Cellular Uptake of Gold Nanoparticles Passivated with BSA-SV40Large T Antigen Conjugates
Joph A.Ryan,†K.Wesley Overton,†Molly E.Speight,†Christine N.Oldenburg,†LiNa Loo,†
Wayne Robarge,‡Stefan Franzen,†and Daniel L.Feldheim*,§
Departments of Chemistry and Soil Science,North Carolina State University,Raleigh,North Carolina27695,and Department of Chemistry and Biochemistry,University of Colorado,Boulder,Colorado80309
Internalization and subcellular localization in HeLa cells of gold nanoparticles modified with the SV40large T antigen were quantified using inductively coupled plasma optical emission spectroscopy(ICP-OES).Internalization was monitored as a function of incubation time,temper-ature,nanoparticle diameter,and large T surface cover-age.Increasing the amount of large T peptides per gold nanoparticle complex,by either increasing the coverage at constant nanoparticle diameter or by increasing the nanoparticle diameter at constant large T coverage,re-sulted in more cellular internalization.In addition,nuclear fractionation was performed to quantify nuclear localiza-tion of the complexes as a function of large T coverage. In contrast to our prior qualitative investigations of nuclear localization by video-enhanced color differential interference contrast microscopy(VEC-DI
C),ICP-OES was able to detect nanoparticles inside fractionated cell nuclei.Although increasing the large T coverage was found to afford higher cell internalization and nuclear targeting, quantitative evaluation of cytotoxicity revealed that higher large T coverages also resulted in greater cytotoxicity.The ICP-OES and nuclear fractionation techniques reported here are valuable tools that can add important quantitative information to optical and electron imaging methods such as VEC-DIC and transmission electron microscopy re-garding the fate of nanoparticles in cells.
Polyvalent interactions are ubiquitous in biology.The valency of a particle(protein,virus,cell,etc.)is the number of connections it can make with another particle.It has been propod that biological systems exploit polyvalent interactions becau they allow an organism to take advantage of an existing t of monovalent(and perhaps weak)ligands rather than evolving completely new,higher affinity ligands for a given function.1-3 Indeed,polyvalent interactions can be very favorable;binding of a trivalent oligosaccharide ligand to its asialoglycoprotein cell surface receptor occurs with a binding constant of108M-1even though the binding constant of the corresponding monovalent interaction is only103M-1.4
Many synthetic systems are being explored for their ability to rve as polyvalent binders.The include dendrimers,5lipo-somes,6and gold surfaces(both planar and nanoparticles).7,8 Additionally,it
has been obrved that many virus u multiple binding sites in order to facilitate membrane fusion and enhance endocytosis.9Gold is advantageous as a multifunctional and polyvalent synthetic architecture as it can be synthesized in colloidal form with excellent control over shape and size and its surfaces are readily modified with small molecules,peptides, oligonucleotides,and polymers.10-22The visible light extinction properties of gold nanoparticles also make them traceable inside
*To whom correspondence should be addresd.E-mail:Daniel.Feldheim@ Colorado.edu.
†Department of Chemistry,North Carolina State University.
‡Department of Soil Science,North Carolina State University.
§University of Colorado.
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9150Analytical Chemistry,Vol.79,No.23,December1,200710.1021/ac0715524CCC:$37.00©2007American Chemical Society
Published on Web10/31/2007
of cells using video-enhanced color differential interference contrast microscopy(VEC-DIC).For example,we have ud VEC-DIC previously to monitor the subcellular localization of gold nanoparticles modified with cell and nuclear targeting peptides. VEC-DIC has revealed that particle internalization is dependent upon peptide quence and cell type10and that a combination of receptor-mediated endocytic(RME)and nuclear localization(NLS) peptides displayed on a single nanoparticle qualitatively afford more efficient nuclear targeting than particles modified with either peptide alone or even the analogous full-length(RME-NLS) quence.11
Our prior work illustrated qualitatively the potential benefits of a multipeptide approach to cell targeting.It was then of interest to develop protocols for quantifying the effects of peptide coverage and gold nanoparticle size on cell uptake and nuclear localization. The peptide chon for the studies was the large T antigen of the SV40virus.It was known previously that large T is an effective NLS peptide if injected directly into the cytoplasm.12During the cour of our investigations we have found that it also behaves as an RME signal,affording rapid nanoparticle internalization into veral cell lines ,HeLa,3T3,HepG2).Here we have characterized nanoparticle internalization into HeLa cells using inductively coupled plasma optical emission spectroscopy(ICP-OES).While ICP analytical techniques have been previously ud to study nanoparticle complexes of a different nature,19in this rearch ICP-OES has revealed an increa in the number of nanoparticles internalized as the number of large T peptides per particle and nanoparticle size was incread.In addition,by fractionating cell nuclei following nanoparticle delivery it was possible to determine the ability of large T-modified nanoparticles to target the nucleus from outside of the cell.In contrast to prior work using VEC-DIC,which suggested that large T/gold nano-particle conjugates were internalized into HeLa cells but were unable to escape the endosomal/lysosomal pathway and translo-cate the nuclear membrane,11ICP-OES was able to detect nanoparticles inside the nucleus.ICP-OES and the nuclear fractionation techniques reported here are thus valuable tools that can add important quantitative information to optical imaging methods such as VEC-DIC.
EXPERIMENTAL SECTION
Materials.HeLa(human cervical cancer cells)cell line was purchad from the American Type Culture Collection(Rockville, MD).Minimal esntial medium Eagle’s(EMEM),fetal bovine rum(FBS),Dulbecco’s phosphate-buffered saline(DPBS),T-25 and T-75cell culture flasks,12-well cell culture plates,and trypsin were purchad from Bio-Whittaker,Inc.(Walkersville,MD).All gold nanoparticles were purchad from Ted Pella,Inc.(Redding, CA).The modified large T peptide quence(rhodamine-Cys-Gly-Gly-Gly-Pro-Lys-Lys-Lys-Arg-Lys-Val-Gly-Gly-OH)was syn-thesized at the University of North Carolina Microprotein Se-quencing and Peptide Synthesis Facility(Chapel Hill,NC).Bovine rum albumin(BSA)and subcellular fractionation kit were purchad from Pierce Co.(Rockford IL).[3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide](MTT)assay was pur-chad from Promega Corp.Dimethylformamide(DMF),hep-arin sulfate,4-(N-maleimidomethyl)-cyclohexane-1-carboxylic acid N-hydroxysuccinimide ester(SMCC),sodium chloride,mono basic sodium phosphate(NaH2PO3),dibasic sodium phosphate (Na2HPO3),Optima-grade HCl,Optima-grade HNO3,Centricons (MWCO:30000),and glass coverslips(18mm)were all pur-chad from Fisher Scientific.FluorSave cell mounting media was purchad from Calbiotech(San Diego,CA).
All UV-vis absorption measurements were acquired using a Hewlett-Packard8453Chemstation photo
diode array spectropho-tometer with attached Chemstation software.Fluorescence spectra were acquired using a BioTek FL-600plate reader with550(40 nm(excitation)and590(40nm(emission)filters.All ICP experiments were performed with a Perkin-Elmer Optima2100DV optical emission spectrometer equipped with a Meinhard type C glass nebulizer,unbaffled cyclonic chamber,and an alumina injection tube(2mm opening).Other instrument ttings include the following:18L/min plasma flow,0.2L/min auxiliary flow, 0.62L/min nebulizer flow,1.00L/min pump rate,and1500 W rf power.All ICP standards were made from100mg/mL SpexCertiPrep stock solution(lot no.CL3-19AU),and all ICP samples were analyzed at242.795nm with a read delay of80s and an integration time from2to5s.
Methods.Conjugation of Peptide to BSA.Conjugation was performed in accordance with a previously published protocol.10 Briefly,15.2µL aliquots of a20.0mg/mL SMCC solution(in DMF) were added to six individual1.0mL BSA samples(1mg/mL,in sodium phosphate buffer,pH)7.8)to achieve an SMCC/BSA molar ratio of60:1and allowed to mix at room temperature for 60min.Excess unreacted SMCC was then removed from each of the samples via centrifugation(Centricon,MWCO:30000),and each sample was diluted to1mL with sodium phosphate buffer (pH)7.0).Varying volumes(49.5,99.1,148.6,198.1,247.6,and 297.2µL)of rhodamine-labeled modified large T peptide(rh
o-damine-CGGGPKKKRKVGG;3mg/mL in sodium phosphate buffer;pH)7.0)were subquently added to each BSA-SMCC sample to achieve six large T/BSA-SMCC molar ratios(5:1, 10:1,15:1,20:1,25:1,and30:1,respectively),and all samples were allowed to mix at room temperature for24h.Excess unreacted peptide was removed via centrifugation(Centricon,MWCO: 30000).All samples were then diluted to1mL with sodium phosphate buffer(pH)7.0).Following dilution,the degree of peptide conjugation was then determined via examination of rhodamine fluorescence at595nm for each sample.
Synthesis of Large T-BSA/Gold Nanoparticle Complexes.Each large T-BSA conjugate was added to parate1mL aqueous aliquots of15nm diameter gold nanoparticles(1.15nM final concentration)in a large T-BSA conjugate/nanoparticle molar ratio of250:1and allowed to mix at room temperature for30min.
The stability of large T-BSA/gold nanoparticle complexes was assd by measuring the critical flocculation concentration.This assay was accomplished by adding10µL of a10%NaCl solution to each sample of large T-BSA/gold nanoparticle complex.The samples were allowed to equilibrate for30min at room temper-ature and were then mixed thoroughly with a vortex mixer.For each sample,the visible spectrum of each sample was acquired and compared to the spectrum acquired i
n pure water.A critical flocculation concentration(CFC)value for each sample was then generated by repeated additions of10%NaCl until either a significant red shift in theλmax was obrved(indicating floccula-tion of the nanoparticles)or stability was obrved at1M concentration of NaCl.
Analytical Chemistry,Vol.79,No.23,December1,20079151
Cell Culture.HeLa cells were maintained in T-75cell culture flasks using EMEM growth media containing10%FBS at37°C and at5%CO2.Prior to experimentation,cells were either plated on sterile glass coverslips in12-well plates or eded into T-25 cell culture flasks.In either ca,cells were grown to ap-proximately75%confluency before beginning experiments.It should be noted that6h was chon as a maximum incubation time for all experiments in this rearch to avoid potential effects on any cellular internalization due to overconfluency of the HeLa cells.Cell counting was performed using a fluorescent-activated cell sorting(FACS)instrument.
ICP-OES Experiments.Four ts of control experiments were performed to probe the utility of ICP-OES as an analytical technique for u in subquent determinations of nanoparticles internalized by HeLa cells.The first t of experiments was done to ensure that ICP-OES could accurately determine the concentra-tion of gold nanoparticles in solution and that no matrix effects were introdu
ced by the u of aqua regia in sample preparation. This was accomplished by placing100µL of1nM citrate-stabilized 20nm diameter gold nanoparticles in each well of a12-well cell culture plate and allowing the solutions to evaporate to dryness at room temperature.Six of the wells were then further treated with0.5mL of aqua regia while the remaining six wells received 0.5mL of ultrapure water.The well plate was allowed to remain at room temperature for2h,after which0.4mL of each sample was placed in a15mL centrifuge tube,diluted to3.9mL with ultrapure water,and analyzed via ICP-OES.
A cond t of experiments was conducted to examine the relative degree to which nanoparticle complexes might associate with the glass coverslips in the abnce of HeLa cells.Sterile glass coverslips were individually placed in each well of two12-well cell culture plates.Six of the wells then received150µL of ultrapure water and were diluted to1.5mL with EMEM growth media,six wells received150µL of citrate-stabilized15nm diameter gold nanoparticles and were then diluted to1.5mL with growth media (0.23nM final concentration),six wells received1.5mL of growth media containing10%15nm diameter nanoparticle complexes stabilized with native BSA(0.23nM final complex concentration), and the remaining six wells received1.5mL of EMEM growth media containing10%15nm diameter nanoparticle complexes stabilized with a large T-BSA conjugate(15:1large T/BSA experimentally determined molar ratio;0.23nM final complex concentratio
n).Both well plates were then incubated at37°C and 5%CO2for6h thus simulating the conditions ud in later incubations with HeLa cells.After incubation,each well was rind with1mL of DPBS three times,and the coverslips were removed from their wells and allowed to air-dry in a sterile cell culture hood. The dried coverslips were then placed in a new well plate, treated with0.5mL of aqua regia for2h,after which0.4mL of each sample was placed in a15mL centrifuge tube,diluted to3.9 mL with ultrapure water,and analyzed via ICP-OES.
The third t of ICP-OES control experiments was performed to determine both if the HeLa cells themlves would interfere with the resulting obrved ICP-OES signal and if the ICP-OES signal detected from combining HeLa cell samples prior to treatment with aqua regia was linear.The15nm gold nanopar-ticles were passivated with large T-BSA conjugate(15:1large T/BSA experimentally determined molar ratio)using a large T-BSA/nanoparticle molar ratio of250:1,and the final concentra-tion of gold nanoparticles in the solution was approximately2.3 nM.HeLa cells which had been previously cultured on sterilized coverslips placed in two12-well plates were then allowed to incubate with either1.5mL of pure growth media(6wells)or1.5 mL of growth media containing10%large T-BSA/gold nanopar-ticle complexes(18wells;0.23nM nanoparticle concentration) per well for6h.Each well was then rind with1mL of DPBS three times,and the coverslips were removed from their wells
and allowed to air-dry in a sterile cell culture hood.The dried coverslips were then placed in a new well plate;each of the6the coverslips containing HeLa cells which had not been incubated with the nanoparticle complexes was placed in its own well,6of the remaining coverslips were similarly placed in their own well, while the remaining12coverslips were placed in wells in pairs. Each well was then treated with0.5mL of aqua regia for2h, after which0.4mL of each sample was placed in a15mL centrifuge tube,diluted to3.9mL with ultrapure water,and analyzed via ICP-OES.
A final t of experiments was performed to evaluate binding of nanoparticles to the exterior of the outer cell membrane.Gold nanoparticles,15nm in diameter,were passivated with large T-BSA conjugate(15:1large T/BSA experimentally determined molar ratio)using a large T-BSA/nanoparticle molar ratio of250:1 and a final concentration of gold nanoparticles of2.3nM.HeLa cells,which had been previously cultured on sterilized coverslips and placed in one12-well plate,were allowed to incubate with1.5 mL of growth media containing10%large T-BSA/gold nanopar-ticle complexes(0.2nM nanoparticle concentration)per well for 6h.After incubation,each well was rind three times with1mL of DPBS,once with1mL of DPBS containing heparin sulfate(5 U/mL),and one final time with1mL of DPBS.Heparin sulfate is commonly ud to desorb ,molecules,biomol-ecules,particles)adhered to cell outer membranes.The samples were then treated with aqua regia and analyzed by ICP-OES as described above.
Cellular Internalization of Large T-BSA/Gold Nanoparticle Complexes as a Function of Incubation Time.Gold nanoparticles 15nm in diameter were passivated with large T-BSA conjugate (15:1large T/BSA experimentally determined molar ratio)using a large T-BSA/nanoparticle molar ratio of250:1,and the final concentration of gold nanoparticles in the solution was ap-proximately 2.3nM.HeLa cells which had been previously cultured on sterilized coverslips placed in12-well plates were then allowed to incubate with1.5mL of growth media containing10% large T-BSA/gold nanoparticle complexes(0.23nM nanoparticle concentration)per well for varying amounts of time from0to6 h.Six wells of HeLa cells were ud for every time period under scrutiny.After the desired incubation time had elapd,each well was rind with1mL of DPBS three times,and the coverslips were removed from their wells and allowed to air-dry in a sterile cell culture hood.The dried coverslips were then placed in a new well plate,combining two coverslips of identical samples per well.Each well was then treated with0.5mL of aqua regia for2 h,and the resulting solution was prepared for ICP-OES analysis.
Cellular Internalization as a Function of Large T/Gold Nano-particle Ratio.Separate15nm diameter gold nanoparticle samples were passivated with one of six large T-BSA conjugates,each
9152Analytical Chemistry,Vol.79,No.23,December1,2007
one containing a different molar ratio of large T per BSA.The molar ratio of large T-BSA conjugates/gold nanoparticles was 250:1,and the final concentration of gold nanoparticles in the solution was2.3nM.HeLa cells which had been previously cultured on sterilized coverslips and placed in12-well plates were then allowed to incubate with1.5mL of growth media containing 10%large T-BSA/gold nanoparticle complexes(0.23nM nano-particle concentration)per well for1,3,or6h.Additional well plates of HeLa cells which had been previously cultured on sterilized coverslips and placed in12-well plates were removed from the incubator and cultured at4°C for3h prior to experimentation(along with the corresponding aliquots of DPBS and growth media containing nanoparticle complexes)in order to allow one replicate t of incubations to take place for6h at this temperature.Six wells of HeLa cells were ud for every time period under scrutiny.After the desired incubation time had elapd,each well was rind with1mL of DPBS three times, and then the coverslips were removed from their wells and allowed to air-dry in a sterile cell culture hood.The dried coverslips were then placed in a new well plate,combining two coverslips of identical samples per well.Each well was then treated with0.5 mL of aqua regia for2h,and the resulting solution was prepared for ICP-OES analysis.Additional experiments under identical conditions were performed on parate samples of cells for the purpos of obtaining cell count and toxicity data.
Cellular Internalization as a Function of Gold Nanoparticle Size. Experiments designed to probe the effect of nanoparticle size on cellular internalization were performed under two different ex-perimental conditions.In both ts of conditions,parate samples of gold nanoparticles of varying diameter(10,15,and20nm)but identical concentration(1.2nM)were passivated with a large T-BSA conjugate(15:1peptide/BSA experimentally determined molar ratio)prepared in the same fashion as described earlier. However,in one t of experiments,the large T-BSA conjugate was added to each nanoparticle sample in a large T-BSA/ nanoparticle molar ratio of500:1irrespective of nanoparticle size, while in the other t of experiments,conjugate was added to the samples of nanoparticles in a large T-BSA/nanoparticle molar ratio commensurate with nanoparticle surface area;that is,20nm diameter colloids were expod to large T-BSA conjugates in a large T-BSA/nanoparticle molar ratio of500:1,while the molar ratio ud for15and10nm diameter colloids was250:1and 125:1,respectively.An additional sample of20nm diameter gold nanoparticles was prepared using native BSA in a BSA/nano-particle molar ratio of500:1.HeLa cells,which had been pre-viously cultured on sterilized coverslips and placed in12-well plates,were allowed to incubate with1.5mL of growth media containing10%large T-BSA/gold nanoparticle complexes(0.12 nM nanoparticle concentration)per well for6h.Six wells of HeLa cells were ud for every nanoparticle size under scrutiny. After the desired incubation time had elapd,each well was rind with1mL of DPBS three
times,and then the coverslips were removed from their wells and allowed to air-dry in a sterile cell culture hood.The dried coverslips were then placed in a new well plate,combining two coverslips of identical samples per well.Each well was then treated with0.5mL of aqua regia for2h,and the resulting solution was prepared for ICP-OES analysis.
Cellular Internalization as a Function of Excess Passivating Conjugate.In this experiment,parate samples of15nm gold nanoparticles were passivated with a peptide-BSA conjugate(15:1 peptide/BSA experimentally determined molar ratio)in varying large T-BSA/nanoparticle molar ratios(250:1to3000:1),and the final concentration of gold nanoparticles in the solution was2.3 nM.Additional nanoparticle complexes were prepared using native BSA instead of large T-BSA conjugates.HeLa cells,which had been previously cultured on sterilized coverslips and placed in 12-well plates,were allowed to incubate with1.5mL of growth media containing10%large T-BSA/gold nanoparticle complexes (0.23nM nanoparticle concentration)per well for6h.Six wells of HeLa cells were ud for every time period under scrutiny. After the desired incubation time had elapd,each well was rind with1mL of DPBS three times,and then the coverslips were removed from their wells and allowed to air-dry in a sterile cell culture hood.The dried coverslips were then placed in a new well plate,combining two coverslips of identical samples per well.Each well was then treated with0.5mL of aqua regia for2 h,and the resulting solution was prepared for ICP-OES analysis.
Pul-Cha Experiments.Gold nanoparticles15nm in diam-eter were passivated with a large T-BSA conjugate(15:1peptide/ BSA experimentally determined molar ratio)in a large T-BSA/ nanoparticle molar ratio of250:1,and the final concentration of gold nanoparticles in the solution was2.3nM.HeLa cells,which had been previously cultured on sterilized coverslips and placed in12-well plates,were allowed to incubate with1.5mL of growth media containing10%large T-BSA/gold nanoparticle complexes (0.23nM nanoparticle concentration)per well for3h.After this “pul,”each well was rind with1mL of DPBS three times,1.5 mL of fresh growth media without nanoparticles was applied to each well,and the cells were allowed to further incubate for varying amounts of time(“cha”times)over a period of12h. Six wells of HeLa cells were ud for every time period under scrutiny.After the desired cha time had elapd,each well was again rind with1mL of DPBS three times,and then the coverslips were removed from their wells and allowed to air-dry in a sterile cell culture hood.The dried coverslips were then placed in a new well plate,combining two coverslips of identical samples per well.Each well was then treated with0.5mL of aqua regia for2h,and the resulting solution was prepared for ICP-OES analysis.
Subcellular Fractionation and Nuclear Targeting Efficiency. Separate5nm diameter gold nanoparticle samples were passivated with one of the six previously prepared large T-BSA conjugates containing
varying amounts of large T per BSA in a peptide-BSA/nanoparticle molar ratio of10:1,and the final concentration of gold nanoparticles in the solution was16.6nM.HeLa cells, which had been previously cultured in T-25cell culture flasks, were allowed to incubate with5mL of growth media containing 10%large T-BSA/gold nanoparticle complexes(1.6nM nano-particle concentration)per well for6h.Nanoparticles5nm in diameter were chon for the experiments to minimize the potential accumulation of nanoparticles in nuclear fractions due solely to high-speed centrifugation(ud in the cell fractionation protocol).T-25flasks were chon for u in the experiments in the interest of increasing the number of cells per experiment and thereby increasing the resulting gold emission signal detect-
Analytical Chemistry,Vol.79,No.23,December1,20079153
able via ICP-OES.Conquently,six T-25flasks of cells were ud for each large T-BSA/nanoparticle complex under scrutiny.After the desired incubation time had elapd,the cells in each flask were rind with1mL of DPBS three times and then trypsinized. Small aliquots(100µL)of cells were removed from each flask for cell counting and toxicity assays using FACS,and the remaining cells were concentrated via centrifugation.Three of the six resulting samples per construct were then air-dried in a sterile cell culture hood,treated with1.0mL of aqua regia for2h,and each subquent solutio
n was prepared for ICP-OES analysis.The
remaining three samples were subjected to fractionation using the Pierce Co.subcellular fractionation kit to obtain nuclear and cytosolic fractions of each sample.Each individual fraction was then air-dried in a sterile cell culture hood,treated with1.0mL of aqua regia for2h,and each resulting solution was prepared for ICP-OES analysis.Last,12additional T-25flasks of HeLa cells, which had been incubated for6h with standard growth media (not containing colloidal constructs),were trypsinized and con-centrated via centrifugation.The12flasks were divided into four groups containing three flasks each.All three of the flasks in a particular group had one of four different5nm nanoparticle complexes(passivation layers:native BSA,7:1,11:1,or15:1large T/BSA experimentally determined molar ratios)introduced to the fractionated cells prior to the final paration(via centrifugation) of nuclei from nonnuclear material.The samples created in such fashion are referred to as“spiked”samples.All of the fractions were then allowed to air-dry in a sterile cell culture hood,treated with1.0mL of aqua regia for2h,and each resulting solution was prepared for ICP-OES analysis.
RESULTS
Characterization of Large T-BSA/Gold Nanoparticle Com-plexes.In order to determine trends in the number of nanopar-ticles internalized into HeLa cells versus large T peptide coverage, it was first necessary to establish methods for modulating and quantifying the number of large T peptides per nanoparticle.This was accomplished by measuring the molar ratio of large T/BSA using rhodamine-labeled large T and fluorescence spectroscopy (Table1).
The number of large T-BSA conjugates per nanoparticle was subquently calculated using a previously determined value for the number of BSA molecules per20nm diameter gold nanopar-ticle.13In that work,it was determined that160(8[Ru(2,2′-bipyridine)3]2+-labeled BSA complexes were associated with each 20nm diameter gold nanoparticle using time-correlated single-photon counting(TCSPC).For the experiments described here, it was presumed that large T-BSA conjugates associate with gold nanoparticles with a constant surface density irrespective of particle diameter and should thus scale in proportion to the total nanoparticle surface area.
竹韵山庄The first t of measurements was designed to test the reproducibility of gold nanoparticle analysis by ICP-OES,asss matrix effects potentially introduced by aqua regia sample diges-tion,and examine the statistical correlation between the number of nanoparticles detected by ICP-OES and the known value of nanoparticles contained in a standard.Table2shows the results of the analys.
语无伦次
One area of concern when using glass coverslips with protein-coated gold nanoparticles is nonspecific binding of particles to the glass substrate.To quantify this interaction,standard samples were incubated with bare ,no cells)and then analyzed by ICP-OES.The data in Table3show that nonspecific binding of gold nanoparticles to the glass coverslips does occur to a small degree and increas with the addition of large T-BSA conjugates to the particles.
The next t of experiments was designed to optimize ICP-OES signals and determine the extent of nanoparticle nonspecific binding to the exterior cell membrane.In order to optimize ICP-OES signals,the effects of combining two identically treated coverslips into one ICP-OES sample were examined.It was expected that coverslips containing identical cell coverage and incubated with an identical concentration of nanoparticle com-
Table1.Number of Large T Peptides Per BSA and Per Gold Nanoparticle Complex
large T/BSA mole ratio in reaction
mixture
large T/BSA
学期个人总结
determined
experimentally a
est no.of
large T/15nm
diameter
particles b
est no.of
large T/5nm
diameter
particles b
5.03(1300(10030(10
10.07(2600(10070(20
15.08(1700(10080(10
20.011(21000(200110(20
25.013(21200(200130(20
30.015(21300(200150(20
a Uncertainty reprents the result of three measurements from three independent samples.
b Estimate was bad on the number of BSA molecules per20nm diameter gold nanoparticle determined using both fluorescence labeling and time-correlated single-photon counting (TCSPC)(ref13)and adjusting for surface area.The standard deviation provided was calculated from the%error in the corresponding entry in column2and is not a propagated error.Table2.ICP-OES Intensities and Calculated Number of Citrate-Stabilized20nm Diameter Nanoparticles for Standards Prepared with and without Acid Digestion a analyte
intensity
家常糖醋鱼(×105)
number of
nanoparticles
(×1010)
Au nanoparticles 1.537(0.005 6.220(0.002 Au nanoparticles(aqua regia) 1.550(0.009 6.264(0.004
a The standards contained7.0×1010gold nanoparticles.The intensity values from the two ts of samples were compared using Student’s t test and were obrved to be statistically different from each other at the95%confidence limit(t calc)2.99).The uncertainty listed is the standard deviation of six replicate samples.
Table3.ICP-OES Intensities of15nm Diameter Nanoparticle Complexes Constructed with Varying Passivating Layers and Incubated on Glass Coverslips for6h at37°C and in5%CO2in the Abnce of HeLa Cells a
nanoparticle passivating layer intensity(×103)
18MΩH2O(no Au)0.05(0.03
citrate0.18(0.08
native BSA 1.2(0.1
large T-BSA conjugate(14.6:1) 4.0(0.1
a The uncertainty listed is the standard deviation of six replicate samples.
9154Analytical Chemistry,Vol.79,No.23,December1,2007
