ARTICLESNATUREMATERIALSAddendum:Plasmonicnanosensorswithinversesensitivitybymeans ofenzyme-guidedcrystalgrowthLaura Rodriguez-Lorenzo,Robertodela Rica,Ramon A.Alvarez-Puebla, LuisM.Liz-Marzan and MollyM.StevensNature Materials11, 604-607 (2012); published online 27 May 2012; corrected after print 15 December 2017.Prompted by queries on the potential mechanismfor the inverse sensitivityof the biosensing assayreported in the published Letter, aswell as about the protocols that we used to obtain the results, herewe provideextended discussion ofthe signal-generation mechanismas well as additional methodological details.Signal-generationmechanismAs described in the published Letter, the signal is generated by the reduction of silver ions on gold nanostars.Thereduction of silvertriggers a shift in thelocalized surface plasmon resonance (LSPR) of the dispersed gold nanostars (which act as sensors). The reductionofsilver on thegoldnanostars isknowntobeaffected bythefollowing factors:(i)The concentration of hydrogen peroxidegenerated by glucose oxidase (GOx),which can reduce silver ions.(i)The concentration of ammonia,which dictates the pH of the solution (it is well established that the reduction potential of achemical species depends on thepHofthesolution2.Also,ammonia coordinates silver ions,therebychanging theirreduction potential°).(ii)The presence ofglucose, which is a weak reducingagent and isfound in vast excess(iv)Thepresenceof2-(N-morpholino)ethanesulfonicacid (MES), which can also actas a weak reducing agents(v)The presence of light (it is well known that UV lightcan reduce silver ions).(vi)The concentration of poly(vinylpyrrolidone)(PVP)around the nanostars.PVPcan block the gold surface and thereforechange thereduction potentialofsilverions on gold.Also,PVPhas alsobeen shown to act asa weakreducing agentcapable ofreducing silverions?Furthermore, PVP can coordinate silver ions.The nitrogen and oxygen atoms in the pyrrolidyl groups of PVP molecules may donate theirlone unpaired electron to sp hybrid orbitals of Ag.Two kinds of coordination bond (PVP molecular intra- and inter-chain interactions)can take places, whichmay significantly decreasethe chemical potential and further facilitatethereduction of PVP-attached silver ionsto generate a silver coating around the nanostars.The reduction of silver ions on gold nanoparticles can also be affected bythe presenceof PVP becausePVP can selectively adsorb silver nanostructures.Indeed, in gold-silver core-shell nanoparticles with cubic,triangular-bipyramidal, and rod or wire silver shells, (100)-typedominant facets were formed upon epitaxial growth on the (111)-type facets of thegold corelaulThe morphology change between the Au core and Ag shells may have its origin in the change ofadsorption selectivity ofPVPmoleculesfromAu(111)-typefacetstoAg(100)-typeonesin ethyleneglycol solvent.Such selectiveadsorptionofPVPmoleculescan alsoaffectthegrowthrateoftheadsorbedAgfacets.Finally,itshouldbenoted thatthePVParoundthenanostarsmayplayacrucial roleinotherprocesses not relatedto thegrowth of silver nanoparticles.For example, thereshaping of nanostars depends on theconcentration of PVParound them2. Moreover,different batches of nanostars may have slightly different LSPRs, owingto slightly different concentrations of PVParound these colloids. Since the concentration of PVP around the nanostars depends on the number of washing steps to which the nanostarsare subjected,the LSPR of the nanostars may also change depending on the number of intermediate steps preceding the silver reduction onthe nanostars. For allthese reasons, we reported changes in the LSPR ofthe nanostars resulting from the reduction of silver ions on gold asa shift withrespectto ablank experiment performed in thesame conditions andwithnanostars obtainedfrom thesamebatch.(vii)The presence of unreacted aldehyde groups around the nanostars.Aldehydes have been previously used to reducemetal ions onnanoparticle seedsis. Unreacted aldehyde groups did not affect the reported data (Fig. 4) because they were blocked with bovine serumalbumin (BSA)and ethanolamine to avoid non-specific interactions in the immunoassay.However,aldehyde groups werenotblocked in theexperiments shown inFig.2c,becausenobiorecognitionexperiments wereperformedwiththenanostarsbearingcovalentlybound GOx.Ourexperiments wereperformed in excess ofglucose,MESandlightWeobservedthatthereaction did nottakeplace (toan extentthat would have been observable) in the absence of GOx when aldehyde groups were blocked (blue dots in Fig. 4a and green dots inFig. 4b),because glucose, MES and light are weak reducing agents. Background signal in the presence ofglucose (Fig. 2c, red dots) mayhave resulted from the reduction of Agtby unblocked aldehyde groups, even in the absence of GOx,as observed when GOx was diluteddownto 1o-2gmL- (atthis concentrationwe would expectthepossibilityofless than onemolecule of enzymetobepresent inthesolution).However,as the concentration of GOx increases,the effects onthe signalfromthebiocatalyticproduction ofhydrogenperoxidearemuchstrongerthanthosefromunblockedaldehydes.Backgroundsignalisnotpresentintheabsenceofglucose(Fig.2c,blackdots)Andforthe actual assay (Fig.4)there isno background signal because aldehydegroups wereblocked,and therefore Agtcannotbereducedin the absence of the hydrogen peroxide produced by GOx (as is evidenced bythefact that the shiftfalls to zero when there is no prostate-specific antigen(PSA),andbythe absence ofa shift whenBSA is substitutedforPSA).Pleasenote thatfor SupplementaryFig.S5thegoldnanostars were not modified with GOx and therefore also did not haveglutaraldehyde added.Thereduction of silver ions on gold,even intheabsence of GOx, is also affected bythe concentration ofammonia and bythe concentration of PVP around thenanostars.For example,silver ions may be reduced on gold nanostars by glucose in the absence ofGOx when the concentration of ammonia is much higherthanthe 40 mM concentration reported, because the reduction potentials of glucose and silver ions are different at higher pH valuesi4Hydrogen peroxide is a stronger reducing agentthan glucose, MES and light.This means that hydrogen peroxide can reduce silverionson gold in the presence of low concentrations of ammonia.In the signal-generationmechanism proposed in theLetter,the productionofalow concentration of hydrogen peroxideby GOx triggers the reduction of silver ions ongold.In other words,a small concentrationof hydrogen peroxide can overcome the energybarrier required to reduce silver ions on gold.Once this energy barrier is overcome,the system has enough reactive species to continue reducing silver ions, since there is an excess ofglucose, MES and light.Therefore, asmall concentration ofenzyme-generated hydrogen peroxide can trigger the formation of a silver coating around gold nanostars.Thisphenomenon is likelyresponsiblefor theultralowlimitof detectionreported intheLetter.The inverse-sensitivity phenomenon is related to thekinetics of metal reduction.It is generally accepted thatfast kinetics ofreduction205NATUREMATERIALS|VOL17|FEBRUARY2O18www.nature.com/naturematerial@ 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved
NATURE MATERIALS | VOL 17 | FEBRUARY 2018 | www.nature.com/naturematerials 205 NATURE MATERIALS ARTICLES Prompted by queries on the potential mechanism for the inverse sensitivity of the biosensing assay reported in the published Letter, as well as about the protocols that we used to obtain the results, here we provide extended discussion of the signal-generation mechanism as well as additional methodological details. Signal-generation mechanism As described in the published Letter, the signal is generated by the reduction of silver ions on gold nanostars. The reduction of silver triggers a shift in the localized surface plasmon resonance (LSPR) of the dispersed gold nanostars (which act as sensors). The reduction of silver on the gold nanostars is known to be affected by the following factors: (i) The concentration of hydrogen peroxide generated by glucose oxidase (GOx), which can reduce silver ions1 . (ii) The concentration of ammonia, which dictates the pH of the solution (it is well established that the reduction potential of a chemical species depends on the pH of the solution2 . Also, ammonia coordinates silver ions, thereby changing their reduction potential3 ). (iii) The presence of glucose, which is a weak reducing agent and is found in vast excess4 . (iv) The presence of 2-(N-morpholino)ethanesulfonic acid (MES), which can also act as a weak reducing agent5 . (v) The presence of light (it is well known that UV light can reduce silver ions6 ). (vi) The concentration of poly(vinylpyrrolidone) (PVP) around the nanostars. PVP can block the gold surface and therefore change the reduction potential of silver ions on gold. Also, PVP has also been shown to act as a weak reducing agent capable of reducing silver ions7 . Furthermore, PVP can coordinate silver ions. The nitrogen and oxygen atoms in the pyrrolidyl groups of PVP molecules may donate their lone unpaired electron to sp hybrid orbitals of Ag+. Two kinds of coordination bond (PVP molecular intra- and inter-chain interactions) can take place8 , which may significantly decrease the chemical potential and further facilitate the reduction of PVP-attached silver ions to generate a silver coating around the nanostars9 . The reduction of silver ions on gold nanoparticles can also be affected by the presence of PVP because PVP can selectively adsorb silver nanostructures. Indeed, in gold–silver core–shell nanoparticles with cubic, triangular– bipyramidal, and rod or wire silver shells, {100}-type dominant facets were formed upon epitaxial growth on the {111}-type facets of the gold core10,11. The morphology change between the Au core and Ag shells may have its origin in the change of adsorption selectivity of PVP molecules from Au{111}-type facets to Ag{100}-type ones in ethylene glycol solvent. Such selective adsorption of PVP molecules can also affect the growth rate of the adsorbed Ag facets. Finally, it should be noted that the PVP around the nanostars may play a crucial role in other processes not related to the growth of silver nanoparticles. For example, the reshaping of nanostars depends on the concentration of PVP around them12. Moreover, different batches of nanostars may have slightly different LSPRs, owing to slightly different concentrations of PVP around these colloids. Since the concentration of PVP around the nanostars depends on the number of washing steps to which the nanostars are subjected, the LSPR of the nanostars may also change depending on the number of intermediate steps preceding the silver reduction on the nanostars. For all these reasons, we reported changes in the LSPR of the nanostars resulting from the reduction of silver ions on gold as a shift with respect to a blank experiment performed in the same conditions and with nanostars obtained from the same batch. (vii) The presence of unreacted aldehyde groups around the nanostars. Aldehydes have been previously used to reduce metal ions on nanoparticle seeds13. Unreacted aldehyde groups did not affect the reported data (Fig. 4) because they were blocked with bovine serum albumin (BSA) and ethanolamine to avoid non-specific interactions in the immunoassay. However, aldehyde groups were not blocked in the experiments shown in Fig. 2c, because no biorecognition experiments were performed with the nanostars bearing covalently bound GOx. Our experiments were performed in excess of glucose, MES and light. We observed that the reaction did not take place (to an extent that would have been observable) in the absence of GOx when aldehyde groups were blocked (blue dots in Fig. 4a and green dots in Fig. 4b), because glucose, MES and light are weak reducing agents. Background signal in the presence of glucose (Fig. 2c, red dots) may have resulted from the reduction of Ag+ by unblocked aldehyde groups, even in the absence of GOx, as observed when GOx was diluted down to 10–20 g mL–1 (at this concentration we would expect the possibility of less than one molecule of enzyme to be present in the solution). However, as the concentration of GOx increases, the effects on the signal from the biocatalytic production of hydrogen peroxide are much stronger than those from unblocked aldehydes. Background signal is not present in the absence of glucose (Fig. 2c, black dots). And for the actual assay (Fig. 4) there is no background signal because aldehyde groups were blocked, and therefore Ag+ cannot be reduced in the absence of the hydrogen peroxide produced by GOx (as is evidenced by the fact that the shift falls to zero when there is no prostatespecific antigen (PSA), and by the absence of a shift when BSA is substituted for PSA). Please note that for Supplementary Fig. S5 the gold nanostars were not modified with GOx and therefore also did not have glutaraldehyde added. The reduction of silver ions on gold, even in the absence of GOx, is also affected by the concentration of ammonia and by the concentration of PVP around the nanostars. For example, silver ions may be reduced on gold nanostars by glucose in the absence of GOx when the concentration of ammonia is much higher than the 40 mM concentration reported, because the reduction potentials of glucose and silver ions are different at higher pH values14. Hydrogen peroxide is a stronger reducing agent than glucose, MES and light. This means that hydrogen peroxide can reduce silver ions on gold in the presence of low concentrations of ammonia. In the signal-generation mechanism proposed in the Letter, the production of a low concentration of hydrogen peroxide by GOx triggers the reduction of silver ions on gold. In other words, a small concentration of hydrogen peroxide can overcome the energy barrier required to reduce silver ions on gold. Once this energy barrier is overcome, the system has enough reactive species to continue reducing silver ions, since there is an excess of glucose, MES and light. Therefore, a small concentration of enzyme-generated hydrogen peroxide can trigger the formation of a silver coating around gold nanostars. This phenomenon is likely responsible for the ultralow limit of detection reported in the Letter. The inverse-sensitivity phenomenon is related to the kinetics of metal reduction. It is generally accepted that fast kinetics of reduction Addendum: Plasmonic nanosensors with inverse sensitivity by means of enzyme-guided crystal growth Laura Rodríguez-Lorenzo, Roberto de la Rica, Ramón A. Álvarez-Puebla, Luis M. Liz-Marzán and Molly M. Stevens Nature Materials 11, 604–607 (2012); published online 27 May 2012; corrected after print 15 December 2017. © 2 0 1 7 M a c mill a n P u bl i s h e r s Li mit e d, p a rt o f S p ri n g e r N a t u r e. All ri g h t s r e s e r v e d
ARTICLESNATUREMATERIALSfavour nanoparticlenucleation.Inother words,theyfavourtheformation of newnanoparticles.Instead,slowkinetics ofmetal reductionfavour thereduction ofsilver ions on pre-existing nanoparticles.This results intheformationoflargernanoparticles.That is,slowkineticsofmetal reductionfavourgrowth over nucleation.Thekineticsofreductionofnoblemetal nanoparticlesare influenced,amongotherfactors,by:(i)Thereduction potentialofthereducing agent as well as anyfactor affecting thisreduction potential (such asthepHofthe solution,and the presence ofcapping ligands or chelating agents)"s.(ii)Thetemperature of the solution*6(ii) The concentration ofthe reducing agent.Higher concentrations ofreducing agent lead to fasterkinetics ofmetal reduction?In ourexperiments,thepHwasbuffered,theconcentration ofammonia fixed,thetemperatureconstant,andvariationsintheconcentration of the weak reducing agent glucose are irrelevant.Therefore, thekey factor that governs the kinetics of crystal growth is theconcentration of the stronger reducing agent hydrogen peroxide.When the concentration of hydrogen peroxide is low (near the limit ofdetection),thekineticsofreductionareslow.Thisfavoursgrowthovernucleation,andthesilverionsarereducedasacoatingaroundthenanostars.ThesilvercoatinggeneratesablueshiftintheLSPRofthegoldnanostars,whichisthebasisofthedetectionmechanismdescribedin the Letter.When the concentration ofhydrogen peroxide increases,thekinetics ofreduction isfast.Thisfavours nucleation over growth,and separate silver nanoparticles are formed (it is important to stress that the assay measures changes in the LSPR of the nanostars; separatesilver nanoparticles do not change the LSPR of the nanostars but can be detected with TEM).Therefore, faster kinetics of reduction leadto smaller LSPR shifts because less silver is deposited as a coating around the nanostars.This relationship between the concentration ofhydrogen peroxide and the LSPR shift enables the analyst to quantifythelevels ofthe target molecule,since its concentration is related to theconcentration of peroxide through thebiocatalytic cycle of GOx bound to the nanostars.The sensitivity of an analytical method is usuallydefinedastheslopeofthecalibrationcurve, that is,thechangein signalregisteredforaparticularchange in the concentrationoftheanalyte.In ourcase, the slope ofthecalibration curve is negative, and therefore the sensitivityis inversed when compared to other analytical methodswith apositivecalibration curve.Negative slopes arenot uncommon in analytical chemistry;for example,they can befound in competitiveimmunoassays.Ourmethod is different fromthese approaches in that thehighest signal possible (larger differencebetween the signal oftheanalyte and the blank signal) is registered at thelowestconcentration of analyte within the inverse sensitivity regime.Noise levels in Fig. 4a,b were shown as error bars that represent the standard deviation ofthree measurements performed on threedifferent samples. This is standard methodology for variability measurements in analytical chemistryis-2o. The origin of the error maydepend onalargenumberofvariables(dilution error,analystproficiency,pipettecalibration,instrumental error andmanyothers)Figure 4a,b shows a log-linear response between analyte concentration and the blueshift of the LSPR; that is, the LSPR blueshift varieswith the logarithm of the number ofmolecules.As a consequence,variabilityin the concentration of PSA (horizontal axis), includingthat attributableto Poisson sampling,would not be expected to result in proportionatevariation in the phase shift (vertical axis).Suchchanges (or uncertainties)in the number of molecules will correspond to substantiallysmaller changes in theblueshift,as observed inthe data reported in the Letter (it should be noted that the only data for which noise is relevant is those in Fig.4, as these experimentsrepresentthe actual performance for the assay and were carried out in triplicate).DetailedprotocolsThe detection of PSA in Fig. 4 was carried out by following these protocols.Modification ofgold nanostars withpolyclonal anti-PSA,Gold nanostars (NSs)were synthesized according to a previously publishedmethod with a modification in the molecular weight of PVP (M,= 8,000, Alfa-Aesar 41626)21.After removing excess layers of PVP bycentrifugationandredispersionin isopropanol(2,950×g,3times),theconcentrationsofgoldandPVPwere0.80mMand~3mM,respectively7mLofNSs ([Au]~0.80mM)wascentrifuged at2,950×gfor 30min at25°C.TheNSs wereredispersed in7mLsodiumbicarbonatebuffer solution(10mM,pH9).Thegold concentration was~0.70mM.0.7mL of 50%glutaraldehyde solution (Sigma G7651)wereadded.The final concentrations of glutaraldehyde and gold were5% and-0.65mM, respectively.The solution was shaken gently(on aroller)for3h at 25°C.TheNSs werethen centrifuged at2,380×gfor 25min.Theresultingpellet wasredispersed in7mLofa sodiumbicarbonatebuffer solution (10 mM,pH9).Thefinal gold concentration was~0.50mM.20μL of a 1 mg mL-'anti-PSA solution wereadded to~0.50mM of gold nanostars and 2mM ofNaCNBH,in sodium bicarbonatebuffer (10mM,pH 9).Thefinal reaction volumewas 7mL.The reaction was allowed to proceed for 3 h at room temperature.Non-reacted aldehyde groups were blocked by adding 1ooμLofa100mMethanolaminesolution (bicarbonatebuffer10mMpH9)for1h.100μLof1mgmL-BSAinbicarbonatebuffer (10mM,pH 9)were then added.The reaction was allowed to proceed overnight at 4°C.TheNSs were centrifuged at 1,450xg for 20 min.Theresultingpellet was redispersed in 7mLPBSbuffer (10 mM,pH7.4).Thefinal gold concentration was~0.5mM.Immunoassay.APSA stock solution (PBSbuffer 10 mM,pH7.4) with the concentration of 10 μg mL-was serially diluted (1:10).Thiswasdonebyadding100μL(usingamicropipette)of10ugmL-lPSAsolutionto900uLofPBS,andthenmixing.Therestofthecalibrating solutionswereprepared in the sameway:byadding100μLofa10-fold moreconcentratedPSA solution to900μLof PBS.An appropriate volume of thediluted samples was added to500μL of anti-PSA-conjugated NSs in order to obtain the desired finalconcentrations of PSA.The final volume was 1 mL.The dispersions were incubated for 90 min at room temperature.The NSs werecentrifuged at 450 ×gfor10min.The resulting pellet was redispersed in 500μLPBS.10 μL ofmonoclonal anti-PSA antibody were added.The dispersions were incubated for2 h at room temperature.TheNSs were centrifuged at 450×gfor 10 min.The pellet was redispersedin500μLof PBS (10mM,pH7.4).10μLofGOx-conjugatedanti-mouseIgGwereadded to eachsample.Thedispersions were incubatedfor 2hat room temperature.TheNSs werecentrifuged at 450×gfor5min.The pellet was redispersed in 400μLMES (10mM,pH 6).For thereductionof silver,a1Mglucosesolution inMES buffer(10mM,pH6)was prepared.100μLofthe1Mglucose solution wasadded to 400μLNSs in MES buffer.Thereaction was allowed to proceedfor1h at room temperature.During this period GOxgenerateshydrogenperoxide.A fresh solution of AgNO, (0.2 mM) in 80 mM NH(aq) was prepared and filtered with a syringe filter (0.2 μm).500 μL of theresulting [Ag(NH,)] solution was added to the mixture of protein-covered NSs and glucose. Visible-near-infrared (vis-NIR) spectra206NATUREMATERIALS/VOL17|FEBRUARY2018|www.nature.com/naturematerials@ 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved
206 NATURE MATERIALS | VOL 17 | FEBRUARY 2018 | www.nature.com/naturematerials ARTICLES NATURE MATERIALS favour nanoparticle nucleation. In other words, they favour the formation of new nanoparticles. Instead, slow kinetics of metal reduction favour the reduction of silver ions on pre-existing nanoparticles. This results in the formation of larger nanoparticles. That is, slow kinetics of metal reduction favour growth over nucleation. The kinetics of reduction of noble metal nanoparticles are influenced, among other factors, by: (i) The reduction potential of the reducing agent as well as any factor affecting this reduction potential (such as the pH of the solution, and the presence of capping ligands or chelating agents)15. (ii) The temperature of the solution16. (iii) The concentration of the reducing agent. Higher concentrations of reducing agent lead to faster kinetics of metal reduction17. In our experiments, the pH was buffered, the concentration of ammonia fixed, the temperature constant, and variations in the concentration of the weak reducing agent glucose are irrelevant. Therefore, the key factor that governs the kinetics of crystal growth is the concentration of the stronger reducing agent hydrogen peroxide. When the concentration of hydrogen peroxide is low (near the limit of detection), the kinetics of reduction are slow. This favours growth over nucleation, and the silver ions are reduced as a coating around the nanostars. The silver coating generates a blue shift in the LSPR of the gold nanostars, which is the basis of the detection mechanism described in the Letter. When the concentration of hydrogen peroxide increases, the kinetics of reduction is fast. This favours nucleation over growth, and separate silver nanoparticles are formed (it is important to stress that the assay measures changes in the LSPR of the nanostars; separate silver nanoparticles do not change the LSPR of the nanostars but can be detected with TEM). Therefore, faster kinetics of reduction lead to smaller LSPR shifts because less silver is deposited as a coating around the nanostars. This relationship between the concentration of hydrogen peroxide and the LSPR shift enables the analyst to quantify the levels of the target molecule, since its concentration is related to the concentration of peroxide through the biocatalytic cycle of GOx bound to the nanostars. The sensitivity of an analytical method is usually defined as the slope of the calibration curve, that is, the change in signal registered for a particular change in the concentration of the analyte. In our case, the slope of the calibration curve is negative, and therefore the sensitivity is inversed when compared to other analytical methods with a positive calibration curve. Negative slopes are not uncommon in analytical chemistry; for example, they can be found in competitive immunoassays. Our method is different from these approaches in that the highest signal possible (larger difference between the signal of the analyte and the blank signal) is registered at the lowest concentration of analyte within the inverse sensitivity regime. Noise levels in Fig. 4a,b were shown as error bars that represent the standard deviation of three measurements performed on three different samples. This is standard methodology for variability measurements in analytical chemistry18–20. The origin of the error may depend on a large number of variables (dilution error, analyst proficiency, pipette calibration, instrumental error and many others). Figure 4a,b shows a log-linear response between analyte concentration and the blueshift of the LSPR; that is, the LSPR blueshift varies with the logarithm of the number of molecules. As a consequence, variability in the concentration of PSA (horizontal axis), including that attributable to Poisson sampling, would not be expected to result in proportionate variation in the phase shift (vertical axis). Such changes (or uncertainties) in the number of molecules will correspond to substantially smaller changes in the blueshift, as observed in the data reported in the Letter (it should be noted that the only data for which noise is relevant is those in Fig. 4, as these experiments represent the actual performance for the assay and were carried out in triplicate). Detailed protocols The detection of PSA in Fig. 4 was carried out by following these protocols. Modification of gold nanostars with polyclonal anti-PSA. Gold nanostars (NSs) were synthesized according to a previously published method with a modification in the molecular weight of PVP (Mw = 8,000, Alfa-Aesar 41626)21. After removing excess layers of PVP by centrifugation and redispersion in isopropanol (2,950 × g, 3 times), the concentrations of gold and PVP were 0.80 mM and ~3 mM, respectively. 7 mL of NSs ([Au] ~ 0.80 mM) was centrifuged at 2,950 × g for 30 min at 25 °C. The NSs were redispersed in 7 mL sodium bicarbonate buffer solution (10 mM, pH 9). The gold concentration was ~0.70 mM. 0.7 mL of 50% glutaraldehyde solution (Sigma G7651) were added. The final concentrations of glutaraldehyde and gold were 5% and ~0.65 mM, respectively. The solution was shaken gently (on a roller) for 3 h at 25 °C. The NSs were then centrifuged at 2,380 × g for 25 min. The resulting pellet was redispersed in 7 mL of a sodium bicarbonate buffer solution (10 mM, pH 9). The final gold concentration was ~0.50 mM. 20 μL of a 1 mg mL–1 anti-PSA solution were added to ~0.50 mM of gold nanostars and 2 mM of NaCNBH3 in sodium bicarbonate buffer (10 mM, pH 9). The final reaction volume was 7 mL. The reaction was allowed to proceed for 3 h at room temperature. Non-reacted aldehyde groups were blocked by adding 100 μL of a 100 mM ethanolamine solution (bicarbonate buffer 10 mM pH 9) for 1 h. 100 μL of 1 mg mL–1 BSA in bicarbonate buffer (10 mM, pH 9) were then added. The reaction was allowed to proceed overnight at 4 °C. The NSs were centrifuged at 1,450 × g for 20 min. The resulting pellet was redispersed in 7 mL PBS buffer (10 mM, pH 7.4). The final gold concentration was ~0.5 mM. Immunoassay. A PSA stock solution (PBS buffer 10 mM, pH 7.4) with the concentration of 10 μg mL–1 was serially diluted (1:10). This was done by adding 100 μL (using a micropipette) of 10 μg mL–1 PSA solution to 900 μL of PBS, and then mixing. The rest of the calibrating solutions were prepared in the same way: by adding 100 μL of a 10-fold more concentrated PSA solution to 900 μL of PBS. An appropriate volume of the diluted samples was added to 500 μL of anti-PSA-conjugated NSs in order to obtain the desired final concentrations of PSA. The final volume was 1 mL. The dispersions were incubated for 90 min at room temperature. The NSs were centrifuged at 450 × g for 10 min. The resulting pellet was redispersed in 500 μL PBS. 10 μL of monoclonal anti-PSA antibody were added. The dispersions were incubated for 2 h at room temperature. The NSs were centrifuged at 450 × g for 10 min. The pellet was redispersed in 500 μL of PBS (10 mM, pH 7.4). 10 μL of GOx-conjugated anti-mouse IgG were added to each sample. The dispersions were incubated for 2 h at room temperature. The NSs were centrifuged at 450 × g for 5 min. The pellet was redispersed in 400 μL MES (10 mM, pH 6). For the reduction of silver, a 1 M glucose solution in MES buffer (10 mM, pH 6) was prepared. 100 μL of the 1 M glucose solution was added to 400 μL NSs in MES buffer. The reaction was allowed to proceed for 1 h at room temperature. During this period GOx generates hydrogen peroxide. A fresh solution of AgNO3 (0.2 mM) in 80 mM NH3(aq) was prepared and filtered with a syringe filter (0.2 μm). 500 μL of the resulting [Ag(NH3)2] solution was added to the mixture of protein-covered NSs and glucose. Visible–near-infrared (vis–NIR) spectra © 2 0 1 7 M a c mill a n P u bl i s h e r s Li mi t e d, p a rt o f S p ri n g e r N a t u r e. Al l ri g h t s r e s e r v e d. © 2 0 1 7 M a c mil l a n P u bl i s h e r s Li mit e d, p a rt o f S p ri n g e r N a t u r e. Al l ri g h t s r e s e r v e d
NATUREMATERIALSARTICLESwererecorded after 2h.The LSPR shift was calculated with respect to a blank experiment in the absence ofthe targetmoleculeThe reduction of silver ions byGOxcovalently attached to the nanostars (Fig.2)was achievedbyfollowing thefollowingprotocolModification of gold nanostars withglucose oxidase.NSs were synthesized according to a previously published method withamodification in themolecular weight of PVP(M,=8,000,Alfa-Aesar41626)21.Afterremoving excessPVPby.centrifugation andredispersion in isopropanol (2950xg,3times),thegold and PVPconcentrationswere0.85mMand~3mM,respectively10mLofNSs (Au)=0.86mM)were centrifuged at2,950×gfor30min at 25°C.TheNSs wereredispersed in10mLof sodiumbicarbonatebuffer (10mM,pH9).Thegold concentration was0.74mM.1mLof 50%glutaraldehyde solution (Sigma G7651)wereadded to the NS solution.The final concentrations of glutaraldehyde and gold were5%and 0.61 mM, respectively.The dispersionwas then shakengently (on a roller)for3h at 25°C.Then the NSs were centrifuged at 1,880×gfor 20min.The resulting pellet wasredispersedin10mLsodiumbicarbonatebuffer(10mM,pH9).Thefinalgoldconcentrationwas0.57mMAstock solutionof2mgmL-of GOxwasprepared in sodiumbicarbonatebuffer(10mM,pH9).TheGOxstock solutionwas seriallydiluted in sodiumbicarbonatebuffer (10mM pH 9).An appropriatevolume of thediluted GOx solutions was added to 0.57mM goldnanostars and 2 mM NaCNBH, in the sodium bicarbonate buffer (10 mM, pH 9) in order to obtain the desired final enzyme concentration.Thefinal volume was 1mL.The reaction was let to proceed for 3 h at room temperature.The resulting protein-modified nanostars werecentrifuged at1,000×gfor 20 min.Theresulting pellet was redispersed in 400 μLofMES buffer10 mM,pH6.Thefinal gold concentrationwas 0.5mM.For the reduction of silver,a solution containing 1 M glucose in MES buffer (10 mM,pH 6) was prepared.100 μLofa 1 M glucosesolution was added to 400μLof0.5mMprotein-coated NSs in MES bufferfor1h at room temperature.The addition of glucose resultedin the production of hydrogen peroxideby theenzymeAfreshsolution ofAgNO, (0.2mM)in 80mM NHs(aq)was prepared andfiltered witha syringefilter (0.2μmcut-off).It is importanttonotethatthe80mMofNH.(aq)solutionwaspreparedfromacommercialsolutioncontaining28-30%ammoniumhydroxide,andthat theNH, concentration in this solution rapidlychanged,owingto evaporation.Therefore,theexact concentration oftheNH,(aq)solution required to coordinate silver ions and to modify the pH from 6 to 9 in the proposed experimental conditions needs to beoptimized. 5o0 μL of the resulting [Ag(NH,)] solution was added to the mixture of protein-coated nanostars and glucose. Vis-NIRspectrawererecordedafter2h.TheshiftinLSPRwascalculatedwithrespecttoablankexperimentintheabsenceofGOx.ReferencesZhang, Y.et al.A quantitative colorimetric assayofH,O,andglucose using silver nanoparticles induced byH,O, and UV.ChineseChem. Lett.24,10531058 (2013)2.Bastis, N.G., Merkoci, E, Piella, J.& Puntes,V. Synthesis ofhighly monodisperse citrate-stabilized silver nanoparticles ofup to 200 nm: kinetic control andcatalyticproperties.Chem.Mater.26,2836-2846 (2014)3.Wang, C., Liu, G, Liu, R,Li, W. & Zhang, W. Stability improving effect of silver diamminohydroxide precursor in green hydrothermal synthesis of silvernanoparticlecolloids.MicroNano Lett.9,320-324 (2014).4harnSatija,&MujGucoemediad syhisofgodnashlscile andfrndyappoachconfrring highllodal stabilityRC4, 3984 (2014)5. Lopez, A. & Liu, J. DNA-templated fluorescent gold nanoclusters reduced by Good's buffer: from blue-emitting seeds to red and near infrared emitters. Can.J. Chem. 93, 615620 (2015)6.Darroudi, M, Ahmad, M.B., Zak, A. K., Zamiri, R.& Hakimi, M. Fabrication and characterization of gelatin stabilized silver nanoparticles under UV-light, Int.LMoLSci12.6346-6356(2011)7.Hoppe, C. E, Lazzari, M, Pardinas-Blanco, IL & Lopez-Quintela, M. A, One-step synthesis of gold and silver hydrosols using poly(N-vinyl-2-pyrrolidone) as areducingagent.Langmuir22,7027-7034(2006).8.Wang, H., Qiao, X, Chen,J., Wang,X.& Ding,S. Mechanisms ofPVP in the preparation of silver nanoparticles.Mater. Chem.Phys.94,449-453 (2005).9.Jiang,C.,Nie,J.& Ma, G.Apolymer/metal coreshell nanofber membrane by electrospinning with an electric field, and its application for catalyst support.RSCAdv.6,22996-23007(2016)10. Tsuj, M. et al. Shape-dependent evolution of Au@Ag coreshell nanocrystals by PVP-assisted N,N-dimethylformamide reduction. Cryst. Growth Des.8,25282536 (2008)I1. Tsuj, M.et al. crystal structures and growth mechanisms of icosahedral Au@Ag coreshell and Au/Ag twin nanocrystals prepared by PVP-assisted N,N.dimethylformamide reduction. Cryst. Growth Des. 10, 4085-4090 (2010)12. Rodriguez-Lorenzo,Avarz-Puba,RGarcia dAbajo& LizMarzan,MSurfaceehanced Rmanscatering ung starshapedgold lloidnanoparticles.J.Phys.Chem.C114,7336-7340(2010),13. Brinson, B. E, et al. Nanoshells made easy: improving Au layer growth on nanoparticle surfaces. Langmuir 24, 14166-14171 (2008).14.Li,T.et al.Sensitive detection of glucosebased on gold nanoparticlesassisted silver mirror reaction.Analyst136,2893-2896 (2011)15.Fukui,R,Katayama,Y.& Miura,T,The influence ofpotential on electrodeposition ofsilver andformation of silver nanoparticles in some ionic liquidsJ.Electrochem.Soc.158,D567 (2011).16. Jiang, X. C., Chen, W. M, Chen, C. Y,Xiong S. X. & Yu, A. B. Role of temperature in the growth of silver nanoparticles through a synergetic reduction approach.Nanoscale Res. Lett. 6, 32 (2011)17. Piella,J. Bastis, N. G.& Puntes, V. Size-controlled synthesis of sub-10-nanometer citrate-stabilized gold nanoparticles and related optical properties. Chem. Mater28, 10661075 (2016).18. Luo, X,Xu, M, Freeman, C., James, T & Davis,J.J Ultrasensitive label free electrical detection ofinsulin in neat blood serum. Anal. Chem. 85, 41294134 (2013)19. Averseng,O.,Hagege,A,Taran &Vidaud,C.Surfaceplasmon resonancefor rapid screening ofuranyl affine proteins.Anal. Chem.82,9797-9802 (2010)20. Zhu,H,Dale,P. S., Caldwell,C. W. &Fan,X. Rapid and label-free detection of breast cancer biomarker CA15-3 in clinical human serum samples with optofluidicrs.AnalChem.81.9858-9865(2009)ingresona21, Kumar, P, S., Pastoriza-Santos,, Rodriguez-Gonzalez,B,Garcia de Abajo,J.& Liz-Marzan,L M, High-yield synthesis and optical response ofgold nanostarNanotechnology19,015606 (2008).207NATUREMATERIALS|VOL17|FEBRUARY2O18www.nature.com/naturematerials@ 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved
NATURE MATERIALS | VOL 17 | FEBRUARY 2018 | www.nature.com/naturematerials 207 NATURE MATERIALS ARTICLES were recorded after 2 h. The LSPR shift was calculated with respect to a blank experiment in the absence of the target molecule. The reduction of silver ions by GOx covalently attached to the nanostars (Fig. 2) was achieved by following the following protocol. Modification of gold nanostars with glucose oxidase. NSs were synthesized according to a previously published method with a modification in the molecular weight of PVP (Mw = 8,000, Alfa-Aesar 41626)21. After removing excess PVP by centrifugation and redispersion in isopropanol (2950 × g, 3 times), the gold and PVP concentrations were 0.85 mM and ~3 mM, respectively. 10 mL of NSs ([Au] = 0.86 mM) were centrifuged at 2,950 × g for 30 min at 25 °C. The NSs were redispersed in 10 mL of sodium bicarbonate buffer (10 mM, pH 9). The gold concentration was 0.74 mM. 1 mL of 50% glutaraldehyde solution (Sigma G7651) were added to the NS solution. The final concentrations of glutaraldehyde and gold were 5% and 0.61 mM, respectively. The dispersion was then shaken gently (on a roller) for 3 h at 25 °C. Then the NSs were centrifuged at 1,880 × g for 20 min. The resulting pellet was redispersed in 10 mL sodium bicarbonate buffer (10 mM, pH 9). The final gold concentration was 0.57 mM. A stock solution of 2 mg mL–1 of GOx was prepared in sodium bicarbonate buffer (10 mM, pH 9). The GOx stock solution was serially diluted in sodium bicarbonate buffer (10 mM pH 9). An appropriate volume of the diluted GOx solutions was added to 0.57 mM gold nanostars and 2 mM NaCNBH3 in the sodium bicarbonate buffer (10 mM, pH 9) in order to obtain the desired final enzyme concentration. The final volume was 1 mL. The reaction was let to proceed for 3 h at room temperature. The resulting protein-modified nanostars were centrifuged at 1,000 × g for 20 min. The resulting pellet was redispersed in 400 μL of MES buffer 10 mM, pH 6. The final gold concentration was 0.5 mM. For the reduction of silver, a solution containing 1 M glucose in MES buffer (10 mM, pH 6) was prepared. 100 μL of a 1 M glucose solution was added to 400 μL of 0.5 mM protein-coated NSs in MES buffer for 1 h at room temperature. The addition of glucose resulted in the production of hydrogen peroxide by the enzyme. A fresh solution of AgNO3 (0.2 mM) in 80 mM NH3(aq) was prepared and filtered with a syringe filter (0.2 μm cut-off). It is important to note that the 80 mM of NH3(aq) solution was prepared from a commercial solution containing 28–30% ammonium hydroxide, and that the NH3 concentration in this solution rapidly changed, owing to evaporation. Therefore, the exact concentration of the NH3(aq) solution required to coordinate silver ions and to modify the pH from 6 to 9 in the proposed experimental conditions needs to be optimized. 500 μL of the resulting [Ag(NH3)2] solution was added to the mixture of protein-coated nanostars and glucose. Vis–NIR spectra were recorded after 2 h. The shift in LSPR was calculated with respect to a blank experiment in the absence of GOx. References 1. Zhang, Y. et al. A quantitative colorimetric assay of H2O2 and glucose using silver nanoparticles induced by H2O2 and UV. Chinese Chem. Lett. 24, 1053–1058 (2013). 2. Bastús, N. G., Merkoçi, F., Piella, J. & Puntes, V. Synthesis of highly monodisperse citrate-stabilized silver nanoparticles of up to 200 nm: kinetic control and catalytic properties. Chem. Mater. 26, 2836–2846 (2014). 3. Wang, C., Liu, G., Liu, R., Li, W. & Zhang, W. Stability improving effect of silver diamminohydroxide precursor in green hydrothermal synthesis of silver nanoparticle colloids. Micro Nano Lett. 9, 320–324 (2014). 4. Tharion, J., Satija, J. & Mukherji, S. Glucose mediated synthesis of gold nanoshells: A facile and eco-friendly approach conferring high colloidal stability. RSC Adv. 4, 3984 (2014). 5. Lopez, A. & Liu, J. DNA-templated fluorescent gold nanoclusters reduced by Good’s buffer: from blue-emitting seeds to red and near infrared emitters. Can. J. Chem. 93, 615–620 (2015). 6. Darroudi, M., Ahmad, M. B., Zak, A. K., Zamiri, R. & Hakimi, M. Fabrication and characterization of gelatin stabilized silver nanoparticles under UV-light. Int. J. Mol. Sci. 12, 6346–6356 (2011). 7. Hoppe, C. E., Lazzari, M., Pardiñas-Blanco, I. & López-Quintela, M. A. One-step synthesis of gold and silver hydrosols using poly(N-vinyl-2-pyrrolidone) as a reducing agent. Langmuir 22, 7027–7034 (2006). 8. Wang, H., Qiao, X., Chen, J., Wang, X. & Ding, S. Mechanisms of PVP in the preparation of silver nanoparticles. Mater. Chem. Phys. 94, 449–453 (2005). 9. Jiang, C., Nie, J. & Ma, G. A polymer/metal core–shell nanofiber membrane by electrospinning with an electric field, and its application for catalyst support. RSC Adv. 6, 22996–23007 (2016). 10. Tsuji, M. et al. Shape-dependent evolution of Au@Ag core−shell nanocrystals by PVP-assisted N,N-dimethylformamide reduction. Cryst. Growth Des. 8, 2528–2536 (2008). 11. Tsuji, M. et al. crystal structures and growth mechanisms of icosahedral Au@Ag core−shell and Au/Ag twin nanocrystals prepared by PVP-assisted N,Ndimethylformamide reduction. Cryst. Growth Des. 10, 4085–4090 (2010). 12. Rodríguez-Lorenzo, L., Álvarez-Puebla, R. A., García de Abajo, F. J. & Liz-Marzan, L. M. Surface enhanced Raman scattering using star-shaped gold colloidal nanoparticles. J. Phys. Chem. C 114, 7336–7340 (2010). 13. Brinson, B. E. et al. Nanoshells made easy: improving Au layer growth on nanoparticle surfaces. Langmuir 24, 14166–14171 (2008). 14. Li, T. et al. Sensitive detection of glucose based on gold nanoparticles assisted silver mirror reaction. Analyst 136, 2893–2896 (2011). 15. Fukui, R., Katayama, Y. & Miura, T. The influence of potential on electrodeposition of silver and formation of silver nanoparticles in some ionic liquids. J. Electrochem. Soc. 158, D567 (2011). 16. Jiang, X. C., Chen, W. M., Chen, C. Y., Xiong S. X. & Yu, A. B. Role of temperature in the growth of silver nanoparticles through a synergetic reduction approach. Nanoscale Res. Lett. 6, 32 (2011). 17. Piella, J. Bastús, N. G. & Puntes, V. Size-controlled synthesis of sub-10-nanometer citrate-stabilized gold nanoparticles and related optical properties. Chem. Mater. 28, 1066−1075 (2016). 18. Luo, X., Xu, M., Freeman, C., James, T. & Davis, J. J. Ultrasensitive label free electrical detection of insulin in neat blood serum. Anal. Chem. 85, 4129–4134 (2013). 19. Averseng, O., Hagège, A., Taran F. & Vidaud, C. Surface plasmon resonance for rapid screening of uranyl affine proteins. Anal. Chem. 82, 9797–9802 (2010). 20. Zhu, H., Dale, P. S., Caldwell, C. W. & Fan, X. Rapid and label-free detection of breast cancer biomarker CA15–3 in clinical human serum samples with optofluidic ring resonator sensors. Anal. Chem. 81, 9858–9865 (2009). 21. Kumar, P. S., Pastoriza-Santos, I., Rodríguez-González, B., García de Abajo, F. J. & Liz-Marzán, L. M. High-yield synthesis and optical response of gold nanostars. Nanotechnology 19, 015606 (2008). © 2 0 1 7 M a c mill a n P u bli s h e r s Li mit e d, p a rt o f S p ri n g e r N a t u r e. All ri g h t s r e s e r v e d. © 2 0 1 7 M a c mill a n P u bl i s h e r s Li mit e d, p a rt o f S p ri n g e r N a t u r e. All ri g h t s r e s e r v e d