/ac Naked Eye Detection of Glucose in Urine Using Glucose Oxidase Immobilized Gold NanoparticlesChangerath Radhakumary and Kunnatheeri Sreenivasan*Laboratory for Polymer Analysis,Biomedical Technology Wing,Sree Chitra Tirunal Institute for Medical Sciences and Technology, Thiruvananthapuram695012,Indiab Supporting InformationC hemical sensors are miniaturized devices that can deliverreal-time and online information on the presence of specific compounds or ions in complex matrixes.1Metallic nanoparticle based sensing has emerged as an important colorimetric tool for the detection of biomolecules linked to the onset of diseases to aid in early diagnosis.Among them,colloidal gold is extensively used for molecular sensing due to the wide opportunities it offers in the design of easy to perform methods.2The synthesis of gold colloid with an organic monolayer in a one-step process is also exploited, and this monolayer provides the extraordinary stability to the nanoparticles along with the additional surface properties.3À8 Optical sensing based on the plasmon resonance absorption exhibited by nanoparticles has been used with a view to develop analytical tools in clinical diagnosis.9À11It is well-known that the gold nanoparticles display surface plasmon resonance(SPR) absorption bands at a specific wavelength(∼519nm).The frequency and width of the SPR absorption depend on the size and shape of the metal nanoparticle as well as on the dielectric constant of the metal itself and of the medium surrounding it.12 On aggregation of the gold nanoparticles,the absorption maxima shifts to longer wavelengths resulting in the color change of the colloid from wine red to blue due to mutually induced dipoles that depend on interparticle distance and aggregate size.13In the present study,we use functionalized gold nanoparticles for urine glucose sensing.We focus on urine because it is the most informativefluid that can be obtained noninvasively and is used routinely to diagnose and monitor a variety of medical conditions.14The glucose level in blood is used as a clinical indicator of diabetes.The presence of glucose in urine is a more dangerous condition,as it is an indication of worsening of diabetes. According to the World Health Organization,over150million people in the world were affected with diabetes in the year2004 and it is expected to climb further to366million in2030.The affected population has to be tested for blood glucose levels daily for an effective treatment.In order to avoid the inconveniences caused by drawing blood intravenously or by hand pricking,a preliminary screening of the patients with high level diabetes (having renal glycosuria)15,16can be done instantly by checking their urine glucose levels.The visible color change of the functionalized gold nanopar-ticles on interacting with urine glucose enable the patients to have a self-checking method at home and seek immediate medical attention.Recently,Malhotra et al.and Li et al.have studied in length the characteristics of glucose oxidase(GOD) immobilized GNPs.17,18These authors have shown enhanced stability of GOD by the process of immobilization.Ma and Ding have reported that,while free GOD in solution only retains about 22%of its relative activity at90°C,the immobilized GOD on gold nanorods retains about39.3%activity.19Li et al.also suggested that such GOD/GNPs bioconjugates can be consid-ered as a catalytic nanodevice to construct a nanoreactor basedReceived:December18,2010Accepted:February25,2011inaccessible to the bulk of the population.on a glucose oxidation reaction for a biotechnological purpose.18 Interestingly,the feasibility of the use of these entities in the sensing of glucose has not been attempted.Zhang et al.have reported the use of modified glassy carbon electrode for the application of glucose sensing.The GNPs was deposited on the electrode surface through the glucose oxidase catalyzed oxidation of glucose which in turn was measured by differential pulse stripping voltammetry.20Very recently,Jiang et al.have demon-strated the application of a AuNP based colorimetric assay for the simple but effective detection of glucose in the rat brain by taking advantage of the cascade reactions of GOD catalyzed oxidation of glucose and the Fenton reaction of H2O2,as well as the oxidative cleavage of ssDNA with OH radicals.21There has been an ever increasing demand for the develop-ment of simple,cost-effective methodologies in an easy to read out format for the detection of clinically relevant molecules to aid in diagnosis.Such approaches are extremely important,particu-larly in third world countries where high tech diagnostics aids are inaccessible to bulk of the population.14Simple procedures which could be performed at home without the need of sophisticated expensive instrumentation possibly bring radical changes in rural health care management.Visual detection of disease specific marker molecules in biologicalfluids such as urine,saliva,or blood is an attractive approach to address these issues.A color change observable by the naked eye in response to the concentration of an analyte can be an indication of a disease condition warranting further medical attention.Herein,we report conjugation of GOD,the enzyme specific toβ-D glucose onto thiol capped GNPs,using carbodiimide chemistry and its use in the colorimetric detection of glucose in urine.’EXPERIMENTAL SECTIONMaterials.Gold chloride,glucose oxidase(GOD),trisodium citrate,Tween20,16-mercaptohexadecanoic acid(16-MHDA), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide(EDC),and N-hydroxysuccinimide(NHS)were obtained from Sigma-Al-drich,Bangalore,India.The glucose standard used is from the glucose kit supplied by Enzyme Technologies Pvt.Ltd., Mumbai,India.Preparation of Gold Nanoparticles.GNPs were synthesized as reported by Turkevich et al.22Briefly,to a boiling solution of 20mL of1.0mM HAuCl4,2mL of a1%solution of trisodium citrate dihydrate was added under constant stirring.The contents were removed from the hot plate when the solution turned red. The cooled contents were kept under refrigeration until its usage.Preparation of Alkane Thiol Modified Gold Colloids.The surface modification of gold colloids using alkane thiols was done as per Aslan and Luna.2Briefly,equal volumes of gold colloid and Tween20(2mg/mL)in phosphate buffer at pH7.0were gently mixed and allowed to stand for a minimum of20min to allow for the physisorption of Tween20to the gold nanoparticles.Then,a solution of0.5mM16-MHDA in methanol was added to the above solution and allowed to stand for4h.Excess Tween20and thiols were removed from the surface modified gold colloids by centrifugation for20min at14000rpm with an ultracentrifuge (Sigma3À30K,Germany).The absorbance spectra of the colloidal gold and the modified colloids with Tween20,16-MHDA was taken with a UVÀvisible spectrophotometer (Varian,Cary-Win Bio,Melbourne,Australia)using1cm path length polystyrene cuvettes.Conjugation of GOD on Alkanethiol Modified Gold Col-loids.Immobilization of GOD with16-MHDA capped gold nanoparticles was done on the basis of the EDC/NHS chemistryvia the formation of an amide linkage between the carboxyl groups of the16-MHDA and the primary amine groups of the GOD.The thiolated gold colloids(3mL)were activated with 5mL of a mixture of EDC and NHS,both10mM in phosphate buffer at pH7.0by incubating the mixture at room temperature for30min.Then,2mL of GOD solution(1mg/mL in phosphate buffer)was added to the above mixture and incubated at room temperature for2h and then kept overnight at4°C. After centrifugation at14000rpm,the GOD immobilized gold nanoparticles were resuspended in3mL of phosphate buffer(pH 7.0)and kept refrigerated until use.Fourier Transform Infrared Spectroscopy(FT-IR)of the Nanoparticles.The FT-IR spectra of the citrate stabilized GNPs and GOD capped GNPs were recorded in the range of 600À4000cmÀ1on a Nicolet5700FT-IR spectrometer (Nicolet Inc.,Madison,USA)using a Diamond ATR accessory.Particle Size and Zeta Potential Determination.The tech-nique of dynamic light scattering(DLS;Malvern Instruments Ltd.,Malvern,UK)was used for the determination of the size of the nanoparticles.All measurements were performed at a fixed angle of90°at25°C.The zeta potential of a particle is the overall charge that the particle acquires in a particular medium and is also measured using the same equipment at25°C.Transmission Electron Microscopy.Transmission electron microscopy(TEM)images were obtained on a Hitachi,H7650 microscope(Tokyo,Japan).The gold colloid and the GOD capped gold colloid were deposited onto a200mesh copper grid coated with a Formvar film and dried overnight.Glucose Sensing Using GOD Functionalized Gold Col-loids.The shift in the SPR absorption maxima of the GOD functionalized GNPs on adding predetermined quantities of glucose standard is recorded using a UVÀvisible absorption spectrophotometer,and a calibration curve was plotted with the wavelength against the optical density.The urine samples,which did not contain glucose,were collected from a clinical laboratory. These fluids were spiked with glucose and used as the test samples.The corresponding shift in the absorption maxima was used to calculate the amount of glucose present.Selectivity of the Method.The selectivity of the method was investigated by checking the shift in SPR absorption maxima of the GOD functionalized GNPs on interacting with normal urine samples and the same spiked with albumin/creatinine in the ratio of40mg/mM and cysteine200μM/L.To confirm the precision and recovery of the probe,each set of experiments was carried out in triplicate,and similar results within the maximum error of 2À3%were obtained.’RESULTS AND DISCUSSIONThe surface plasmon resonance makes the absorption cross section of the nanoparticles several orders of magnitude stronger than the most strongly absorbing molecules and the light scattering cross section several orders of magnitude stronger than the organic dyes.23Hence,most of the applications of gold nanoparticles as sensors are based on detecting the shift in surface plasmon peak either due to change in the local dielectric constant of the nanoparticles by adsorbed biomolecules or due to analyte induced aggregation of the nanoparticles.24À29Both these effects rely on the selectivity provided by the functionalized capping agents.Functionalization of GNPs.The typical plasmon absorptionpeak of the gold colloid was observed at 519nm (Supporting Information,Figure S1),indicating the formation of spherical GNPs.30,31This is further substantiated in Figure S2(Supporting Information)which shows the TEM images of the GNPs at a magnification of 500nm,and the nanoparticles displayed an average size of 11to 12nm.After adding Tween 20,the SPR absorption maximum of the gold colloid was shifted to 523(1nm due to the physical adsorption of the surfactant on the GNPs and was consistent with the reported shifts of the band upon formation of dielectric layers around colloidal metals.2The absorption maxima further shifted to 524(1nm upon chemisorption of 16-MHDA,indicating the formation of a thicker monolayer around the nan-oparticles,as reported by Aslan etal.2The presence of a physisorbed layer of Tween 20on the surface of colloidal gold could prevent irreversible aggregation of gold nanoparticles during and after chemisorption of alkane thiols.2On conjugating the ÀCOOH group of the 16-MHDA with the amino group of GOD,the SPR was further red-shifted to 534(1nm.The spectral shift is not accompanied by any broadening,confirming nonaggregation of the particles at this stage.Fourier Transform Infrared Spectroscopy (FT-IR).We re-corded FT-IR spectra of the nanoparticles to get further insight on the surface modification (Supporting Information,Figure S3).Citrate stabilized GNPs showed peaks at 1509and 1399cm À1,characteristic of citrate ions.GOD conjugated GNPs showed intense peaks at 1654and 1549cm À1,typical of amide I (ÀC d O)and amide II (N ÀH bending)bands of the enzyme and a strong ÀC ÀO Àstretching band at 1074cm À1.The peak at 3278cm À1was assigned the N ÀH/O ÀH stretching frequency of the GOD.Effect of Glucose on the SPR Absorption Maximum of the GOD Conjugated GNPs.On adding varied amount of glucose (from 10to 100μg/mL),the SPR absorption band was found to shift from 535to 569nm,reflecting the formation of aggregates having enhanced sizes.The reaction is instant,and no incubation time is required between glucose and GOD conjugated GNPs.The corresponding spectra are shown in Figure 1.The red shift in the plasmon peak was found to vary almost linearly with the concentration of glucose,suggesting the possi-bility of the use of this methodology for the quantitative estimation(inset in Figure 1).The magnitude of wavelength shift was con-comitantly increased with concentration of glucose,and beyond 90μg/mL,it is leveled o ff,indicating the saturation of the reactive sites.The lowest amount of glucose that can produce a red shift in the SPR absorption maxima was found to be 10À90μg/mL with a detection limit of 5μg/mL.Particle Size and Zeta Potential Determinations.The particle sizes and zeta potentials of the GNPs before and after the addition of glucose (50and 100μg/mL)are given in Table 1.The average particle size of GNPs was 22.5(0.7nm,and the same was increased to 158nm on conjugating with GOD.On adding 50μg/mL glucose,the particles size was increased to 231.7nm and the size was enlarged to 1202nm on the addition of 100μg/mL glucose,showing the tendency toward the formation of aggregates.The formation of aggregated assembly is reflected again in the color change of the solution from red to blue (Figure2).As shown in Table 1,the zeta potential of the GNPs was reduced from À48(0.41mV to À21.4mV,on conjugating with GOD.When 50μg/mL glucose was added to GOD functionalized GNPs,the charge was reduced to À14.1mV and subsequently to À5.85mV on the addition of 100μg/mL glucose.The considerable reduction in zeta potential also indicates the tendency for the formation of aggregates.GOD oxidizes glucose to gluconic acid and H 2O 2.H þions resulted from the formation of gluconic acid could reduce anionic character of the enzyme,bringing down the negative zeta potential.At acidic pH,nanoparticles have shown less zeta potential due to the increased chemical potential of H þions.32The magnitude of the measured zeta potential is an indication of the net charge over the nanoparticles and can be used toFigure 1.UV Àvisible absorption spectra of GOD conjugated GNP showing red shift on reacting with di fferent quantities of glucose standard.(Inset is the graphical relationship of wavelength shift against increasing quantities of glucose.)Table 1.Size and Charge of the Nanoparticlessampleparticle size (nm)zeta potential (mV)gold nanoparticles 22.5(0.7À48(0.41Au-GOD158(2À21.4(0.1Au-GOD-glucose 50μg/mL 231.7(9À14.1(0.3Au-GOD-glucose 100μg/mL1202(26À5.9(0.5Figure 2.Color of (A)GNPs and (B)GOD-GNP on reacting with g 100μg/mL glucose.predict the long-term stability of the particles.If all the particles in suspension have either a large negative or a positive zetapotential,there will not be any tendency for them to come closer due to strong repulsion.33However,if the particles have low zeta potential values,the probability of them coming together to form an aggregate is higher.The GOD functionalized GNPs synthe-sized in our laboratory did not show any sign of aggregation even after 2months under refrigerated conditions.Transmission Electron Microscopic (TEM)Analysis.We measured the size of the particles using TEM.Figure 3A shows the size of the GOD conjugated GNPs as 44À47nm.Figure 3B ÀD displays the sizes of the GOD functionalized GNPs on interaction with 5,100,and 150μg/mL glucose,respectively.The size of the nanoparticles increased on adding increasing quantities of glucose.When 50μg/mL glucose was added,the size was increased to 73À75nm;with 100μg/mL glucose,it became 102to 145nm,and a total aggregate was formed on adding 150μg/mL glucose.TEM,however,showed lower values for the size of the nanoparticles compared to DLS measurements (Table 1).DLS measurement records higher values since the light is scattered by the core particle and the layers formed on the surface of the particles.TEM,on the other hand,shows the size of metallic core only.He et al.have also made similar observations earlier.34Glucose induced aggregation of GOD conjugated GNPs leading to color change is well supported by the absorption,particle size measurements,and TEM investigations.Glucose Sensing in Urine Samples.We spiked a urine sample with glucose (50μg/mL),and the absorption peaks are shown in Figure 4.The peak maximum showed a shift of 21nmexactly as in the case of the aqueous standard sample (see Figure 1),demonstrating the feasibility of extending the method for the estimation of glucose in real samples.Rather than quantitative estimation,it seems that the method provides a quick qualitative screening of samples.It is apparent from Figure 2that a concentration of 100μg/mL glucose showed a visible color change of the solution to conclude that the glucose content of the test solution is g 100μg/rmation of this kind could be used for further assessment of the health status of the individual.The optical detection method discussed here may be useful for rural populations where clinical laboratory facilities are limited.Figure 3.TEM images of (A)GOD conjugated GNP,(B)50μg/mL glucose added,(C)100μg/mL glucose added,and (D)150μg/mL glucose added.Figure 4.Wavelength shift with a urine sample spiked with 50μg/mL glucose.导致纳米金聚集的原因Selectivity of the Method.To check the cross reactivity of the GOD functionalized GNPs,we conducted the following studies.Diabetic patients are at increased risk of renal diseases. The principal feature of diabetic nephropathy is proteinuria which is defined as the albumin/creatinine ratio being g30 mg/mmol.35We spiked the urine sample with100μL of aqueous solution containing albumin/creatinine in the ratio40mg/mM to mimic the urine samples of patients with diabetic nephro-pathy.It is also reported that the concentration of cysteine in urine is200μM/L.36A urine sample(100μL)spiked with cysteine is also added to Au-GOD to check its selectivity toward glucose.The SPR absorption maximum of the Au-GOD shifted only on adding50μL of100mg/dL glucose standard.No red shift for albumin/creatinine or cysteine was found,confirming that the GOD functionalized GNPs were highly specific to glucose and had no interference with any other molecules present in the urine.The SPR absorption pattern and the corresponding color images in the inset are shown in Figure S4 (Supporting Information).’CONCLUSIONSWe devised a simple method for the detection of glucose in fluids like urine using GOD immobilized gold nanoparticles.The color of the solution was found to change from red to blue in the presence of∼100μg/mL glucose.Plasmon absorption peak was red-shifted proportionally up to100μg/mL glucose,and good correlation was obtained between the wavelength shift and concentration pointing out the feasibility of the use of the method for the quantitative measurement of glucose in urine. The color change observable by the naked eye can advanta-geously be used for a preliminary screening at home itself and may be referred for more focused investigations.The method seems to be a potential one,particularly for the rural population in third world countries.’ASSOCIATED CONTENTb Supporting Information.Additional information as noted in text.This material is available free of charge via the Internet at .’AUTHOR INFORMATIONCorresponding Author*E-mail:sreeni@sctimst.ac.in.Phone:091À471À2520248.Fax: 091À471À2341814.’ACKNOWLEDGMENTWe are grateful to the Director of SCTIMST and Head of the Biomedical Technology Wing,SCTIMST,for providing all the facilities for conducting this study.We are also thankful to Ms.S. 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