Cao-2015-Protein-inorganic hy

Protein-inorganic hybrid nanoflowers as ultrasensitive electrochemical cytosensing Interfaces for evaluation of cell surface sialic acidHongmei Cao a,Da-Peng Yang b,n,Daixin Ye a,Xianxia Zhang a,Xueen Fang a,Song Zhang a,Baohong Liu a,Jilie Kong a,na Department of Chemistry and Institute of Biomedical Sciences,Fudan University,Shanghai200433,PR Chinab College of Chemistry and Life Science,Quanzhou Normal University,Quanzhou,PR Chinaa r t i c l e i n f oArticle history:Received9October2014Received in revised form22December2014Accepted2January2015Available online3January2015Keywords:Electrochemical cytosensorsBovine serum albumin(BSA)incorporatedAg nanoflowersSambucus nigra agglutinin(SNA)DLD-1cellsSialic acida b s t r a c tThe identification of biocompatible nanomaterials with high conductivities as sensing interfaces is im-portant in developing novel electrochemical cytosensors.We prepared a novel protein-inorganic nano-material-bovine serum albumin(BSA)incorporated Ag nanoflowers with three-dimensional porous ar-chitectures,using a simple biomimetic method.The BSA-incorporated Ag nanoflowers were modified ona glassy carbon electrode(GCE)surface and conjugated with a targeting lectin molecule,i.e.,Sambucusnigra agglutinin(SNA),for sensing DLD-1human colon cancer cells.The BSA-incorporated Ag nano-flowers were a suitable platform,and showed improved cell-immobilization capacity,and good bio-compatibility,with retention of activity of the immobilized cells.These properties are attributed to thelarge surface area of the porous structure and the natural BSA layer acting as a biocompatible support.The attachment of DLD-1cells to the GCE increased the electron-transfer resistance,with a good cor-relation with the logarithm of the concentration from1.35Â102to1.35Â107cells mLÀ1,with a lowdetection limit of40cells mLÀ1.Based on the affinity between SNA and sialic acid(SA),the UV–visabsorption spectrum of the one-step reaction between SA and acidic ninhydrin indicated that theaverage number of SA molecules on a single living DLD-1cell surface was approximately2.16Â1012.Thisproposed cytosensing strategy had good reproducibility,acceptable precision,and high specificity for SA-over-expressed cells,indicating that it has potential applications for the early monitoring of tumor cellsand convenient evaluation of SA on living cells.&2015Elsevier B.V.All rights reserved.1.IntroductionGlycoproteins on cell surfaces play essential roles in a widevariety of cell activities,including cell proliferation and differ-entiation,cell–cell communication,and immune response mod-ulation(Raman et al.,2005).Sialic acid(SA),a clinically approvedtumor marker,is a glycoprotein containing a nine-carbon back-bone,and is commonly found at the terminal positions of sugarchains(Liu et al.,2011;Matsumoto et al.,2009).Accumulatingevidence shows that over-expression of SA on cell surfaces isclosely associated with the malignant and metastatic phenotypesof many cancers such as colorectal,breast,liver,and ovarian can-cers(Ang et al.,2006;Chen et al.,2007a;Wang et al.,2003;Zhanget al.,2010).Therefore,the study of SA on the cell surface is criticalboth to understand its role in disease development and to provideeffective diagnostic tools to help prompt therapy.Currently,there are various methods for SA analysis,includingfluorescence analysis,mass spectrometry(MS),and liquid chro-matography(LC)(Shen et al.,2007;Wang et al.,2014a,2014b;Xiong et al.,2013).However,these techniques need long analyticaltimes and stringent laboratory conditions,and are not suitable forliving cell analysis.The design and development of rapid,non-invasive,highly sensitive,and specific techniques for evaluating SAexpression level are therefore important.In recent years,electro-chemical impedance spectroscopy(EIS)has become a popularapproach in a broad range offields,including semiconductorbatteries,corrosion,fuel cells,and biosensors(Chang and Park,2010;Sherif and Park,2006;Venkatanarayanan et al.,2013;Wanget al.,2014c).In particular,EIS has been widely applied in livingcell measurements;for example,Zhu et al.reported a highlysensitive impedance sensor for HL-60cells,based on carboxylicsingle-walled carbon nanotube/Au composites(Zhang et al.,2009),Ju et ed electrochemical impedance detection of K562leu-kemia cells(Ding et al.,2007),and Wang et al.prepared folateconjugated-polyethylenimine carbon nanotubes for HeLa cell de-tection using EIS(Wang et al.,2013).Thus,we intend to fabricateEIS cytosensor to evaluate SA expression level of cell membrane.Contents lists available at ScienceDirectjournal homepage:/locate/biosBiosensors and Bioelectronics/10.1016/j.bios.2015.01.0030956-5663/&2015Elsevier B.V.All rightsreserved.n Corresponding authors.Biosensors and Bioelectronics68(2015)329–335However,interfacial effects play a key role in EIS measurements, therefore selecting appropriate and biocompatible interface ma-terials is crucial in fabricating EIS cytosensors.Moreover,cell im-mobilization based on nanomaterials with good biocompatibility also provides a favorable platform for evaluating SA expression on the living cell surface.Recently,protein-mediated synthesis of inorganic nanomater-ials for the sensing interfaces of electrochemical cytosensors is attractive for a number of reasons:the synthetic conditions are mild,i.e.,near room temperature,in aqueous solutions,and at neutral pH;the sizes,shapes,morphologies,and crystal structures are complex and controllable;and the resulting products are multifunctional and biocompatible.In our previous work,we synthesized a series of metal@protein nanocomposites via a pro-tein-mediated method,and used them as sensing platforms for the sensitive detection of cancer cells(Hu et al.,2013a,2013b).These hybrid nanocomposites have a range of advantages such as ex-cellent conductivities,large surface areas,and nontoxicity.Con-sidering the effects of the substrate morphology and structure on the cytosensor performance,we preparedflower-like Ag nano-materials using bovine serum albumin(BSA)as a -pared with previously reported Ag@BSA microspheres(Huang et al.,2011),the BSA-incorporated Ag nanoflowers have larger surface areas and open nanoporous structures,providing an elec-trochemical sensing matrix that is better for cytosensor fabrication for the following reasons.First,the three-dimensional(3D)porous architecture promotes cell anchoring and increases the number of cells that adhere to the substrate.Secondly,the incorporated BSA molecules provide a multifunctional interface for conjugation of targeting molecules,improving the water solubility and main-taining the biomolecule bioactivity and reducing nonspecific in-teractions.Finally,Ag has excellent electrical conductivity and can amplify the electrochemical signal,improving the cytosensing ability.Because of these advantages,BSA-incorporated Ag nano-flowers are expected to provide an effective sensing platform for monitoring SA change on tumor cells surface.However,the de-signed substrate should achieve specific target-cell recognition, while preventing nonspecific adhesion of non-target cells,there-fore modification of specific recognition molecules is required. Sambucus nigra agglutinin(SNA)with a highly specific binding affinity with SA(Chen et al.,2007a)is an excellent candidate for biosensor fabrication,because of its ease of production and la-beling,and good stability during long-term storage(Cho et al., 2014).Herein,we report the synthesis of novel BSA-incorporated Ag nanoflowers as biomimetic3D nanoporous sensing interfaces for electrochemical measurements of tumor cells and SA on the DLD-1cells surface.Human colon cancer DLD-1cells,which have SA expression patterns on the cellular surface,were used as model SA-over-expressed cells.Lectin(SNA),which was successfully conjugated with the sensing platform using glutaraldehyde(GA), was employed as the targeting molecule for specifically re-cognizing SA groups on cell surfaces.The DLD-1cells were cap-tured on the cytosensor surface by specific binding between the SA groups on the cell surfaces and SNA,i.e.,the amount of cells captured on the cytosensor surface was related to the expression of SA groups on the cell surfaces.On the basis of this,we devel-oped a novel approach for conveniently and sensitively quantify-ing the SA expression value on the DLD-1cell surface by com-bining a simple UV–vis measurement with one-step reaction. Owning to achieving highly specific recognition and utilizing ul-trasensitive interfaces,the proposed cytosensor had a broad de-tection range,with a lower detection limit,than other reported electrochemical cytosensors.Our proposed EIS cytosensing therefore offers a potential protocol for tumor cells detection and quantitative evaluation of the cell surface SA in clinical diagnosis.Furthermore,our results highlight the importance of nano-bio interfaces in optimizing biosensor performances.2.Experimental2.1.Chemicals and materialsAgNO3,ascorbic acid,and ethanol GA solution(25%)were purchased from the Sinopharm Chemical Reagent Co.,Ltd.(China). SNA,3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-mide(MTT,ultrapure grade),lyophilized99%BSA(molecular mass ∼68kDa),and dimethyl sulfoxide(DMSO)were acquired from the Sigma-Aldrich Corporation.A10mM phosphate buffer solution (PBS,pH7.4)was used as the rinsing solution and SNA diluent.A [Fe(CN)6]3À/4Àsolution containing10mM K3Fe(CN)6,10mM K4Fe (CN)6,and0.1M KCl(as the supporting electrolyte)was prepared as a redox probe for the measuring system.Doubly distilled water was used in all experiments.All reagents were analytical grade and used as received,without further purification.2.2.Apparatus and measurementsThe morphologies of the BSA-incorporated Ag nanoflowers were investigated using transmission electron microscopy(TEM; JEOL2011)andfield-emission scanning electron microscopy (FESEM;Philips XL30,the Netherlands),at accelerating voltages of 200and10kV,respectively.EIS was performed,using an Autolab PGSTST30analyzer(Metrohm Autolab BV,Switzerland),in0.1M KCl solution containing10mM[Fe(CN)6]3À/4À(1:1)as the redox probe,at an open-circuit potential.The impedance spectra were recorded in the frequency range10À1–105Hz,with a signal am-plitude of5mV.Cyclic voltammetry(CV)was performed using a CHI1030electrochemical workstation with a conventional three-electrode system.A modified electrode was used as the working electrode,with a platinum wire as the auxiliary electrode and a saturated calomel electrode as the reference.Zeta potential was measured in Zeta potential analyzer(Malvern Instruments ZS90). MTT assays were performed using an enzyme-labeled microplate reader(SUNOSTIK SPR-960).2.3.Cell culture and collectionHuman colon cancer DLD-1cells were purchased from the Cell Bank of the Chinese Academy of Sciences.The DLD-1cells were cultured in RPMI1640medium supplemented with10%fetal bo-vine serum(FBS),and incubated at37°C in a humidified atmo-sphere containing5%CO2(Matsumoto et al.,2005).The DLD-1 cells were trypsinized for2–3d in0.25%trypsin solution,collected from the culture medium by centrifugation at1000rpm for5min, and then washed twice with1ÂPBS(pH7.4).The sediment was resuspended in the1ÂPBS to obtain a0.5mL homogeneous cell suspension.Cell suspensions of different concentrations were prepared from this stock.The cell numbers were determined usinga Petroff–Hausser counter.2.4.Fabrication of BSA-incorporated Ag nanoflowers/GA-modified electrochemical cytosensorThe method for synthesizing the sensingfilm(BSA-in-corporated Ag nanoflowers)is key to our research.Briefly,BSA (5mg mLÀ1,10mL)and AgNO3(10mM,10mL)aqueous solutions were mixed in a50mL beaker.The mixture was stirred at room temperature for10min,and then the solution was placed in a water-bath at55°C for5min.Ascorbic acid(50mg)was then rapidly added to the solution.The mixture was kept at55°C forH.Cao et al./Biosensors and Bioelectronics68(2015)329–335 3305min.Magnetic stirring was used throughout the process.The resulting products were collected and washed three times with water and ethanol,respectively.The final precipitate was redis-persed in deionized water for subsequent use.The assembly and recognition process of the electrochemical cytosensor based on BSA-incorporated Ag nano flowers is shown in Scheme 1.Glassy carbon electrodes (GCEs,diameter 3.0mm)were used as the substrates for modi fication with the prepared BSA-incorporated Ag nano flowers.Prior to modi fication,the GCEs were polished to a mirror-like appearance with 1.0,0.3,and 0.05μM αalumina powder.The electrodes were successively sonicated in ethanol and doubly distilled water,and then allowed to dry in a stream of nitrogen.BSA-incorporated Ag nano flower solution (0.625mg/ml,3μL)was dropped onto the pretreated GCE surface,and the GCE was kept in a refrigerator (4°C)for 2h.To attach the targeting molecules,i.e.,SNA,freshly prepared GA solution (25%,2μL)was added to the substrate,followed by incubation for 1.5h at room temperature.Herein a high molar ratio of GA was used to ensure that only one aldehyde group of GA reacted with the amino groups of the BSA-incorporated Ag nano flowers.After the reaction,the electrode was washed with PBS (pH 7.4)and then the modi fied electrode surface was coated with SNA solution (3μL)and in-cubated at 4°C under a wettish atmosphere for at least 4h to prepare the SNA-based biosensor.Finally,above modi fied elec-trode was then rinsed thoroughly with PBS (pH 7.4)to remove nonspeci fic bound species.In the process of preparation,the so-lutions of SNA and BSA-incorporated Ag nano flowers reacted with GA,forming a Schiff base structure (Cozzi,2004;Olde Damink et al.,1995),according to the following equation:SNA –NH 2þBSA –NH 2þO ¼CH(CH 2)3HC ¼O -BSA –N ¼CH (CH 2)3HC ¼N –SNA þ2H 2OThe cytosensor exploited the signal ampli fication effect of the BSA-incorporated Ag nano flowers for the mass loading of SNA,to achieve highly sensitive recognition of cell surface SA by lectin SNA.EIS was used to record the electrochemical response of the [Fe(CN)6]3À/4Àprobe.The electron-transfer resistance (R et )re-flected the amount of cells captured by the cytosensor,providing a potential strategy for the analysis of SA expression in living cells.2.5.MTT assayFirst,DLD-1cells were cultured in RPMI 1640supplemented with 10%FBS.The cells were incubated for 2–3d in a humidi fied 5%-CO 2-containing balanced air incubator at 37°C.Cells (100μL),at a density of 1Â105cells/well,were seeded in each well of a 96-well plate.The cells were incubated for 24and 48h at 37°C,re-spectively.The medium was then removed and the cells were in-cubated with different concentrations of BSA-incorporated Agnano flowers.The controls contained only cells.The medium con-taining the matrix was removed,and 20μL of a stock solution containing 5mg mL À1of MTT were added,followed by incubation for 4h.Finally,an MTT-solubilizing agent (150μL,DMSO)was added,and the mixture was shaken thoroughly for 15min (Zhang et al.,2013).3.Results and discussion3.1.Characterization of BSA-incorporated Ag nano flower sensing layerThe sensing substrate (BSA-incorporated Ag nano flowers)plays a crucial role in the cytosensor detection performance.The structure and morphology of the BSA-incorporated Ag nano-flowers were investigated using FESEM and TEM.Fig.1A shows the overall morphology of the BSA-incorporated Ag nano flowers;the product consists of large quantities of flower-like structures of average diameter 500nm.The enlarged FESEM image in Fig.1B shows that numerous thin petals assemble into 3D flower-like structures.Fig.1C displays a typical example of nano flower mor-phology,wrapped with a thin layer of BSA;this con firms the FESEM results.The TEM image of a single nano flower (Fig.1D)clearly shows that the nano flower is porous.The porous nanos-tructure and large number of self-assembled Ag nanoparticles signi ficantly increase the effective electrode surface for biomole-cule loading,promoting electron transfer.The thin layer of BSA molecules acts as a multifunctional platform for conjugating the targeting reagent,i.e.,SNA,and blocking nonspeci fic interaction sites.Cytosensors are used to investigate the viability and natural physiological states of cells.The cells usually need to be im-mobilized on the cytosensor surface during constructing of the cytosensor,therefore the biocompatibility of the nano-biointerface materials is important in the subsequent electrochemical detec-tion.MTT assays were performed to evaluate the cytotoxicity of the BSA-incorporated Ag nano flowers at various concentrations (Fig.2).SA-over-expressed DLD-1cells were chosen as the cell model.The formazan absorbance,which is proportional to the number of living cells,was measured at a wavelength of 492nm (Zhu et al.,2012).The relative cell viability was measured by comparison with the control well containing only cells.The MTT assay showed no obvious cytotoxic effect on DLD-1cells after 48h treatment,even at a concentration of 1.25mg/ml,suggesting good biocompatibility of the BSA-incorporated Ag nano flowers in cellproliferation.Scheme 1.Fabrication of EIS Cytosensor Based on BSA-Incorporated Ag nano flowers.H.Cao et al./Biosensors and Bioelectronics 68(2015)329–3353313.2.EIS characterization of cytosensor fabricating stepsEIS is generally used to characterize the assembly of modi fied electrodes,because it gives sensitive and immediate responses to changes in electroactive substances (K ’Owino and Sadik,2005).In impedance spectra,the semicircle (in the high-frequency region)represents the electron-transfer process and the straight line (in the low-frequency region)corresponds to the diffusion process.Fig.3A shows the detailed changes in the R et value during layer-by-layer assembly,using [Fe(CN)6]3À/4Àas the electroactive redox probe.After assembly of the BSA-incorporated Ag nano flowers into a 3D sensing layer,the nyquist plots displayed an almost straight line,with an R et value of 77.5Ω(curve b,inset),which is lower than that of the pretreated GCE (119Ω),indicating sig-ni ficant electrochemical signal ampli fication by the BSA-in-corporated Ag nano flower sensing layer,and a diffusion-limitedelectron-transfer process.This indicates that nano flowers ac-celerated electron transfer.GA acted as a mediator to achieve cross-links between the BSA-incorporated Ag nano flowers and SNA,to give covalent conjugation with SNA.GA is an ideal reagent,because it reacts immediately and quantitatively with primary amino groups to give stable imines,giving chemical activation.After the immediate and quantitative reaction with GA,the R et value sharply increased to 832Ω(curve c),because of the for-mation of Schiff base structures and steric hindrance of kinetic solvent effect (Hu et al.,2013b ).BSA,which has a large number of functional groups (e.g.,ÀSH and ÀNH 2)(Hu et al.,2012),can be converted to a reactive interface by GA,where another layer of target biomolecules SNA was covalently immobilized on the sur-face of the BSA-incorporated Ag nano flowers via Schiff base structures,the R et value increased to 1943Ω(curve d).This large increase in R et could be caused by SNA limiting access of the redox probe to the electrode surface.Highly speci fic recognition of DLD-1cells attached to the biosensor (1.35Â103cells mL À1)increased the R et value to 4004Ω(curve e),indicating a larger barrier,caused by cell membranes hindering the redox couple [Fe(CN)6]3À/4Àfrom getting close to the electrode surface.In addition,based on an equivalent circuit model (inset in Fig.3B),the successive changes in the impedance data were fitted to the R et values using Autolab software;the results are shown in a histogram.The equivalent circuit includes the following elements:(a)R s ,the oh-mic resistance of the electrolyte solution;(b)Z w ,the Warburg impedance;and (c)C dl ,the interfacial double layer capacitance between the solution and electrode,related to the surface condi-tion of the electrode.3.3.Optimization of biosensing conditionsWe investigated the effects of SNA concentration and DLD-1cells incubation time,which are related to the cytosensor sensi-tivity,on the modi fied electrode in detail,to construct an EIS cy-tosensor with good cells capture capabilities andrecognitionFig.1.FESEM (A,B,from lower magni fication to higher magni fication)and TEM (C,D)images of the BSA-incorporated Ag nano flowers.Fig.2.Cytotoxicities of various concentrations of BSA-incorporated Ag nano flowers incubated with DLD-1cells for 24and 48h.H.Cao et al./Biosensors and Bioelectronics 68(2015)329–335332ef ficiency.The CV peak current decreased to a minimum response at 1.5mg mL À1(Fig.S1.B in the Supporting Information (SI))with increasing SNA concentration.This demonstrated that the amount of adhered DLD-1cells gradually increased with increasing SNA concentration,which would effectively restrain penetration of the redox probe.The optimum SNA concentration for maximum DLD-1cell capture was 1.5mg mL À1,and was used for target conjugation.For SA recognition,the incubation time is also an important factor,because it affects SA-SNA combination.The fabricated cy-tosensor was immersed in [Fe(CN)6]3À/4Àsolutions for various reaction times from 30to 180min to further explore the effect of incubation time on the EIS response.The EIS R et response in-creased (Fig.S1.A in the Supporting Information (SI))with in-creasing binding time,indicating capture of increasing amounts of DLD-1cells on the sensing platform.After incubation for 120min,the R et reached a steady value,demonstrating that the amount of cells anchored on the cytosensor surface reached saturation.Ac-cordingly,120min should be used for binding of DLD-1cells to achieve the best cytosensor performance.3.4.EIS detection of DLD-1cells on SNA/BSA-incorporated Ag na-no flower sensing interfaceEIS re flects the ef ficiency of electron transfer between an electroactive substance and the electrode surface.Quantitative monitoring of tumor cells was performed using impedance mea-surements.The optimized cytosensor was used to detect DLD-1cells.As shown in Fig.4A,the semicircle diameter in the Nyquist diagram for the captured DLD-1cells/SNA/BSA-incorporated Ag nano flower electrode increased with increasing amount of DLD-1cells in the range 1.35Â102–1.35Â107cells mL À1.The R et increase is de fined as ΔR et ¼R cell ÀR SNA ,where R cell is the R et value after cell capture and R SNA is the impedance after conjugation with SNA but without cell adhesion.The linear equation is given as ΔR et (Ω)¼1092.68Âlog C [DLD À1](cells mL À1)À1287.68(R ¼0.993);the de-tection limit is 40cells mL À1(signal-to-noise ¼3)(Fig.4B),which is much lower than,or comparable with,the values of 40cells mL À1for a MUC-1conjugated CNS interface for detection of DLD-1cells (Cao et al.,2014),90cells mL À1based on a folate-PEI-CNT cytosensor for detection of HeLa cells (Wang et al.,2013)and 80cells mL À1at PANI-NFs/AuNPs/anti-P-gp for detection of K562/ADM cells (Zhang et al.,2014)[see Table S1in the Sup-porting Information (SI)].The proposed cytosensor was also more sensitive than these reported EIS sensors,because of the large surface area of the porous structure,the good electroconductivity and biocompatibility of the BSA-incorporated Ag nano flowers,and high af finity between SNA and SA,with high expression levels on the DLD-1cell membranes.We investigated the performance of a BSA-incorporated Ag nano flower/GCE biosensor without SNA.Pure BSA-incorporated Ag nano flowers alone were used for DLD-1cell detection.As shown in Fig.S2.A ,the Nyquist diagrams gra-dually changed with increasing adhered concentrations of DLD-1cells;the change was proportional to the logarithm of cell con-centration in the range 2.8Â103–2.8Â105cells mL À1.The ΔR et value was de fined as the difference between the values before and after cell adhesion for the BSA-incorporated Ag nano flowers/GCE.The linear relationship is ΔR et (Ω)¼421.96Âlog C [DLD-1](cells mL À1)À568.05(R ¼0.987)(Fig.S2.B ).The analytical perfor-mance of the BSA-incorporated Ag nano flower sensing layerwasFig.3.(A)Nyquist diagrams of EIS recorded from 0.1to 105Hz (a)bare GCE pretreated electrochemically,(b)formation of BSA-incorporated Ag nano flowers film,(c)conjugation with GA,(d)immobilization of SNA,and (e)after DLD-1cell adhesion in 10mM [Fe(CN)6]3À/[Fe(CN)6]4À(10mM,1:1)containing 0.1M KCl.(B)Histogram shows fitting impedance data for R et during assembly (inset:equivalent circuitmodel).Fig.4.(A)EIS responses of the electrode modi fied with BSA-incorporated Ag nano flowers/GA/SNA after recognition with different concentrations (cells mL À1)of DLD-1cells for 120min:(a)0;(b)1.35Â102;(c)1.35Â103;(d)1.35Â104;(e)1.35Â105;(f)1.35Â106;and (g)1.35Â107.(B)Calibration curve for change in electron-transfer resistance with logarithm of DLD-1cell concentration.H.Cao et al./Biosensors and Bioelectronics 68(2015)329–335333attributed to electrostatic adhesion between the BSA-incorporated Ag nano flowers,which had excellent electroconductivity and biocompatibility,and the negatively charged tumor cells.In addi-tion,the charge of BSA-incorporated Ag,SNA and BSA were char-acterized by zeta-potential measurements.Based on the different isoelectric points of each substance,the obvious changes of zeta-potential could be observed from Fig.S3(SI),providing a favour-able evidence for electrostatic adhesion.The introduction of SNA reduced the detection limit of the BSA-incorporated Ag/SNA cy-tosensor by almost two orders of magnitude;therefore,the novel cell biosensor have a better capability for targeting tumor cells and hinder non-speci fic binding,and greatly improved sensitivity in detecting tumor cells.3.5.Evaluation of sialic acid expression on DLD-1cellsSA on cell surfaces has been implicated in many different types of cancer,and its expression on cancer cells can provide rational indexes of phenomena,such as dynamic changes in tumor ma-lignancy,metastatic phenotypes,and diabetic symptoms.It is therefore important to monitor cell surface SA expression.We used UV –vis absorption spectroscopy to determine the amount of SA on the cell surface.As can be seen in Fig.S4(SI),SA and acidic ninhydrin solution form a stable yellow complex,generating a distinct maximum absorption peak at about 470nm.The standard curve of peak absorbance vs.SA concentration from 10to 70μg mL À1was drawn (Fig.S4,inset,SI).An electrode modi fied with SNA/BSA-incorporated Ag nano flowers was incubated in SA solution of concentration 70μg mL À1for 2h,until saturated.Then acidic ninhydrin solution was added to the SA solution,and the UV –vis absorption spectrum of the yellow complex was recorded.The intensity of absorbance of the SA solution incubated on the electrode modi fied with SNA/BSA-incorporated Ag nano flowers decreased (curve b)(Fig.S5,SI)compared with that of the SA solution (70μg mL À1).Based on the difference between the peak absorbances,the SA loading on the SNA/BSA-incorporated Ag na-no flowers was calculated to be 15μg mL À1.The number of im-mobilized cells on the cytosensor surface was calculated to be 4.05Â104.The amount of SA on each DLD-1cell was therefore 2.16Â1012molecules.3.6.Speci ficity,stability,and reproducibility of cytosensor strategy To evaluate the selectivity of the BSA-incorporated Ag-based cytosensor,differential pulse voltammetry (DPV)and EIS techni-que were applied in our study.DPV were performed to monitorthe current signal changes after incubating different cells.Human embryonic kidney 293cells (HEK 293),human astrocyte 1800cells,and DLD-1cells,at the same concentrations,were used to study the speci ficity of our cytosensor under the optimized conditions.As shown in Fig.5A,no obvious peak current changes were detected after incubation of the cytosensor in HEK 293cells and 1800cells,whereas the DLD-1cells gave a signi ficant DPV peak current.EIS result (Fig.5B)was consistent with DPV.They were therefore expected that the prepared cytosensor could dis-tinguish between cancer cells and noncancerous ones.The good selectivity of this cytosensor is attributed to the speci fic binding between cell surface SA and lectin SNA.The stability of the sensing interface was further investigated by storing the prepared cyto-sensor in a refrigerator at 4°C for 20d.The peak current only decreased by 8.7%.The cytosensor was therefore suf ficiently stable for detection of cancerous DLD-1cells.The intra-assay precision of the cytosensor was estimated by detecting DLD-1cells at two le-vels for five replicate determinations.At DLD-1cell concentrations of 1.35Â106and 1.35Â107cells mL À1,the relative standard de-viations (RSDs)of the intra-assay using the fabricated biosensor were 4.7%and 4.8%,respectively,signifying acceptable precision.The reproducibility of the cytosensor for DLD-1cells was evaluated using five replicate measurements from the batch;the RSD was 4.3%,indicating that the developed cytosensor gives excellent reproducibility.4.ConclusionsIn summary,we developed a novel electrochemical cytosensor based on a 3D nano-bio interface,with BSA-incorporated Ag na-no flowers as a new platform with excellent biocompatibility and cell capture capability.The BSA-incorporated Ag nano flowers provide an ideal sensing layer for cell adhesion,because of the excellent conductivity of Ag nanoparticles and high stability and biocompatibility of the BSA layer.With the help of GA,SNA was covalently immobilized on the BSA-incorporated Ag,for speci fic recognition and binding of SA on cell surfaces.The proposed bio-sensor was successfully used to quantify DLD-1cells and evaluate the average expressed amount of SA on a single cell surface.The cytosensing strategy based on BSA-incorporated Ag nano flowers had a wide linear range,low detection limit,good fabrication re-producibility,detection precision,and high speci ficity for SA-po-sitive tumor cells,and will help to achieve early diagnosis and treatment of humancancer.Fig.5.Speci ficity of the fabricated EIS biosensor.(A)Typical DPV response changes in peak current with modi fied electrode after respective incubations with same con-centration of DLD-1,HEK-293,and 1800cells in [Fe(CN)6]3À/4À(10mM,1:1)containing 0.1M KCl.(B)Histogram shows relative data of ΔR et ,inset:EIS responses of (a)BSA-incorporated Ag nano flowers/GA/SNA electrode after respective incubations with same concentration of DLD-1(d),HEK-293(b),and 1800cells (c)in [Fe(CN)6]3À/4À(10mM,1:1)containing 0.1M KCl.Error bars are standard deviations of three replicate determinations.H.Cao et al./Biosensors and Bioelectronics 68(2015)329–335334。