research article highly sensitive …downloads.hindawi.com/journals/ijac/2015/347621.pdfhighly...

Research ArticleHighly Sensitive Immunochromatographic Identification ofTetracycline Antibiotics in Milk

N A Taranova1 A S Kruhlik2 E A Zvereva1 V V Shmanai2 I I Vashkevich3

D A Semyonov3 S A Eremin14 A V Zherdev1 and B B Dzantiev1

1AN Bach Institute of Biochemistry Research Centre of Biotechnology of the Russian Academy of Sciences Leninsky Prospect 33Moscow 119071 Russia2Institute of Physical Organic Chemistry Surganov Street 13 220072 Minsk Belarus3Institute of Bioorganic Chemistry Acad Kuprevich Street 52 220141 Minsk Belarus4Chemical Department MV Lomonosov Moscow State University Leninskie Gory Moscow 119991 Russia

Correspondence should be addressed to B B Dzantiev dzantievinbirasru

Received 17 September 2015 Accepted 4 November 2015

Academic Editor Josep Esteve-Romero

Copyright copy 2015 N A Taranova et alThis is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

A rapid immunochromatographic assay was developed for the control of tetracycline (TC) The assay is based on the competitionbetween immobilized TC-protein conjugate and TC in a tested sample for binding with polyclonal anti-TC antibodies conjugated tocolloidal gold during the flow of the sample along a membrane strip with immobilized reactants Conjugation of colloidal gold andthe total immunoglobulin (IgG) fraction of polyclonal antibodies was used to increase the assay sensitivity to ensure low contentof specific antibodies in the conjugate This allowed effective inhibition of free TC and conjugate binding in the strip test zonePhotometric marker registration allows control of the reduction of binding thereby enhancing detection sensitivity The proposedassay allows TC to be detected at concentrations up to 20 ngmL exceeding the limit of detection of the known analogues in a wideworking range (more than two orders) of 60 pgmL to 10 ngmL ensured through the use of polyclonal antibodies The assay timeis 10minThe efficiency of the designed assay is shown to identify TC in milk the degree of recovery of TC ranges from 90 to 112The precision of the concentrations measurements was no more than 10

1 Introduction

Tetracyclines (TCs) are a groupof broad-spectrumantibioticsrepresenting polyketones in their chemical structure [1] Byblocking the binding of transferred ribonucleic acid aminoa-cyl to ribosome they inhibit protein synthesis in bacterialcells [2 3] TCs are widely used in veterinary medicine bothfor therapeutic and preventive purposes due to their highactivity towards a large number of Gram-positive and Gram-negative bacteria small doses and broad spectrum of actionIn addition TCs are used as growth promoters of animal bodyweight destroyers of pathogens in drinking water sourcesfodder and food and phytopathogenic agents in crop pro-duction [4] The most widely used tetracycline antibioticsare tetracycline (TC) chlortetracycline and oxytetracycline(Figure 1)

Because of their intensive and diverse uses tetracyclinesmay enter the human body not only in the treatment ofdiseases but also with food This can cause toxic and allergiceffects dysfunction of the gastrointestinal tract renal insuffi-ciency and deformation of mucous tissues [5 6] The emer-gence of bacteria resistant to tetracycline antibiotics is due totheir massive clinical veterinary and agricultural use [7] It istherefore important to monitor the presence of tetracyclinesand other antibiotics in food

Most countries of the world have regulations concerningacceptable levels of tetracyclines in food According to theregulations of the CustomsUnion the concentration of tetra-cyclines inmilk dairy productsmeat and prefabricatedmeatproducts should not exceed 10 ngmL (ngg) in Russia [8]The European Commission established maximum residuelevels (MRLs) for the amounts of tetracyclines namely

Hindawi Publishing CorporationInternational Journal of Analytical ChemistryVolume 2015 Article ID 347621 10 pageshttpdxdoiorg1011552015347621

2 International Journal of Analytical Chemistry

NH2

CH3

CH3

H3CR1 R2HO N

OH

OH OHOH

OO O

Figure 1 The overall structure of tetracycline antibiotics tetracy-cline (R

1 H R

2 H) chlortetracycline (R

1 Cl R

2 H) oxytetracycline

(R1 H R

2 OH)

oxytetracycline chlortetracycline and their stereoisomers of100 pgg for meat 600 ngg for kidneys 200 ngg for eggsand 100 ngg for milk [9] Tetracycline is not allowed in somefood products (honey and baby food) that is the level oftetracycline should be below the detection threshold of therecommended analytical methods In the United States themaximum permissible level of tetracycline is 300 120583gg formeat 6 120583gg for liver and 12 120583gg for kidneys [10] In ChinaMRLs for the total content of tetracycline hydrochloride andoxytetracycline are 50 ngmL for milk 600 ngg for kidneys300 ngg for liver and 100 ngg for muscular tissue [11]

Low MRLs necessitate highly sensitive methods fordetecting antibiotics Control of TCs in food productsrequires rapid analysismethods and needs to be implementeddirectly at sampling points However stationary methods arestill predominant in the state-of-the-art analytical practice

Relatively simple and low-budget microbiological meth-ods are widely used to identify TC residues in foods [12 13]However they lack specificity (different classes of antibioticsare identified) and are time-consuming (15ndash3 h) Methodsbased on liquid chromatography are also used to identifyTCs [14 15] These methods are highly specific and highlysensitive but they are also time-consuming moreover theyrequire large amounts of solvent and an expensive specializedequipment base None of thesemethods allow field analysis tobe performed

Methods based on the use of specific cell receptors [16 17]including their rapid variants have gained widespread cur-rency because of the difficulty of obtaining antibodies specificto TCs TC receptor tests are characterized by their rapidity(10ndash20min) but the test system needs to be incubated in aconstant-temperature bath at 45∘C

A number of immunochemical detection methods forTCs have also been successfully developed Implementationof these methods does not require expensive equipment andis therefore of interest for mass contamination screeningThemost common methods of microplate enzyme immunoassay(ELISA) for TCs [18ndash20] are implemented in a laboratoryenvironment but with the use of relatively inexpensive andeasy to use instrumentation Unlike ELISA immunochro-matography (IC) test systems are nonlaboratory analyticaltools All processes in the IC test system are carried outwithout operator intervention or additional devices contactof the test strip with the sample initiates all subsequent

interactions There are several options for IC test systems toidentify tetracycline antibiotics based on the use of mono-clonal antibodies Existing test systems allow visual detectionof TCs at 30 ngmL and above [21 22] but this satisfies onlypart of the above regulations Therefore the creation of ahighly sensitive IC assay is an extremely necessary and urgenttask

Two approaches for lowering the limit of detection ofLFTS are implemented in this paper The first approach isbased on the coating gold nanoparticles (GNPs) by polyclonalantibodies allowing low content of specific antibodies againstTC in the conjugate [23] As we have previously shown[24] for such conjugates less antigen in the sample causessaturation of the antibody valency and binding inhibitionin the strip test zone The second approach is based onthe use of video digital detectors to register quantitativelydecrease of the label binding These detectors [25 26] allowthe quantization of the reduction in the intensity of colorationof the strip test zone and identification of the presence ofantigen in the sample for concentrations that do not causethe complete disappearance of coloration

Thus the aims of theworkwere to obtain and characterizeGNP conjugates with polyclonal antibodies against TC toapply the described above approaches for immunochro-matographic assay of TC to characterize sensitivity of theproduced test strips and to study their application for theidentification of TC in milk

2 Experimental

21 Chemicals Sodium azide bovine serum albumin (BSA)331015840551015840-tetramethylbenzidine (TMB) dimethyl sulfoxide(DMSO) formaldehyde Tween-20 and Triton X-100 wereobtained from Sigma (St Louis MO) Gold chloride andtetracycline hydrochloride (TC-HCl) were obtained fromFluka (Buchs Switzerland) Sephadex G-25 was obtainedfrom MP Biomedicals (Santa Ana CA) The preparationused in this work comprised the anti-TC rabbit sera andimmunoglobulin (IgG) fraction described in [23] Goatanti-mouse IgG antibodies were purchased from AristaBiologicals (Allentown PA) Peroxidase-labeled anti-mouseimmunoglobulins were from the Gamaleya Institute ofMicrobiology and Epidemiology Russia All other chemicals(salts and solvents of analytical grade) were from Chimmed(Moscow Russia)

All solutions for synthesis were prepared using waterthat was purified using Milli-Q system (Millipore Bed-ford MA httpwwwmilliporecom) Stock solutions of TC(1 120583gsdotmLminus1) were prepared in water and stored at minus20∘C

22 Tetracycline-BSA Conjugate Synthesis The procedure in[20] was amended and applied In this procedure 30mg ofBSA was dissolved in 20mL of H

2O and 26mg TC-HCl

solutionwas added to 200120583LDMSO and 120 120583L of 37 aque-ous formaldehyde solution (BSA molar ratio TC formalde-hyde = 1 120 3600) and stirred at room temperature over-nightThe product was purified from unreacted low-molecu-lar compounds by means of gel permeation chromatographyon the Sephadex G-25 carrier in phosphate-buffered saline

International Journal of Analytical Chemistry 3

(PBS 50mM pH 74 with 01M NaCl) Spectrophotometrywas used to evaluate the composition of the TC-BSA conju-gate The resulting product was stored at 4∘C

23 Microplate Enzyme Immunoassay for TC Analysis wascarried out using the same procedure described in [24]The TC-BSA conjugate was immobilized in microplate wells(4 120583gmLminus1 in PBS 100 120583L per well) and incubated overnightat 4∘C The stock antibiotic solution was diluted in PBScontaining 005 Triton X-100 and 1 BSA (PBST) to obtaina series of solutions in the range of 100 to 014 ngmLminus1 andadded to themicroplate wells (50 120583L per well)The antibodieswere diluted in PBST (dilution 1 40000 from the initialantiserum volume) and the microplate was incubated for 1 hat 37∘C After washing with PBST peroxidase-labeled anti-mouse antibodies were added (dilution of the commercialproduct in PBST was 1 6000 100 120583L per well) and incubatedfor 1 h at 37∘C Finally the microplate was washed three timeswith PBST and once with distilled water

To detect the activity of the bound peroxidase label asubstrate solution containing 042mM of TMB and 18mMof H2O2in 01M sodium citrate buffer pH 40 was added

(100 120583L per well) After 15min of incubation at room tem-perature the reaction was stopped with 1M H

2SO4(50 120583L

per well) The optical density of the reaction product wasmeasured at 450 nm using a Zenyth 3100 microplate reader(Anthos Labtec Instruments Salzburg Austria)

24 Preparation of Gold Nanoparticles Gold nanoparticles(GNPs) with an average diameter of 30 nm were preparedaccording to the protocol described in [25] Briefly 10mLof a 1 water solution of HAuCl

4was added to 975mL of

water The mixture was heated to reflux and 15mL of 1sodium citrate solutionwas added After refluxing for 30minthe preparation was cooled and then stored at 4∘C

25 Generation of a FlocculationCurve to Immobilize Antibod-ies for GNPs The procedure in [27] was applied with modi-fications In this procedure 200120583L of GNPs (119860

520= 1) was

placed in microplate slots and 20mcL of antibody solutionsin water at concentrations ranging from 200 to 05 120583gmLwasadded After incubation 20120583L of 10 NaCl solution at roomtemperature was added to each slot within 10min followedby measurement of 119860

580and the creation of its dependence

on the final concentration of antibodies (flocculation curve)

26 Synthesis of Conjugate of Antibodies with GNPs Synthe-sis was carried out using the procedure in [27 pp 931-932]as amended in [28] Antibodies were added to 10mM Trisbuffer pH 85 The GNP solution (119860

520= 1) was adjusted

with 1M of potassium carbonate to pH 85ndash90 followedby the introduction of antibodies in an amount selectedbased on the flocculation curve and under thorough mixingThe mixture was incubated at room temperature for 15minfollowed by the introduction of 10 aqueous solution ofBSA (v v = 40 1) and incubation for 10min under thoroughmixing Gold particles with antibodies immobilized on theirsurface were pelleted by centrifugation at 13000 g and 4∘C for15min The supernatant was removed and the residue was

dissolved in 10mV Tris buffer pH 85 with 1 BSA and 1sucrose (TBSA) and subjected to centrifugation in the sameenvironment The resulting deposit was dissolved in TBSAwith the introduction of sodium azide to a final concentrationof 005 and storage at 4∘C

27 Sizing of GNPs and Their Conjugates with AntibodiesTo characterize the particle size images of the GNPs wereobtained with a CX-100 transmission electron microscope(Jeol Tokyo Japan) at an accelerating voltage of 80000V anda magnification of 3300000 The images were processed byTotalLab software (Nonlinear Dynamics Newcastle UK)

A Zetasizer Nano ZSP (Malvern UK) was used to eval-uate the hydrodynamic diameter of free GNPs and theirconjugates Dynamic light scattering (DLS 10 per sample)was measured at 20∘C with the preliminary incubation ofsamples The angle of light scattering was 103∘

28 Determination of the Amount of Antibodies Adsorbed onthe Surface of GNPs Polarization fluoroimmunoassay wasused to compare concentration of antibodies in the for-mulation added for immobilization and the formulation ofunbounded antibodies Formulations were characterized bybinding with the BSA-TC conjugate

The TC-BSA conjugate was immobilized in microplatewells (5 120583gmLminus1 in PBS 100 120583L per well) and incubated over-night at 4∘CAfter washingwith PBST the anti-TC antibodieswere diluted in PBST to obtain a series of solutions in therange of 10 to 0 120583gmLminus1 After centrifugation antibody solu-tions and supernatants were added to the microplate wells(100 120583L per well) and the microplate was incubated for 1 hat 37∘C Subsequent reaction with peroxidase-labeled anti-rabbit antibodies andmeasurement of the peroxidase activityassociated with the label was carried out as described above(see Section 23) Optical densities measured for standardantibody solutions and supernatants were compared

29 Production of Immunochromatographic Tests The MdiEasypack (AdvancedMicrodevices AmbalaCantt India) kitsof membranes were used to manufacture the immunochro-matographic tests They included a plastic support workingnitrocellulosemembrane CNPCwith a 12 120583mpore size GFB-R4 separation membrane `f-R7 glass fiber membrane and0`045 adsorption membrane

Reagents were immobilized on membranes using an Iso-Flow automated dispenser (Imagene Technology HanoverNH)The test zone was formed by the TC-BSA conjugate andthe control zone was formed by the goat anti-mouse IgGThefollowing concentrations and immobilization media wereused TC-BSA conjugate 10ndash20mgsdotmLminus1 in PBS and goatanti-mouse IgGs 1mgsdotmLminus1 in PBS One microliter of bothsolutions was applied per centimeter of strip width After thedispensing the membrane was dried at room temperaturefor at least 20 h The conjugate of GNPs with antibodies ata dilution corresponding to 119863

520= 40 was spotted onto a

glass fiber membrane The conjugate load was 32 120583L for 1 cmof strip width The glass fiber membrane was then dried atroom temperature for at least 20 h


After the assembly ofmembrane components the obtainedsheets were cut with Index Cutter-1 (A-Point TechnologiesAllentown PA) into test strips of 35mm in width The teststrips were hermetically packed in laminated aluminum foilbags containing silica gel as the desiccant using a FR-900miniconveyor The cutting and packing were carried out atroom temperature in a special room with a relative humidityunder 30 The packed test strips were stored at roomtemperature

210 Immunochromatographic Assay Procedure Milk sam-ples were purchased from the local market Spiked milksamples were prepared by mixing stock TC solutions andpretested TC-free milk samples

The assay was performed at room temperature Pure andspiked milk samples were diluted with PBS 119881milk 119881PBS =4 1 A test strip was vertically submerged into an analytesolution or a milk sample for 10min corresponding to thetime required for the fluid front to migrate along the entirelength of the working membrane

211 Registration and Processing of ImmunochromatographicAssay Data The binding of the label in the test and controlzones was recorded with the use of a CanoScan LiDE 90scanner (Canon Tokyo Japan) followed by digital processingof the images with TotalLab software (Nonlinear DynamicsNewcastle UK)This programwas used to determine the spotboundaries sum up the intensities of all pixels belonging toa particular unit and normalize the sums to the spot surfacearea thereby representing the color intensity in relative units(RU) Alternatively a portable detector Reflekom equippedwith Videotest software (Synteco-Complex Moscow Russia)was used for quantitative detection of antigen

Based on the color intensities (119884) for different concentra-tions of the analyte (119909) a calibration curve was constructedusing the four-parameter sigmoid function [29]

119884 = ((119860 minus 119863)

(1 + (119909119862)119861)) + 119863 (1)

where 119860 is the asymptotic maximum (the color intensity inthe absence of the analyte) 119861 is the slope of the curve insemilogarithmic coordinates in the inflection point 119862 is theconcentration of the analyte at the inflection point and 119863is the asymptotic minimum (the intensity of the backgroundcoloration)

The quantitative limit of detection was calculated as theTC content corresponding to a binding inhibition of 10Theworking range was calculated as the TC content correspond-ing to a binding inhibition of 20 (lower limit of workingrange) and 80 (upper limit of working range) [30]

The visual detection limit was 1000 RU

212 Addition-Detection Experiments The test system wascharacterized in addition-detection experiments using

300 400 500 60000

02

04

06

08

10

12

14

16

18

20

1

2

3

120582 (nm)

Figure 2 Absorbance spectra of BSA (1) TC (2) and the TC-BSAconjugate (3)

spiked milk samples The recovery (119877 ) in the addition-detection experiments was calculated as follows

119877 = (119909exp

119909theor) sdot 100 (2)

where 119909theor is the added TC concentration and 119909exp is the TCconcentration obtained with the calibration curve

3 Results and Discussion

31 Preparation and Specification of Immunoreagents Char-acteristics of immunoreagents included in the test systemsincluded determination of the composition of hapten-proteinsynthesized conjugate and assessment of the interaction of theconjugate and polyclonal antibodies

The TC-BSA conjugate was characterized using a spec-trophotometer (Figure 2) According to the comparative dataof the spectra of primary components and the conjugate themolar ratio of TC BSA in the conjugate was 10 1

The interaction of the conjugate and polyclonal anti-bodies to tetracycline was characterized using the microplateenzyme immunoassay method The concentration of anti-bodies absorbency of 10 in the microplate enzyme immu-noassay to be obtained (during interaction with the immo-bilized TC-BSA conjugate) was 015120583gmLThe effectivenessof TC identification was characterized using the competitivemicroplate enzyme immunoassay method the conditions ofwhich (reactant concentration and duration of incubation)were optimized to ensure a minimum detection limit TheEIA calibration curve obtained from the selected optimalconditions (see ldquoSection 2rdquo) is shown in Figure 3

As compared to control levels of tetracycline of 10ndash100 ngmL the tested polyclonal antibodies allowed its deter-mination by means of the ELISA method at concentrations1-2 times lower This indicated their viability for the develop-ment of IC test systems

International Journal of Analytical Chemistry 5A450

08

06

04

02

00

TC (ngmL)001 01 1 10 100 1000

Figure 3 TC-competitive EIA dependence of the recorded absorb-ency on the analyte concentration

Figure 4 Electron microphotograph of GNPs

32 Preparation and Characterization of GoldConjugates for Immunochromatography

321 Specification of Sizes of GNPs Formulation of GNPsobtained by the reduction of HAuCl

4to Au0 [31] was charac-

terized using transmission electron microscopy (TEM) andDLS methods Two methods were used because of their dif-ferent capabilities TEM is employed to determine the sizeof electron-dense structures that is the ldquometal corerdquo of theparticle excluding hydrate citrate and other membranesMeanwhile the DLS method is used to characterize particlesin a water environment considering reactants immobilizedon the surface and the hydration shell to approximate theobtained quantities to the properties of the reactants in a realassay environment

The TEM showed (Figure 4) that the average diameter ofthe GNPs was 202plusmn08 nmTheir shape was nearly sphericalwith an elongation coefficient of 126 plusmn 004 Based on thesevalues the estimated average surface area of a single GNPwasequal to 13000 plusmn 100 nm2

0 20 40 60 80 100 120 140 160 180 200

0

5

10

15

20

25

Inte

nsity

()

Hydrodynamic diameter (nm)

1

23

Figure 5 Hydrodynamic diameters for GNPs (1) and their con-jugates with anti-TC antibodies showing an antibody GNP molarratio of 235 1 (2) and 47 1 (3)

The results of DLS showed that hydrodynamic diameterof GNPs is 316 plusmn 03Nm (Figure 5 1) The differences in thevalues obtained bymeans of the TEM andDLSmethods weredue to the citrate shell on the surface of the GNPs This effectand its degree of manifestation correspond to the results ofour previous studies [32 33]

The narrow monopeak particle distribution by sizeobserved for bothmethods showed a homogeneous nature ofthe formulation of GNPs as well as a lack of aggregates andimpurities therein

322 Preparation and Characterization of Conjugates of GNPswith IgGs The conditions for the conjugation of the anti-bodies to GNPs were chosen based on the photometricdata characterizing the aggregation of the product of thisreaction at a high ionic strength The flocculation curvewas obtained through the process shown in Figure 6 Foradded IgG concentrations of 4120583gmL and above there was aplateau in the recorded optical densityThis demonstrates thatthe stabilizing nanoparticles immobilized protein moleculespreventing the aggregation of nanoparticles under conditionsof high ionic strength Based on Figure 6 the TC antibodieswere taken for conjugation in an amount for the point atwhich OD

580reached the plateau and two times higher at

protein concentrations of 40 and 80120583g per milliliter of thecolloidal solution

The concentration of 4120583gmL corresponded to a molarratio of IgG GNP during the interaction equal to 25 1 whilethe concentration of 8120583gmL corresponded to a ratio 50 1Given the size of GNPs and diameter of the Fc-fragment ofIgG (4Nm) the maximum number of adsorption sites of IgGon the surface of GNP (achieved during their contact withthe surface of Fc-fragment of the molecule) was 100 Thusthe amounts of IgG selected for conjugates were 26 and 52of the maximum possible number covering the entire surfaceof GNP


Table 1 Antibody immobilization characteristics on the GNP surface

Concentration of antibodies when conjugated 120583gmL 40 80Molar ratio of IgG GNP when conjugated 25 1 50 1Residual concentration of antibodies determined in the supernatant 120583gmL 022 047Antibody immobilization degree 945 941Actual molar ratio of IgG GNP in the conjugate 24 1 47 1

0 2 4 6 8 10 12 14 16 18 20 22 24

018

020

022

024

026

028

030

032

034

036

038

C (PAbs) (120583gmL)

A595

Figure 6 Values of GNPs optical density at 580 nmunder high ionicstrength obtained after GNP stabilization by the anti-TC polyclonalantibodies at different concentrations

The accounting estimate of the immobilization processwas compared with experimental data To that effect theconcentration dependences of binding in ELISA were com-pared with the immobilized TC-BSA conjugate obtainedfor the antibody solution to be added when conjugated aswell as supernatants sampled after generating the conjugatesTable 1 shows the experimental data and the data fromcalculations performedusing the experimental results As canbe observed binding of IgGs took place almost quantitativelyfor both formulationsThus at least twice the amount of IgGs(47) may be adsorbed on the GNP surface than what occursduring stabilization of the nanoparticles in accordance withflocculation curve (24)

Dimensioning specifications for the resulting conjugatesand the initial GNPs were determined by means of the TEMand DLS methods

TEM showed an almost spherical shape of GNPs anda high degree of homogeneity The average diameter of theconjugate with a molar ratio of IgG GNP 24 1 was 217 plusmn06 nm and the elongation coefficient was 125 plusmn 004 Theaverage diameter of the conjugate with a molar ratio ofIgG GNP of 47 1 was 223 plusmn 09 nm and the elongationcoefficient was 122 plusmn 006

The hydrodynamic diameter of the conjugate nanopar-ticles varied depending upon their composition equaling554 plusmn 21Nm for the conjugate with a molar ratio of

0 2 4 6 80

2000

4000

6000

8000

10000

12000

14000

Col

or in

tens

ity o

f tes

t zon

e (RU

)3 4

2

1

A520 of GN-AbsTC

Figure 7 Binding of IgG-GNP conjugates (1 and 3mdashconjugateof composition 47 1 2 and 4mdashconjugate of composition 235 1)with the fb-BSA conjugate (curves 1 and 2) and standard BSApreparation (curves 3 and 4) on the immunochromatographicmembrane

IgG GNP of 24 1 and 769 plusmn 28 nm for the conjugate witha molar ratio of 47 1 (Figure 5 curves 2 and 3)

Significant differences in size characteristics obtained bymeans of the TEM and DLS methods reflect the recordingof membranes of immobilized molecules and bound waterusing the DLS method (see above) It was shown that theconjugation was not accompanied by aggregation of thenanoparticles These findings are consistent with the litera-ture data on the immobilization of IgGon the surface ofGNPs[34ndash36]

Preservation of functional activity by IgG-GNP conju-gates was tested by their binding to the TC-BSA conjugateon the immunochromatographic membrane (Figure 7) TheTC-BSA conjugate was replaced with native BSA conjugateto confirm the specific nature of interaction As follows fromFigure 7 the conjugates showed comparable (at equal valuesof absorbency) degrees of binding of a specific nature whichallows their use in the development of IC test systems

33 Development of an Immunochromatographic Assay forTetracycline The immunochromatographic assay protocolwas optimized to achieve a minimum TC detection limitincluding selection of detergent concentration concentra-tions of IgG-GNPs and TC-BSA immobilized conjugates andpreparation ofmilk samplesThe assay optimization using theIgG-GNPs conjugate of 47 1 is described below The IC test


Table 2 The dependence of TC immunochromatographic assay on IgG-GNP conjugate concentration

OD520

of the conjugate Limit of TC detection ngmL Maximal coloration RU Background coloration RU2 016 plusmn 006 11100 plusmn 568 270 plusmn 584 01 plusmn 002 11600 plusmn 251 170 plusmn 258 18 plusmn 018 12000 plusmn 485 5000 plusmn 186

00 02 04 06 08 101000

1500

2000

2500

3000

3500

4000

4500

Max

imum

colo

r int

ensit

y (R

U)

C (Tween-20) ()

Figure 8 Dependence of the intensity of coloration of the testzone of IC test systems on Tween-20 detergent concentration in theabsence of TC

system implemented based on an IgG-GNP conjugate of 24 1showed the worst analytical characteristics therefore it wasexcluded from further study

Use of detergent in immunochromatography (added tothe IgG-GNP conjugate when applied) promotes uniformrapid movement of liquid along the membranes of the teststrip the most complete elution of IgG-GNP conjugate andan increase in the intensity of coloration of the test zone [36]The results obtained with various concentrations of Tween-20 detergent are shown in Figure 8 The concentration rangeunder analysis was 5ndash100 times greater than the critical con-centration for micelle formation (CCM) of Tween-20 whichwas equal to 001 [37] As follows from the results a Tween-20 concentration of 035 provided a uniform movement ofthe liquid front clear boundaries of the binding zone andmaximum intensity of coloration This concentration whichwas chosen as optimal is 35 times greater than the CCMThedetergent concentrations recommended in the literature andused in the immunoassay were at least 5 times greater thanthe CCM [38 39]

The IgG-GNP conjugate concentration was varied inthe range corresponding to the variation of OD

580from 2

to 8 absorbency units As follows from the data obtained(Table 2) the maximum intensity of coloration was constantin this range (ie coloration in the absence of analyte in thesample) within the tolerance For the assay option OD

580=

4 was optimal This dilution provided a minimum detectionlimit and minimal background coloration of the test zone

20 40 60 80 1000

10

20

30

40

50

60

70

Sign

aln

oise

ratio

Volume of milk in the sample ()Buffer

Figure 9 The dependence of the intensity of coloration of the testzone of IC test systems in the absence of TC on the degree of dilutionof milk samples using buffer (PBS)

Maximum intensity of coloration reaching 4300 plusmn 35units at 2mgmL was increased with an increase in the TC-BSA conjugate concentration in the test zone Substantialbackground coloration was typical for higher concentrationsThe optimal TC-BSA conjugate concentration was thereforeselected as 2mgmL

As sample preparation should be maximally simplerapid and non-time-consuming in the IC assay milk sampleswere diluted with buffer (PBS) for this purpose The resultsobtained for different variants of dilution are shown inFigure 9 The optimal volume ratio of the sample and thebufferwas 4 1 (80 content ofmilk in the test sample) In thiscase the sample matrix had minimal effect on the intensityratio of specific and background coloration reaching 50 1and the TC detection limit remained unchangedThe use of alarge sample dilution would reduce the sensitivity of the testsystem

Several parameters of the IC test system including mem-branes used were selected based on our previous data on thedevelopment of test systems for other antigens [24 28 40]

The test strips used in further study were producedin accordance with the optimal immunochromatographicparameters

34 Testing of the Developed Immunochromatographic TestSystem A calibration curve was obtained to identify TC inmilk Figure 10 shows the results of the testing of samples con-taining different concentrations offb inmilkThe test system


Table 3 Description of the test system developed for TC for testing milk samples in ldquoaddition-detectionrdquo experiments

TC added concentration ngmL TC concentration established by means of the testsystem ngmL Recoverylowast

003 0027 plusmn 0001 90 plusmn 303 029 plusmn 001 102 plusmn 805 056 plusmn 002 112 plusmn 415 148 plusmn 004 96 plusmn 32 22 plusmn 01 110 plusmn 7lowast119899 = 3

C (TC) (ngmL)0 005 01 05 1 10 100

(a)

Col

or in

tens

ity (R

U)

C (TC) (ngmL)1E minus 3 001 01 1 10 100 1000 10000

12000

10000

8000

6000

4000

2000

0

(b)

Figure 10 Test strips (a) and calibration curve 119899 = 3 (b) for immunochromatographic determination of the TC in milk samples

developed is characterized by low instrumental detectionthreshold (002 ngmL) and a wide operating range (006ndash10 ngmL) The operating range covering more than twoorders of concentrations is considerably higher than the rangeprovided in the test systems based on monoclonal antibodies(one order) [41] This effect was achieved through the useof polyclonal antibodies characterized by a considerablevariability of individual clones by affinity thereby providinga competitive interaction in a wide range of concentrationsThe precision (standard deviation) of the concentrationsmeasurements was no more than 10 The disappearance ofthe color in the test zone corresponded to the Russian MRLof TC (10 ngmLminus1) The dependence of color intensity fromTC concentration is described by the following equation

119884 = ((11640 minus 1588)

(1 + (119909036)07)) + 1588 (3)

Adequate goodness of fit (1205942) for this equation is 53 and1198772 is 0996The test system developed was validated to identify TC

in milk samples This is characterized in addition-detectionexperiments The data shown in Table 3 are indicative of theefficiency of the test system as a means of quantitative assaythe degree of TC extraction was 102 plusmn 8

4 Conclusions

The developed IC test system is characterized by a lowdetection threshold of TC in milk of 002 ngmL for instru-mental recording as well as a wide (more than two orders)operating range The quantitative assay method proposed is10ndash100 times more sensitive than IC assay [21] and ELISA[18 42] The visual detection threshold of the test system is10 ngmL which is 2ndash5 times greater than that of commercialanalogues (SNAPCharm)The assay time is 10min includingnon-time-consuming sample preparation which is limitedto dilution of the sample under analysis using a buffer Theeffectiveness of the test system for monitoring tetracycline inmilk was confirmedThe characteristics established allow thedeveloped test system to be considered as a promising tool formonitoring the safety of foods

Detection threshold reduction was achieved using twomethods as follows (a) transition from a qualitative assess-ment of results to a quantitative assessment based on videodigital recording and (b) the use of a polyclonal formu-lation containing a small proportion of antibodies specificto TC and nonspecific antibodies stabilizing GNPs Theseapproaches are universal and can potentially be used in thedevelopment of IC test systems to identify a wide range ofcompounds


Conflict of Interests

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

Acknowledgments

This work was supported by the Russian Foundation for Fun-damental Research (Grant no 15-33-50021 Preparation andSpecification of Antibodies) and the Russian Science Founda-tion (Grant no 14-16-00149 Development of Immunochro-matographic Assay)

References

[1] I Chopra and M Roberts ldquoTetracycline antibiotics modeof action applications molecular biology and epidemiologyof bacterial resistancerdquo Microbiology and Molecular BiologyReviews vol 65 no 2 pp 232ndash260 2001

[2] W Hillen and R A Greenwald Tetracyclines in Biology Chem-istry and Medicine Springer New York NY USA 2001

[3] L A Mitscher The Chemistry of the Tetracycline AntibioticsMarcel Dekker New York NY USA 1978

[4] E E Seyfried Tetracycline Resistance Genes in AquacultureEnvironments Genotypic Diversity and Potential ResistanceReservoirs University of Wisconsin-Madison Madison WisUSA 2007

[5] G G Gallo G Lancini and F Parenti Antibiotics A Multidis-ciplinary Approach Springer New York NY USA 2013

[6] J L Bose Acid Sensitivity is Caused by Tetracycline in ResistantStrains of Escherichia coli University of Wisconsin-MadisonMadison Wis USA 2002

[7] D Bansal and V K Purohit ldquoAccessibility and use of essentialmedicines in health care current progress and challenges inIndiardquo Journal of Pharmacology and Pharmacotherapeutics vol4 no 1 pp 13ndash18 2013

[8] T R O C Union TR CU 0212011 On Food Safety 2011[9] Commission Regulation (EU) no 372010[10] CAC FAO andWHO ldquoMaximum residue limits for veterinary

drugs in foodsrdquo in Proceedings of the 34th Session of CodexAlimentarius Commition Geneva Switzerland July 2011

[11] MOANo 235 Bulletin of the Agriculture of the Peoplersquos Repub-lic of China 2002

[12] T J Louie F P Tally J G Bartlett and S L Gorbach ldquoRapidmicrobiological assay for chloramphenicol and tetracyclinesrdquoAntimicrobial Agents and Chemotherapy vol 9 no 6 pp 874ndash878 1976

[13] F M Poonawalla and M R Iyengar ldquoMicrobiological assay ofvitamin B12 in the presence of tetracyclinerdquo Applied Microbiol-ogy vol 13 no 5 pp 755ndash756 1965

[14] E M Hussien ldquoHPLC method validation for modernizationof the tetracycline hydrochloride capsule USP monographrdquoBulletin of Faculty of Pharmacy Cairo University vol 52 no 2pp 239ndash244 2014

[15] K Ng and S W Linder ldquoHPLC separation of tetracycline ana-logues comparison study of laser-based polarimetric detectionwith UV detectionrdquo Journal of Chromatographic Science vol 41no 9 pp 460ndash466 2003

[16] V Gaudin A Rault and E Verdon ldquoValidation of a commercialreceptor kit for tetracycline residues in honey according tothe European guideline for screening methodsrdquo Food andAgricultural Immunology vol 24 no 1 pp 111ndash128 2013

[17] J F M Nouws G Loeffen J Schouten H Van Egmond HKeukens andH Stegeman ldquoTesting of rawmilk for tetracyclineresiduesrdquo Journal of Dairy Science vol 81 no 9 pp 2341ndash23451998

[18] M Jeon and I Rhee Paeng ldquoQuantitative detection of tetra-cycline residues in honey by a simple sensitive immunoassayrdquoAnalytica Chimica Acta vol 626 no 2 pp 180ndash185 2008

[19] A Gaurav J P S Gill R S Aulakh and J S Bedi ldquoELISA basedmonitoring and analysis of tetracycline residues in cattle milkin various districts of punjabrdquoVeterinaryWorld vol 7 no 1 pp26ndash29 2014

[20] M A Burkin and I A Galvidis ldquoImproved group determina-tion of tetracycline antibiotics in competitive enzyme-linkedimmunosorbent assayrdquo Food and Agricultural Immunology vol20 no 3 pp 245ndash252 2009

[21] S F Xu L Hou A J Wubie et al ldquoAnti-oxytetracycline mon-oclonal antibody based detection of oxytetracycline residue inhoneyrdquo Journal of Food Agriculture amp Environment vol 11 no2 pp 249ndash257 2013

[22] T Le H Yu X Wang B Ngom Y Guo and D Bi ldquoDevelop-ment and validation of an immunochromatographic test stripfor rapid detection of doxycycline residues in swine muscle andliverrdquo Food and Agricultural Immunology vol 22 no 3 pp 235ndash246 2011

[23] L A Semyonov O S Kuprienko I I Vashkevich et al ldquoModelsystems of indirect enzyme immunoassay tetracyclinesrdquo in Pro-ceedings of the 7th National Conference on Analytical Chemistrywith international participation (Analytics RB rsquo15) p 78 MinskBelarus 2015

[24] N A Byzova N I Smirnova A V Zherdev et al ldquoRapid immu-nochromatographic assay for ofloxacin in animal original food-stuffs using native antisera labeled by colloidal goldrdquo Talantavol 119 pp 125ndash132 2014

[25] J Gordon andGMichel ldquoAnalytical sensitivity limits for lateralflow immunoassaysrdquoClinical Chemistry vol 54 no 7 pp 1250ndash1251 2008

[26] C Parolo A de la Escosura-Muniz andAMerkoci ldquoEnhancedlateral flow immunoassay using gold nanoparticles loaded withenzymesrdquo Biosensors and Bioelectronics vol 40 no 1 pp 412ndash416 2013

[27] G T Hermanson Bioconjugate Techniques Academic PressNew York NY USA 2008

[28] N A Byzova E A Zvereva A V Zherdev S A Eremin and BB Dzantiev ldquoRapid pretreatment-free immunochromato-graphic assay of chloramphenicol in milkrdquo Talanta vol 81 no3 pp 843ndash848 2010

[29] B I Kurganov A V Lobanov I A Borisov and A N Resh-etilov ldquoCriterion for Hill equation validity for description ofbiosensor calibration curvesrdquo Analytica Chimica Acta vol 427no 1 pp 11ndash19 2001

[30] J Uhrovcık ldquoStrategy for determination of LOD and LOQvaluesmdashsome basic aspectsrdquo Talanta vol 119 pp 178ndash180 2014

[31] G Frens ldquoParticle size and sol stability in metal colloidsrdquoKolloid-Zeitschrift und Zeitschrift fur Polymere vol 250 no 7pp 736ndash741 1972

[32] I V Safenkova A V Zherdev and B B Dzantiev ldquoCorrelationbetween the composition of multivalent antibody conjugateswith colloidal gold nanoparticles and their affinityrdquo Journal ofImmunological Methods vol 357 no 1-2 pp 17ndash25 2010


[33] Z M Li Y Wang J Shen W Liu and X Sun ldquoThe measure-ment system of nanoparticle size distribution from dynamiclight scattering datardquo Optics and Lasers in Engineering vol 56pp 94ndash98 2014

[34] A Hoshino K Fujioka T Oku et al ldquoPhysicochemical prop-erties and cellular toxicity of nanocrystal quantum dots dependon their surface modificationrdquo Nano Letters vol 4 no 11 pp2163ndash2169 2004

[35] W Liu S CHak J P Zimmer E Tanaka J V Frangioni andMBawendi ldquoCompact cysteine-coated CdSe(ZnCdS) quantumdots for in vivo applicationsrdquo Journal of the American ChemicalSociety vol 129 no 47 pp 14530ndash14531 2007

[36] A M Derfus W C W Chan and S N Bhatia ldquoIntracellulardelivery of quantum dots for live cell labeling and organelletrackingrdquoAdvancedMaterials vol 16 no 12 pp 961ndash966 2004

[37] R Hidalgo-Alvarez Structure and Functional Properties of Col-loidal Systems CRC Press Boca Raton Fla USA 2009

[38] R S Wong and H Y Tse Lateral Flow Immunoassay HumanaPress New York NY USA 2009

[39] J R CrowtherTheELISAGuidebook Humana Press NewYorkNY USA 2001

[40] N A Byzova E A Zvereva A V Zherdev S A Eremin P GSveshnikov and B Dzantiev ldquoPretreatment-free immunochro-matographic assay for the detection of streptomycin and itsapplication to the control of milk and dairy productsrdquoAnalyticaChimica Acta vol 701 no 2 pp 209ndash217 2011

[41] S Zhang A Garcia-DrsquoAngeli J P Brennan and Q Huo ldquoPre-dicting detection limits of enzyme-linked immunosorbentassay (ELISA) and bioanalytical techniques in generalrdquoAnalystvol 139 no 2 pp 439ndash445 2014

[42] F Gao G X Zhao H C Zhang P Wang and J P Wang ldquoPro-duction ofmonoclonal antibody against doxycycline for immu-noassay of seven tetracyclines in bovine muscle and milkrdquoJournal of Environmental Science and Health Part B PesticidesFood Contaminants and Agricultural Wastes vol 48 no 2 pp92ndash100 2013

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy


Carbohydrate Chemistry

International Journal of


Journal of

Chemistry


Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of


Chromatography Research International


Applied ChemistryJournal of



Theoretical ChemistryJournal of


Journal of

Spectroscopy

Analytical ChemistryInternational Journal of


Journal of


Quantum Chemistry


Organic Chemistry International

ElectrochemistryInternational Journal of



CatalystsJournal of


NH2

CH3

CH3

H3CR1 R2HO N

OH

OH OHOH

OO O

Figure 1 The overall structure of tetracycline antibiotics tetracy-cline (R

1 H R

2 H) chlortetracycline (R

1 Cl R

2 H) oxytetracycline

(R1 H R

2 OH)

oxytetracycline chlortetracycline and their stereoisomers of100 pgg for meat 600 ngg for kidneys 200 ngg for eggsand 100 ngg for milk [9] Tetracycline is not allowed in somefood products (honey and baby food) that is the level oftetracycline should be below the detection threshold of therecommended analytical methods In the United States themaximum permissible level of tetracycline is 300 120583gg formeat 6 120583gg for liver and 12 120583gg for kidneys [10] In ChinaMRLs for the total content of tetracycline hydrochloride andoxytetracycline are 50 ngmL for milk 600 ngg for kidneys300 ngg for liver and 100 ngg for muscular tissue [11]

Low MRLs necessitate highly sensitive methods fordetecting antibiotics Control of TCs in food productsrequires rapid analysismethods and needs to be implementeddirectly at sampling points However stationary methods arestill predominant in the state-of-the-art analytical practice

Relatively simple and low-budget microbiological meth-ods are widely used to identify TC residues in foods [12 13]However they lack specificity (different classes of antibioticsare identified) and are time-consuming (15ndash3 h) Methodsbased on liquid chromatography are also used to identifyTCs [14 15] These methods are highly specific and highlysensitive but they are also time-consuming moreover theyrequire large amounts of solvent and an expensive specializedequipment base None of thesemethods allow field analysis tobe performed

Methods based on the use of specific cell receptors [16 17]including their rapid variants have gained widespread cur-rency because of the difficulty of obtaining antibodies specificto TCs TC receptor tests are characterized by their rapidity(10ndash20min) but the test system needs to be incubated in aconstant-temperature bath at 45∘C

A number of immunochemical detection methods forTCs have also been successfully developed Implementationof these methods does not require expensive equipment andis therefore of interest for mass contamination screeningThemost common methods of microplate enzyme immunoassay(ELISA) for TCs [18ndash20] are implemented in a laboratoryenvironment but with the use of relatively inexpensive andeasy to use instrumentation Unlike ELISA immunochro-matography (IC) test systems are nonlaboratory analyticaltools All processes in the IC test system are carried outwithout operator intervention or additional devices contactof the test strip with the sample initiates all subsequent

interactions There are several options for IC test systems toidentify tetracycline antibiotics based on the use of mono-clonal antibodies Existing test systems allow visual detectionof TCs at 30 ngmL and above [21 22] but this satisfies onlypart of the above regulations Therefore the creation of ahighly sensitive IC assay is an extremely necessary and urgenttask

Two approaches for lowering the limit of detection ofLFTS are implemented in this paper The first approach isbased on the coating gold nanoparticles (GNPs) by polyclonalantibodies allowing low content of specific antibodies againstTC in the conjugate [23] As we have previously shown[24] for such conjugates less antigen in the sample causessaturation of the antibody valency and binding inhibitionin the strip test zone The second approach is based onthe use of video digital detectors to register quantitativelydecrease of the label binding These detectors [25 26] allowthe quantization of the reduction in the intensity of colorationof the strip test zone and identification of the presence ofantigen in the sample for concentrations that do not causethe complete disappearance of coloration

Thus the aims of theworkwere to obtain and characterizeGNP conjugates with polyclonal antibodies against TC toapply the described above approaches for immunochro-matographic assay of TC to characterize sensitivity of theproduced test strips and to study their application for theidentification of TC in milk

2 Experimental

21 Chemicals Sodium azide bovine serum albumin (BSA)331015840551015840-tetramethylbenzidine (TMB) dimethyl sulfoxide(DMSO) formaldehyde Tween-20 and Triton X-100 wereobtained from Sigma (St Louis MO) Gold chloride andtetracycline hydrochloride (TC-HCl) were obtained fromFluka (Buchs Switzerland) Sephadex G-25 was obtainedfrom MP Biomedicals (Santa Ana CA) The preparationused in this work comprised the anti-TC rabbit sera andimmunoglobulin (IgG) fraction described in [23] Goatanti-mouse IgG antibodies were purchased from AristaBiologicals (Allentown PA) Peroxidase-labeled anti-mouseimmunoglobulins were from the Gamaleya Institute ofMicrobiology and Epidemiology Russia All other chemicals(salts and solvents of analytical grade) were from Chimmed(Moscow Russia)

All solutions for synthesis were prepared using waterthat was purified using Milli-Q system (Millipore Bed-ford MA httpwwwmilliporecom) Stock solutions of TC(1 120583gsdotmLminus1) were prepared in water and stored at minus20∘C

22 Tetracycline-BSA Conjugate Synthesis The procedure in[20] was amended and applied In this procedure 30mg ofBSA was dissolved in 20mL of H

2O and 26mg TC-HCl

solutionwas added to 200120583LDMSO and 120 120583L of 37 aque-ous formaldehyde solution (BSA molar ratio TC formalde-hyde = 1 120 3600) and stirred at room temperature over-nightThe product was purified from unreacted low-molecu-lar compounds by means of gel permeation chromatographyon the Sephadex G-25 carrier in phosphate-buffered saline






2SO4(50 120583L






520= 1) was






















119884 = ((119860 minus 119863)

(1 + (119909119862)119861)) + 119863 (1)





300 400 500 60000

02

04

06

08

10

12

14

16

18

20

1

2

3

120582 (nm)



119877 = (119909exp

119909theor) sdot 100 (2)








08

06

04

02

00

TC (ngmL)001 01 1 10 100 1000








0 20 40 60 80 100 120 140 160 180 200

0

5

10

15

20

25

Inte

nsity

()


1

23











0 2 4 6 8 10 12 14 16 18 20 22 24

018

020

022

024

026

028

030

032

034

036

038


A595






0 2 4 6 80

2000

4000

6000

8000

10000

12000

14000

Col

or in

tens

ity o

f tes

t zon

e (RU

)3 4

2

1

A520 of GN-AbsTC








OD520


00 02 04 06 08 101000

1500

2000

2500

3000

3500

4000

4500

Max

imum

colo

r int

ensit

y (R

U)

C (Tween-20) ()





580from 2


580=


20 40 60 80 1000

10

20

30

40

50

60

70

Sign

aln

oise

ratio












C (TC) (ngmL)0 005 01 05 1 10 100

(a)

Col

or in

tens

ity (R

U)

C (TC) (ngmL)1E minus 3 001 01 1 10 100 1000 10000

12000

10000

8000

6000

4000

2000

0

(b)



119884 = ((11640 minus 1588)

(1 + (119909036)07)) + 1588 (3)



4 Conclusions






Acknowledgments


References




















































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of






2SO4(50 120583L






520= 1) was






















119884 = ((119860 minus 119863)

(1 + (119909119862)119861)) + 119863 (1)





300 400 500 60000

02

04

06

08

10

12

14

16

18

20

1

2

3

120582 (nm)



119877 = (119909exp

119909theor) sdot 100 (2)








08

06

04

02

00

TC (ngmL)001 01 1 10 100 1000








0 20 40 60 80 100 120 140 160 180 200

0

5

10

15

20

25

Inte

nsity

()


1

23











0 2 4 6 8 10 12 14 16 18 20 22 24

018

020

022

024

026

028

030

032

034

036

038


A595






0 2 4 6 80

2000

4000

6000

8000

10000

12000

14000

Col

or in

tens

ity o

f tes

t zon

e (RU

)3 4

2

1

A520 of GN-AbsTC








OD520


00 02 04 06 08 101000

1500

2000

2500

3000

3500

4000

4500

Max

imum

colo

r int

ensit

y (R

U)

C (Tween-20) ()





580from 2


580=


20 40 60 80 1000

10

20

30

40

50

60

70

Sign

aln

oise

ratio












C (TC) (ngmL)0 005 01 05 1 10 100

(a)

Col

or in

tens

ity (R

U)

C (TC) (ngmL)1E minus 3 001 01 1 10 100 1000 10000

12000

10000

8000

6000

4000

2000

0

(b)



119884 = ((11640 minus 1588)

(1 + (119909036)07)) + 1588 (3)



4 Conclusions






Acknowledgments


References




















































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of







119884 = ((119860 minus 119863)

(1 + (119909119862)119861)) + 119863 (1)





300 400 500 60000

02

04

06

08

10

12

14

16

18

20

1

2

3

120582 (nm)



119877 = (119909exp

119909theor) sdot 100 (2)








08

06

04

02

00

TC (ngmL)001 01 1 10 100 1000








0 20 40 60 80 100 120 140 160 180 200

0

5

10

15

20

25

Inte

nsity

()


1

23











0 2 4 6 8 10 12 14 16 18 20 22 24

018

020

022

024

026

028

030

032

034

036

038


A595






0 2 4 6 80

2000

4000

6000

8000

10000

12000

14000

Col

or in

tens

ity o

f tes

t zon

e (RU

)3 4

2

1

A520 of GN-AbsTC








OD520


00 02 04 06 08 101000

1500

2000

2500

3000

3500

4000

4500

Max

imum

colo

r int

ensit

y (R

U)

C (Tween-20) ()





580from 2


580=


20 40 60 80 1000

10

20

30

40

50

60

70

Sign

aln

oise

ratio












C (TC) (ngmL)0 005 01 05 1 10 100

(a)

Col

or in

tens

ity (R

U)

C (TC) (ngmL)1E minus 3 001 01 1 10 100 1000 10000

12000

10000

8000

6000

4000

2000

0

(b)



119884 = ((11640 minus 1588)

(1 + (119909036)07)) + 1588 (3)



4 Conclusions






Acknowledgments


References




















































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


08

06

04

02

00

TC (ngmL)001 01 1 10 100 1000








0 20 40 60 80 100 120 140 160 180 200

0

5

10

15

20

25

Inte

nsity

()


1

23











0 2 4 6 8 10 12 14 16 18 20 22 24

018

020

022

024

026

028

030

032

034

036

038


A595






0 2 4 6 80

2000

4000

6000

8000

10000

12000

14000

Col

or in

tens

ity o

f tes

t zon

e (RU

)3 4

2

1

A520 of GN-AbsTC








OD520


00 02 04 06 08 101000

1500

2000

2500

3000

3500

4000

4500

Max

imum

colo

r int

ensit

y (R

U)

C (Tween-20) ()





580from 2


580=


20 40 60 80 1000

10

20

30

40

50

60

70

Sign

aln

oise

ratio












C (TC) (ngmL)0 005 01 05 1 10 100

(a)

Col

or in

tens

ity (R

U)

C (TC) (ngmL)1E minus 3 001 01 1 10 100 1000 10000

12000

10000

8000

6000

4000

2000

0

(b)



119884 = ((11640 minus 1588)

(1 + (119909036)07)) + 1588 (3)



4 Conclusions






Acknowledgments


References




















































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of




0 2 4 6 8 10 12 14 16 18 20 22 24

018

020

022

024

026

028

030

032

034

036

038


A595






0 2 4 6 80

2000

4000

6000

8000

10000

12000

14000

Col

or in

tens

ity o

f tes

t zon

e (RU

)3 4

2

1

A520 of GN-AbsTC








OD520


00 02 04 06 08 101000

1500

2000

2500

3000

3500

4000

4500

Max

imum

colo

r int

ensit

y (R

U)

C (Tween-20) ()





580from 2


580=


20 40 60 80 1000

10

20

30

40

50

60

70

Sign

aln

oise

ratio












C (TC) (ngmL)0 005 01 05 1 10 100

(a)

Col

or in

tens

ity (R

U)

C (TC) (ngmL)1E minus 3 001 01 1 10 100 1000 10000

12000

10000

8000

6000

4000

2000

0

(b)



119884 = ((11640 minus 1588)

(1 + (119909036)07)) + 1588 (3)



4 Conclusions






Acknowledgments


References




















































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of



OD520


00 02 04 06 08 101000

1500

2000

2500

3000

3500

4000

4500

Max

imum

colo

r int

ensit

y (R

U)

C (Tween-20) ()





580from 2


580=


20 40 60 80 1000

10

20

30

40

50

60

70

Sign

aln

oise

ratio












C (TC) (ngmL)0 005 01 05 1 10 100

(a)

Col

or in

tens

ity (R

U)

C (TC) (ngmL)1E minus 3 001 01 1 10 100 1000 10000

12000

10000

8000

6000

4000

2000

0

(b)



119884 = ((11640 minus 1588)

(1 + (119909036)07)) + 1588 (3)



4 Conclusions






Acknowledgments


References




















































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of





C (TC) (ngmL)0 005 01 05 1 10 100

(a)

Col

or in

tens

ity (R

U)

C (TC) (ngmL)1E minus 3 001 01 1 10 100 1000 10000

12000

10000

8000

6000

4000

2000

0

(b)



119884 = ((11640 minus 1588)

(1 + (119909036)07)) + 1588 (3)



4 Conclusions






Acknowledgments


References




















































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of




Acknowledgments


References




















































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of





















Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of

research article highly sensitive …downloads.hindawi.com/journals/ijac/2015/347621.pdfhighly...

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