraction-in-port derivatization-gaschromatography-mass spectrometryrdquo Journal of Chromatogra-phy A vol 999 no 1-2 pp 51ndash60 2003

[19] J Riu P Eichhorn J A Guerrero T P Knepper and DBarcelo ldquoDetermination of linear alkylbenzenesulfonates in

wastewater treatment plants and coastal waters by automatedsolid-phase extraction followed by capillary electrophoresis-UV detection and confirmation by capillary electrophoresis-mass spectrometryrdquo Journal of Chromatography A vol 889 no1-2 pp 221ndash229 2000

[20] Y An H Bai C Li and G Shi ldquoDisassembly-driven colorimet-ric and fluorescent sensor for anionic surfactants in water basedon a conjugated polyelectrolytedye complexrdquo Soft Matter vol7 no 15 pp 6873ndash6877 2011

[21] J Fan and C Yin ldquoMethylene green SDS detection assayrdquo TechRep Bowdish Lab McMaster University Hamilton Canada2012

[22] A N Reshetilov I N Semenchuk P V Iliasov and L A Tara-nova ldquoThe amperometric biosensor for detection of sodiumdodecyl sulfaterdquo Analytica Chimica Acta vol 347 no 1-2 pp19ndash26 1997

[23] L Taranova I Semenchuk T Manolov P Iliasov and AReshetilov ldquoBacteria-degraders as the base of an amperometricbiosensor for detection of anionic surfactantsrdquo Biosensors andBioelectronics vol 17 no 8 pp 635ndash640 2002

[24] I E Tsybulskii andM A Sazykina ldquoNew biosensors for assess-ment of environmental toxicity based on marine luminescentbacteriardquo Applied Biochemistry and Microbiology vol 46 no 5pp 505ndash510 2010

[25] D Futra S Surif A Ahmad et al ldquoDetermination of Cu(II)toxicity using a biosensor with immobilized recombinantEscherichia coli roGFP cellsrdquo in Environmental Risk Assessmentand Management in Japan and Malaysia Graduate School ofEngineering Kyoto University and Institute for Environmentaland Development (LESTARI) 2009

[26] C R Arias-Barreiro KOkazaki A Koutsaftis et al ldquoA bacterialbiosensor for oxidative stress using the constitutively expressedredox-sensitive protein roGFP2rdquo Sensors vol 10 no 7 pp6290ndash6306 2010

[27] L A Taranova A P Fesaı G V Ivashchenko A N ReshetilovM Winter-Nielsen and J Emneus ldquoComamonas testosteronistrain TI as a potential base for a microbial sensor detectingsurfactantsrdquo Prikladnaia Biokhimiia i Mikrobiologiia vol 40no 4 pp 472ndash477 2004

[28] D McHugh Production and Utilization of Products from Com-mercial Seaweeds Food andAgricultureOrganization of UnitedNations Rome Italy 1987

[29] E R Morris D A Rees and G Robinson ldquoCation-specificaggregation of carrageenan helices domain model of polymergel structurerdquo Journal of Molecular Biology vol 138 no 2 pp349ndash362 1980

[30] C Rochas and S Landry ldquoMolecular organization of kappacarrageenan in aqueous solutionrdquo Carbohydrate Polymers vol7 no 6 pp 435ndash447 1987

[31] S K H Gulrez S Al-Assaf and G O Philips ldquoHydrogelsmethods of preparation characterisation and applicationsrdquo inProgress in Molecular and Environmental Bioengineering FromAnalysis andModeling to Technology Applications A Carpi EdInTech 2011

[32] H J Cha R Srivastava V N Vakharia G Rao andW E Bent-ley ldquoGreen fluorescent protein as a noninvasive stress probein resting Escherichia coli cellsrdquo Applied and EnvironmentalMicrobiology vol 65 no 2 pp 409ndash414 1999

[33] D C Joyner and S E Lindow ldquoHeterogeneity of iron bioavail-ability on plants assessed with a whole-cell GFP-based bacterialbiosensorrdquoMicrobiology vol 146 no 10 pp 2435ndash2445 2000


[34] M T Brandl B Quinones and S E Lindow ldquoHeterogeneoustranscription of an indoleacetic acid biosynthetic gene inErwinia herbicola on plant surfacesrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 98 no6 pp 3454ndash3459 2001

[35] J H J Leveau and S E Lindow ldquoAppetite of an epiphytequantitative monitoring of bacterial sugar consumption in thephyllosphererdquo Proceedings of the National Academy of Sciencesof the United States of America vol 98 no 6 pp 3446ndash34532001

[36] W GMiller M T Brandl B Quinones and S E Lindow ldquoBio-logical sensor for sucrose availability relative sensitivities ofvarious reporter genesrdquo Applied and Environmental Microbiol-ogy vol 67 no 3 pp 1308ndash1317 2001

[37] T Cserhati E Forgacs and G Oros ldquoBiological activity andenvironmental impact of anionic surfactantsrdquo EnvironmentInternational vol 28 no 5 pp 337ndash348 2002

[38] D R Caprette Preparing Protein Samples for Electrophore-sis 2014 httpwwwrufriceedusimbioslabsstudiessds-pagedenaturehtml

[39] M Conte and K Carroll ldquoThe chemistry of thiol oxidation anddetectionrdquo in Oxidative Stress and Redox Regulation U Jakoband D Reichmann Eds pp 1ndash42 Springer Amsterdam TheNetherlands 2013

[40] HNiwa S Inouye THirano et al ldquoChemical nature of the lightemitter of the Aequorea green fluorescent proteinrdquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 93 no 24 pp 13617ndash13622 1996

International Journal of

AerospaceEngineeringHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

RoboticsJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Active and Passive Electronic Components

Control Scienceand Engineering

Journal of



RotatingMachinery


Hindawi Publishing Corporation httpwwwhindawicom

Journal ofEngineeringVolume 2014

Submit your manuscripts athttpwwwhindawicom

VLSI Design



Shock and Vibration


Civil EngineeringAdvances in

Acoustics and VibrationAdvances in



Electrical and Computer Engineering

Journal of

Advances inOptoElectronics


Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

SensorsJournal of


Modelling amp Simulation in EngineeringHindawi Publishing Corporation httpwwwhindawicom Volume 2014


Chemical EngineeringInternational Journal of Antennas and

Propagation




Navigation and Observation



DistributedSensor Networks



water or through direct application such as agrochemicalsprays [7 8] dispersants and pesticides [6] SDS is widelyused in biochemical research in cell lysis in DNA extractionvia SDS-PAGE and as viral biocide [9]

SDS has been reported toxic to aquatic organisms Infish species it enhances organ morphologies such as kidneyand spleen alteration of metabolism rate and swimmingability and changes in growth and death rates [10ndash12]SDS shows acute effect to fertilization of ova and spermdecreasing probability of fertilization in fish [12] SDS is alsoreported to be toxic to mammals such as rodents and human[13] Exposure of SDS to mammals results in physical andbiochemical effects skin irritation hyperplasia alterationof serum lipid composition damage of cells and decreasein cell proliferation [14ndash16] Detection of SDS via chemicalanalytical methods through capillary electrophoresis gaschromatography HPLC and UV-V has drawbacks such asdifficulty to determine LOD with large sample volume lowdetection and volatilization rates production of toxic wasteduring operational analysis high cost and causing damageto sample during detection process [17ndash19] The use of dyeto bind with SDS in water has been suggested for thequantification of SDS concentration but there is challenge toproduce specific binding of the dye with SDS [20 21] Notmany SDS-detection biosensors have been reported Whileseveral bacterial biosensors were produced to assist SDSdetection most of them were reported to have long responsetime (minutes to hours) short dynamic linear range lowreproducibility and short term of stability [22ndash24] In thisstudy we report a sensitive SDS-detection toxicity biosensorfabricated with immobilization of E coli GFP which wasfound to have improved properties

2 Materials and Methods

21 Materials Ampicillin 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES) sodium dodecyl sulfate(SDS) and sodium chloride (NaCl) were purchased fromSigma-Aldrich Nutrient agar and glycerol were purchasedfrom MERCK and HMBC Chemicals respectively Yeastextract and tryptone were purchased from Becton Dickinsonand Company All materials used in this study are of thehighest purity available Solutionswere prepared in deionizedwater (Barnstead ROThermo Scientific)

22 Cultivation of Recombinant Bacteria E coli GFP E coliGFP colonies were produced by spreading the bacterial stockon nutrient agar and incubated under 37∘C for 18 hoursSingle colony of bacteria has been inoculated in 4mL Luria-Bertani medium (LB 10 gL tryptone 5 gL yeast extract10 gL of NaCl pH 70) with 100 120583gmL ampicillin addedPreculturing of E coli GFP bacteria has been carried outin a rotary thermoshaker and has been set at 250 rpm37∘C for 18 hours (until OD

600reached 13 ABS) 500 120583L of

yielded recombinant bacteria from the preculture was furthercultivated in 50mL LB medium for 4 hours (optical density600 nm reached 08ndash084 ABS) [25] Bacteria E coliGFP thathas been cultured was centrifuged under 3000 rpm at 25∘Cfor 10min the supernatant was discharged It was then

Container

E coli GFP bacterial cell

Matrix of k-Carrageenan

Figure 1 The design of E coli GFP toxicity biosensor

washed twice with 5mM 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES) bufferwith 171mMNaCl pH70 under the same condition [26] Bacterial cells in pelletformwere suspended in 10mLHEPES buffer and kept in 4∘CThe bacteria culture is ready to be used in the following steps

E coliGFP bacteria strain was selected because it is moresensitive and fast in giving a response compared with therest of the GFP mutants Wild type GFP was not consideredbecause of its weak fluorescence signal Single colonies wereobtained from the nutrient agar after 20 120583L of E coli GFPfrom glycerol stock was spread equally and allowed to growon the agar Ampicillin was added into the nutrient agar aswell as the LBmedium to ensure plasmidmaintenance of thebacteria E coli GFP The bacteria cultures were washed twicewith HEPES buffer to eliminate excessive LB medium andalso organic wastes that were produced during the growingand duplication process of the bacteria

23 Fabrication of E coli GFP Biosensor Toxicity biosensoris fabricated by immobilizing E coli GFP bacterial cells ink-Carrageenan matrix Immobilization process was carriedout on a hot plate with temperature set to 40ndash45∘C Around container (119889 = 8mm) was used as a mould for theimmobilization to take place Figure 1 illustrates the design ofE coli GFP toxicity biosensor Optimized amount of E coliGFP cells (warmed in 35ndash40∘C water bath) was mixed withoptimized concentration of k-Carrageenan agar (kept in liq-uid form under 40ndash45∘C heating) Temperature for biosensorpreparation was set in between 35 and 45∘C which is asuitable temperature range for active survival of bacteriaMixture was stirred well to make sure there was no formationof gas bubbles in thematrix and to produce a smooth surfaceHomogenous mixture was kept in 4∘C overnight to allowthorough solidification to take place

231 Immobilization Matrix Optimization Six concentra-tions of k-Carrageenan matrix were prepared 10 1214 16 18 and 20 All of themwere prepared bymix-ing the specific amount of k-Carrageenan powderwith deion-ized water in which the temperature was fixed at 75∘C whichis the temperature needed for k-Carrageenan powder to dis-solve homogeneously in the solvent higher temperature willdenature the k-Carrageenan structure Continuous stirringwas set to allow homogenous mixture to form A constantamount of E coli GFP was then immobilized in 100 120583L of k-Carrageenanmatrix for all six concentrationmeasures Eachconcentration was prepared in 3 repetition setsThemixtureswere kept in 4∘C overnight Toxicity biosensors with different









119900) and



RFU =119865119900minus 119865119905

119865119900

times 100 (1)





320

340

360

380

400

420

440

460

480

500

08 1 12 14 16 18 2 22

Fluo

resc

ence

inte

nsity

(au

)







380

400

420

440

460

480

500

520

4 9 14 19 24 29

Fluo

resc

ence

inte

nsity

(au

)



050

100150200250300350400450500

0 10 20 30 40 50

Fluo

resc

ence

inte

nsity

(au

)

Period (day)









0

100

200

300

400

500

600

700

1 2 3 4 5 6 7 8 9 10

Fluo

resc

ence

inte

nsity

(au

)







0100200300400500600700800900

1000

425 450 475 500 525 550 575 600

Without SDS



Fluo

resc

ence

inte

nsity

(au

)







0102030405060708090

100


Inhi

bito

ry (

)


10

30

50

70

90

110

0 1 2 3

020406080

100

0 05 1 15 2 25

y = 63145x minus 34277

R2 = 09925

minus1

Log[SDS] (ppm)

Inhi

bito

ry (

)




























4 Conclusions




Acknowledgment


References















































RoboticsJournal of





Journal of



RotatingMachinery





VLSI Design



Shock and Vibration







Journal of



Volume 2014


SensorsJournal of





Propagation

















119900) and



RFU =119865119900minus 119865119905

119865119900

times 100 (1)





320

340

360

380

400

420

440

460

480

500

08 1 12 14 16 18 2 22

Fluo

resc

ence

inte

nsity

(au

)







380

400

420

440

460

480

500

520

4 9 14 19 24 29

Fluo

resc

ence

inte

nsity

(au

)



050

100150200250300350400450500

0 10 20 30 40 50

Fluo

resc

ence

inte

nsity

(au

)

Period (day)









0

100

200

300

400

500

600

700

1 2 3 4 5 6 7 8 9 10

Fluo

resc

ence

inte

nsity

(au

)







0100200300400500600700800900

1000

425 450 475 500 525 550 575 600

Without SDS



Fluo

resc

ence

inte

nsity

(au

)







0102030405060708090

100


Inhi

bito

ry (

)


10

30

50

70

90

110

0 1 2 3

020406080

100

0 05 1 15 2 25

y = 63145x minus 34277

R2 = 09925

minus1

Log[SDS] (ppm)

Inhi

bito

ry (

)




























4 Conclusions




Acknowledgment


References















































RoboticsJournal of





Journal of



RotatingMachinery





VLSI Design



Shock and Vibration







Journal of



Volume 2014


SensorsJournal of





Propagation










320

340

360

380

400

420

440

460

480

500

08 1 12 14 16 18 2 22

Fluo

resc

ence

inte

nsity

(au

)







380

400

420

440

460

480

500

520

4 9 14 19 24 29

Fluo

resc

ence

inte

nsity

(au

)



050

100150200250300350400450500

0 10 20 30 40 50

Fluo

resc

ence

inte

nsity

(au

)

Period (day)









0

100

200

300

400

500

600

700

1 2 3 4 5 6 7 8 9 10

Fluo

resc

ence

inte

nsity

(au

)







0100200300400500600700800900

1000

425 450 475 500 525 550 575 600

Without SDS



Fluo

resc

ence

inte

nsity

(au

)







0102030405060708090

100


Inhi

bito

ry (

)


10

30

50

70

90

110

0 1 2 3

020406080

100

0 05 1 15 2 25

y = 63145x minus 34277

R2 = 09925

minus1

Log[SDS] (ppm)

Inhi

bito

ry (

)




























4 Conclusions




Acknowledgment


References















































RoboticsJournal of





Journal of



RotatingMachinery





VLSI Design



Shock and Vibration







Journal of



Volume 2014


SensorsJournal of





Propagation













0

100

200

300

400

500

600

700

1 2 3 4 5 6 7 8 9 10

Fluo

resc

ence

inte

nsity

(au

)







0100200300400500600700800900

1000

425 450 475 500 525 550 575 600

Without SDS



Fluo

resc

ence

inte

nsity

(au

)







0102030405060708090

100


Inhi

bito

ry (

)


10

30

50

70

90

110

0 1 2 3

020406080

100

0 05 1 15 2 25

y = 63145x minus 34277

R2 = 09925

minus1

Log[SDS] (ppm)

Inhi

bito

ry (

)




























4 Conclusions




Acknowledgment


References















































RoboticsJournal of





Journal of



RotatingMachinery





VLSI Design



Shock and Vibration







Journal of



Volume 2014


SensorsJournal of





Propagation










0102030405060708090

100


Inhi

bito

ry (

)


10

30

50

70

90

110

0 1 2 3

020406080

100

0 05 1 15 2 25

y = 63145x minus 34277

R2 = 09925

minus1

Log[SDS] (ppm)

Inhi

bito

ry (

)




























4 Conclusions




Acknowledgment


References















































RoboticsJournal of





Journal of



RotatingMachinery





VLSI Design



Shock and Vibration







Journal of



Volume 2014


SensorsJournal of





Propagation
















4 Conclusions




Acknowledgment


References















































RoboticsJournal of





Journal of



RotatingMachinery





VLSI Design



Shock and Vibration







Journal of



Volume 2014


SensorsJournal of





Propagation



















































RoboticsJournal of





Journal of



RotatingMachinery





VLSI Design



Shock and Vibration







Journal of



Volume 2014


SensorsJournal of





Propagation









Research Article Toxicity Biosensor for Sodium Dodecyl...

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