Home Search Collections Journals About Contact us My IOPscience
Ultrahigh sensitivity made simple: nanoplasmonic label-free biosensing with an extremely low limit-of-detection for bacterial and cancer diagnostics
This article has been downloaded from IOPscience. Please scroll down to see the full text article.
2009 Nanotechnology 20 434015
(https://www.doczj.com/doc/422531939.html,/0957-4484/20/43/434015)
View the table of contents for this issue, or go to the journal homepage for more
Download details:
IP Address: 128.178.195.114
The article was downloaded on 21/12/2010 at 21:18
Please note that terms and conditions apply.
IOP P UBLISHING N ANOTECHNOLOGY Nanotechnology20(2009)434015(9pp)doi:10.1088/0957-4484/20/43/434015
Ultrahigh sensitivity made simple: nanoplasmonic label-free biosensing with an extremely low limit-of-detection for bacterial and cancer diagnostics
S Chen,M Svedendahl,M K¨a ll,L Gunnarsson and A Dmitriev
Department of Applied Physics,Chalmers University of Technology,41296G¨o teborg,Sweden
E-mail:alexd@chalmers.se
Received15May2009,in?nal form20July2009
Published2October2009
Online at https://www.doczj.com/doc/422531939.html,/Nano/20/434015
Abstract
We present a simple and robust scheme for biosensing with an ultralow limit-of-detection down
to several pg cm?2(or several tens of attomoles cm?2)based on optical label-free biodetection
with localized surface plasmon resonances.The scheme utilizes cost-effective optical
components and comprises a white light source,a properly functionalized sensor surface
enclosed in a simple?uidics chip,and a spectral analyzer.The sensor surface is produced by a
bottom-up nanofabrication technique with hole mask colloidal lithography.Despite its
simplicity,the method is able to reliably detect protein–protein binding events at low picomolar
and femtomolar concentrations,which is exempli?ed by the label-free detection of the
extracellular adherence protein(EAP)found on the outer surface of the bacterium
Staphylococcus aureus and of prostate-speci?c antigen(PSA),which is believed to be a prostate
cancer marker.These experiments pave the way towards an ultra-sensitive yet compact
biodetection platform for point-of-care diagnostics applications.
S Supplementary data are available from https://www.doczj.com/doc/422531939.html,/Nano/20/434015
(Some?gures in this article are in colour only in the electronic version)
1.Introduction
Quantitative biology and biomedicine are based on the ability to accurately determine biological quantities such as the amount of proteins expressed in an organism or the degree of interaction of a pharmaceutical drug with its target molecule.More speci?cally,clinical diagnostics critically rely on the ability to sensitively detect speci?c proteins in blood and other biological?uids.Naturally,the use of biosensors that are able to measure interactions between prede?ned‘capture’and‘target’biomolecules is a vast and continuously growing research?eld.A biosensor in general is a receptor–transducer device able to quantitatively provide analytical information using a biological recognition element (Nicu and Le¨?chl′e2008).A large effort in biosensors research is directed towards label-free detection schemes, where protein–protein interactions are reported without the need for pre-labeling of the analyte,for example,with ?uorescent markers.In this respect,optical biosensors provide a very promising route towards commercial ultra-sensitive biomolecular detection—they can be realized in a compact setting,have multiplexing capability and are foreseen to be quite cost effective(Cooper2006).A large number of optical biosensing schemes rely on the excitation of surface plasmons—propagating surface plasmon polaritons, commonly referred to as surface plasmon resonances(SPRs), and localized SPRs(LSPRs);both types are essentially the collective charge density oscillations bound to the metal–dielectric interface.The former can be excited on thin metal?lms through grating or prism couplers,and the latter readily couple to the excitation light due to their con?nement in the subwavelength(nanoscopic)metal particles.When excited,such collective oscillations of conduction electrons create regions of enhanced electromagnetic(EM)?elds in the direct proximity of the metal surface that are extremely sensitive to the minute changes of the refractive index—thus,
corresponding refractometric sensing emerges that detects the presence of biomolecular analytes bound to the surface of (L)SPR-supporting metals.SPR-based biodetection is a well-established research and development?eld,where solutions for drug discovery and biomedical diagnostics are readily available in the research laboratory environment(Blow2009, Homola2008).However,point-of-care medical diagnostics is hardly reachable with this method as the instrumentation is bulky,expensive and relatively complex to operate.In contrast,LSPR-based biochemodetection gives the opportunity for the development of a very compact and yet extremely sensitive setup that can be developed into a fully functional clinical diagnostics device.Localized SPRs are supported in metal nanoparticles that can be produced as stand-alone features or in an array format with a variety of nanofabrication methods for use in sensing applications(Stewart et al2008). When properly functionalized with‘capture’biomolecules, such nanoparticles become a powerful tool for optical label-free biodetection(Anker et al2008,Lal et al2008,Willets and Van Duyne2007).Zeptomole sensitivities are directly attainable with single-particle LSPR biosensing(McFarland and Van Dyune2003)and recent reports point towards the possibility of complete integration of the sensing setup into a hand-held device(Neuzil and Reboud2008).
Careful design of nanoplasmonic resonators for biomolec-ular detection employs detailed experimental and theoretical characterization of the enhanced EM?elds around the nanos-tructures(Unger and Kreiter2009,Unger et al2009,Nusz et al 2009).It is recognized that nanostructures containing sharp features,e.g.,nanorice(Wang et al2006),nanostars(Nehl et al2006),nanocrescents(Unger and Kreiter2009,Unger et al2009)or arrangements of strongly interacting nanopar-ticles with tight interstitial gaps(e.g.,nanoparticles dimers (Acimovic et al2009))generally provide high refractive index (RI)sensitivity.In contrast,symmetric nanostructures such as nanodisks present only moderate EM?eld enhancements (Dmitriev et al2008).Nonetheless,here we demonstrate that even the use of a very simple nanoplasmonic sensing platform (i.e.,an array of non-interacting short-range ordered Au nanodisks that covers a large sensor area)yields an extremely low limit-of-detection(LOD)for biomolecular sensing(see below).The latter is achieved without the need for a drastic reduction of the number of probed nanoplasmonic particles(frequently down to the single-particle limit),which is generally considered as a viable route to reduce the number of probed analyte molecules,thus pushing down the detection limit.Notably,the RI sensitivity in symmetric nanostructures such as the high-and low-aspect ratio nanodisks employed here is essentially polarization-independent,which facilitates excitation and detection in the optical biosensing setup.In addition,this platform is produced by the affordable bottom-up nanofabrication.
The present study thus demonstrates a LSPR-based biomolecular detection scheme that is robust,simple and cost effective,and at the same time is characterized by ultrahigh sensitivity down to several tens of attomoles of the analyte detected per cm2of the sensor surface.The chosen clinical target analytes for the detection are the extracellular adherence protein(EAP),found on the outer surface of the bacterium Staphylococcus aureus(S.aureus)(Harraghy et al2003),and prostate-speci?c antigen(PSA).The EAP,a protein of60kDa excreted by S.aureus,has af?nity for both eukaryotic cells and the surface of S.aureus itself.The EAP enhances the adherence of S.aureus to human cells.S.aureus is one of the most common agents causing bacterial infection in humans and is a highly clinically relevant bacterium(Lowy 1998).S.aureus causes a wide range of infections from wound infections to more life-threatening conditions such as endocarditis,osteomyelitis,and septic shock.PSA is a glycol protein of33kDa produced by cells in the prostate gland.A low level of PSA(typically below2.5ng ml?1)is normally constantly present in the blood of a healthy man;however, prostate cancer and benign conditions can result in an increase of the PSA level.Since many factors can cause?uctuations of the PSA level,a detected increase does not necessarily suggest a diagnosis of prostate cancer,rather continuous monitoring of the PSA level is extremely helpful in predicting the prostate cancer risk and aggressiveness and could be used to determine whether a prostate biopsy should be considered(Loeb and Catalona2008).
2.Experimental details
2.1.Optical setup and sensing surface nanofabrication
The optical setup used consists of a compact white light source (20W HL-2000Tungsten Halogen,Ocean Optics),a sensing chip that is an assembly of glass-supported Au nanostructures enclosed in a simple stainless steel?uidics device with the analyte solution kept at static conditions once injected,and a miniature spectral analyzer(B&WTek BRC711E PDA), coupled to a personal computer(see?gure1(a)).The light spot probing the surface of the sensor is～0.13cm2(as measured with a simple ruler).For some of the presented biodetection assays,namely,for those with a low concentration of injected biotinylated bovine serum albumin(bBSA;1and10ng ml?1), EAP and PSA assays,a polarizer was present between the illuminating?ber and the?uidic sensor chip.Normally,it is used for other than normal illumination conditions to bestow the setup with more versatility(not discussed in the present context).The instrument performance in fact experiences a slight improvement in terms of LSPR peak shift upon analyte injection if the polarizer is removed(with normal illumination),thus making the setup more compact.The latter is further supported by the direct comparison of biomolecular adsorption kinetics monitored with and without employing the polarizer(see?gure S1in supporting information available at https://www.doczj.com/doc/422531939.html,/Nano/20/434015).The nanoplasmonic sensor chips were produced by hole mask colloidal lithography (HCL)on regular microscope slide glass substrates(VWR International,thickness200μm).HCL is a bottom-up technique that relies on the self-assembly of negatively charged polystyrene colloidal particles(Interfacial Dynamics Corp.) onto the positively charged surface of a layer of polymer resist(polymethyl methacrylate,PMMA)(Fredriksson et al 2007).Deposition of a thin metallic(Au)?lm over the
Figure1.(a)Experimental setup and a typical LSPR sensor surface: (b)—top view of the typical Au nanodisk array,employed in the current study;(c),(d)—side views of the high-and low-aspect ratio nanodisks,respectively.The scale bar is common for(c)and(d). self-assembled particles,which are subsequently removed by tape-stripping,results in an evaporation mask in the form of a short-range ordered array of nanoscopic holes with the size determined by the size of the colloidal particles.By employing an oxygen plasma treatment,the polymer?lm is then etched through,which means that materials can be evaporated through the mask holes onto the surface.A large variety of nanostructured geometries can be manufactured with HCL(Dmitriev et al2007,Fredriksson et al2007,Pakizeh et al2008),the simplest case being short-range ordered arrays of noble metal nanodisks that have been shown to be excellent refractometric nanosensors and a highly tunable nanoplasmonic system(Dmitriev et al2008,Toftegaard et al 2008).Figures1(b)–(d)show representative SEM micrographs of such arrays,where the nanodisks have a diameter of140nm and a height of20nm(?gures1(b)and(c))and100nm (?gure1(d)).The aspect ratio of the nanodisks is de?ned as the diameter-to-height ratio,so that?gures1(c)and(d)exemplify high-and low-aspect ratio nanostructures,respectively.In the present work we use high-aspect ratio nanodisks with a diameter of120nm and a height of30nm,and low-aspect ratio nanodisks with a diameter of100nm and a height of 70nm.Each nanodisk in the array also features a thin adhesion layer of Cr(1nm)to provide better contact between the Au nanostructures with the surface of the glass slide and to allow for thorough cleaning/recycling of the sensing chip (the cleaning between adsorption cycles is done with TL1 wash).The surface coverage for both types of nanodisks is close to11%.However,in the biosensing experiments, the analytes adsorb on the entirety of the nanodisk surface, which necessarily includes the sidewalls,though certainly some steric hindrance can occur.Thus,the total Au surface coverages of the samples with120nm×30nm and100nm×70nm nanodisks are estimated at20%and32%,respectively. Importantly,such nanodisk arrays produce a distinct LSP resonance in the visible and near-IR range,characterized by the strong extinction(combined absorption and scattering)of the incoming light.For the nanodisk geometries of120nm×30nm and100nm×70nm used in this work,the LSP resonances in ambient atmosphere are centered at645.8nm and590.3nm,respectively(cf,?gures3(a)and(b)).We note that due to the characteristic center-to-center distance of2–3diameters,the nanodisks in the HCL-fabricated array are only weakly interacting,as their lateral separation exceeds the range of plasmon induced near-?eld coupling(Esteban et al2008).Due to the lack of long-range periodicities, there is also only a weak diffractive coupling between the particles.The spectral signature of the arrays is therefore a good representation of the spectral response of a single nanodisk,but with additional inhomogeneous broadening due to the?nite size distribution in the array(Hanarp et al2003). By assembling the glass substrate supporting the nanodisk array into a vertically positioned home-built liquid-tight?ow cell with a total volume of～1.1ml(Dahlin et al2005) (cf,?gure1(a))and exposing the surface with nanodisks to various liquids,the LSPR of the nanodisk array shifts towards longer wavelengths.In particular,in the standard biosensing experiment,protein buffer solution with an appropriate pH is used to inject the target analytes into the?ow cell.
2.2.Preparation of the sensing surface and LSPR biosensing Prior to the protein binding assay,the gold nanodisk substrates were cleaned in TL1(5:1:1,milliQ water:H2O2(30%v/v): NH4OH(25%v/v))at70?C for5min.The LSPR peak position was analyzed in real time with an algorithm previously described by Dahlin et al(2006).All protein adsorption measurements were performed in HEPES buffer(150mM NaCl,pH7.2).Prior to the EAP and PSA antigen detection experiments,the TL1-cleaned substrates were immersed in 1mM16-mercaptohexadecanoid acid(MHDA)in ethanol to form a1.9nm self-assembled monolayer(SAM)on the gold surface.The carboxylic acid end groups were then activated by a mixture of1:10.4M N-(3-dimethylaminopropyl)-N -ethylcarbodiimide hydrochloride(EDC)and0.1M N-hydroxysuccinimide(NHS)to amine-couple the antibody to the surface.In the EAP assay experiments,arrays of100nm×70nm nanodisks were used.An unknown concentration of anti-EAP(protein-puri?ed,provided by Professor Jan-Ingmar Flock,Karolinska Hospital University,Stockholm,Sweden) was injected into the?ow cell followed by deactivation of the surface by ethanolamine to inhibit unspeci?c binding of the antigen(EAP)molecule to the SAM.Concentrations of EAP ranging from8.3nM to2.0μM in buffer were injected into the?ow cell.Both in the bBSA sensing experiment and for PSA detection,arrays of120nm×30nm nanodisks were
Figure2.Probing the spatial extent of the RI sensitivity.The LSPRs of the nanodisk arrays shift upon the deposition of thin alumina(a)and silica(b)?lms on high-aspect ratio(a)and low-aspect ratio nanodisks(b),respectively.(c)The sensitivity decay length is extracted from
?tting(corresponding red and blue solid curves)the LSPR peak shift-versus-dielectric?lm thickness experimental data(blue rhombs—high-aspect ratio nanodisks,red squares—low-aspect ratio nanodisks)by using equation(5)—see the text.Schematic models of the respective nanodisk con?gurations are given on the insets.
used.As anti-PSA,20μg ml?1monoclonal anti-Kallikrein3 (anti-Klk3)was adsorbed on the sensing surface.1ng ml?1 of PSA was subsequently injected into the?ow cell,again after deactivation with ethanolamine.To minimize unspeci?c binding of the antigen molecules(PSA and EAP),the surface was deactivated by ethanolamine following the coupling of the antibodies(anti-EAP and anti-Klk3).All proteins and chemicals were purchased from Sigma-Aldrich unless stated otherwise.
3.Results and discussion
3.1.Probing the extent of the sensing volume by thin dielectric ?lms
As mentioned earlier,excited LSPRs are extremely sensitive to changes in the direct dielectric environment of the nanoparticles.The extent of this environment—i.e.,the ‘sensing volume’of a particular nanoplasmonic sensing platform—can be effectively probed by the sensitivity decay length curves.By depositing a sequence of thin dielectric layers onto the nanodisks,the LSPR peak shift is monitored (see?gure2).Dielectric layers of Al2O3and SiO2were deposited on the arrays of high-aspect ratio(120nm×30nm) and low-aspect ratio(100nm×70nm)nanodisks,respectively, via electron-beam evaporation in a high-vacuum evaporation system(A V AC HVC600).High-aspect ratio nanodisks have LSPR at645.8nm in air that upon the stepwise deposition of 33nm of Al2O3(with the deposition step3nm)eventually shifts to732.3nm(?gure2(a)).Low-aspect ratio nanodisks resonate at590.3nm in air and at610.7nm upon the deposition of31nm of SiO2with the deposition step of5±0.5nm (?gure2(b)).We note that the refractive index change in the proximity of the nanostructures induced by the presence of the thin?lm is intended to model the eventual biomolecular adsorption,though the latter would naturally correspond to considerably lower absolute changes.By plotting the intermediate thicknesses of the respective dielectric?lms, we obtain the relation between the stepwise increase of the effective refractive index and the LSPR response of the nanodisks system(?gure2(c)).Note that both high-and low-aspect ratio nanodisks experience a sensitivity saturation close to30nm above the surface of the nanodisks.Taking into account LSP resonance positions and?lm material/dielectric constants along with the deposition methods,these values further complement previously reported numbers for the extent of a sensing volume for surface-supported nanoplasmonic structures(Dmitriev et al2007,Whitney et al2005).In general,the adsorbed mass on the sensor surface can be approximated by De Freijter’s formula,which is based on the refractive index change:
=d n
?n
?c
(1)
where d is the dimension of the adsorbed species(biomolecule) in nanometers, n is the refractive index difference between
the medium and the species,and?n
?c
is the biomolecule refractive increment.For proteins,the commonly accepted value of the latter is0.182cm3g?1(V¨o ros2004).
The spectral response of the refractometric nanoplasmonic sensors can be described by the simple but general formula:
λ=m(n eff?n medium)(2) where m is the RI sensitivity,expressed in LSPR peak shift per refractive index unit(RIU),and n eff is the effective refractive
Figure3.bBSA assay.(a)LSPR peak shift upon the adsorption of bBSA on high-aspect ratio nanodisks close to saturation coverage. Inset—zoom-in of the top part of the spectra.(b)bBSA adsorption kinetics monitored as the LSPR peak shift with time.Injected bBSA concentrations,corresponding to the solid lines,are given on the right.Dotted line—adsorption kinetics of1mg ml?1of injected bBSA.Inset—?rst5min of the kinetics,collected with pure buffer solution prior to the bBSA injection for the estimation of the
limit-of-detection.
index of the adsorbate layer.In the simplest approximation,the LSPR-induced evanescent?eld decays exponentially from the
surface as:
E(z)=exp
?z
l d
(3)
with a decay length l d.The effective refractive index of the adsorbate layer is described as:
n eff=2
l d
d
n(z)E(z)2d z.(4)
Inserting equations(2)–(4)into equation(1)gives the relationship of the LSPR peak shift with the surface coverage of the adsorbate:
(t)=d λ(t)
m(1?exp(?zd/ld))?n
?c
.(5)
Equations(2)–(4)were used to?t the data in?gure2(c)and to determine the RI sensitivities,m,and the decay lengths, l d,of the two different sensor substrates.The obtained ?tting parameters give l d=39nm and m=173nm/RIU for the high-aspect ratio nanodisks and l d=28nm and m=56nm/RIU for the low-aspect ratio nanodisks.Taking into account that the nanodisks in the present study are directly supported on the surface and thus have reduced RI sensitivities as compared to solution-based nanoplasmonic systems(Dmitriev et al2008),these values come close to the previously reported linear dependence of RI sensitivity on the LSP resonance position(Miller and Lazarides2005).
3.2.LOD for biomolecular sensing
The detection limit for biomolecular sensing with the present setup was probed by the adsorption of bBSA on the sensor surface comprising high-aspect ratio nanodisks(120nm×30nm).Different concentrations of bBSA were injected into the?uidic cell,allowing for1h of incubation under static conditions,i.e.,no?ow was used,followed by a rinsing step with clean buffer.It has been reported previously that at low concentrations also used here the bBSA preferably binds to the gold surface rather than to the surface of the supporting glass slide(Svedhem et al2003).In the present study,this observation was checked with QCMD measurements of the injected1.43μM bBSA on three different surfaces:a plain Au ?lm,a SiO2(glass)surface and high-aspect ratio Au nanodisks (120nm×30nm)fabricated on the QCMD quartz crystal. The data(not shown)evidenced no detectable QC frequency shift for the glass surface,whereas the plain Au?lm and the nanodisks system induced sizable shifts.Upon bBSA adsorption,the frequency shift on the plain Au substrate was approximately5times larger than the one of the nanodisk sample,which corresponds well to the Au-to-glass ratio of the employed nanodisk array:as discussed above,the Au surface area of the high-aspect ratio nanodisks amounts to20%of the total sensor surface.
The LSPR of the sensing surface reacts on the presence of the bBSA with a resonance shift towards longer wavelength (?gure3(a)).Apart from the peak shift detection before and after the completion of adsorption,the adsorption kinetics can be comfortably monitored with LSP resonance shift in real time.The kinetic responses of the nanosensor surface for the different injected bBSA concentrations ranging from 1mg ml?1down to100ng ml?1are plotted in?gure3(b). Most strikingly,the standard deviation of the signal(i.e., LSPR peak position),collected during the?rst5min of the experiment and plotted in the inset of?gure3(b),demonstrates a value as low as9.9×10?5nm(compare this,for example,to a previously reported short-term noise level of5.0×10?4on a similar system(Dahlin et al2006)).This value will be used below for the de?nition of the LOD,which we de?ne here in analogy with SPR biosensors,i.e.as the signal from the blank
sample(no analyte)plus the standard deviation of the signal from the blank sample times three(desired con?dence level) (Homola and Piliarik2006).
After incubation of 1.43μM bBSA(cf?gures3(b) and(c))for60min the LSPR peak shifts by3nm, which corresponds to a fully saturated layer of bBSA on the nanodisks.Indeed,a further increase of the injected concentration does not result in a larger peak shift(?gures3(b) and(c)).Using the previously extracted RI sensitivity and the sensitivity decay length for the120nm×30nm nanodisk array, the absorbed mass of bBSA molecules can be estimated using equation(5).For a peak shift of3nm the calculated adsorbed mass is205ng cm?2of the Au surface or41ng cm?2of the total sensor surface.The thickness of the protein layer was here approximated by4nm.This corresponds to bBSA molecules conformed?at on the surface,and thus having the largest surface footprint(4nm×8nm).If we instead assume that the bBSA molecules are conformed upright with a corresponding footprint of4nm×4nm and thus an adsorbed layer thickness of8nm,the calculated mass for a3nm shift would be 226ng cm?2of the Au surface area and45ng cm?2of the total sensor surface.Most realistically,the true thickness of the adsorbed bBSA layer is in between these two extreme cases. In addition,if bBSA molecules are regarded as hard spheres with a diameter of8nm(the largest dimension of a BSA molecule according to its molecular structure),their adsorption can be described in terms of the random sequential adsorption (RSA)model for non-interacting entities(Adamczyk et al 1994).Despite its simplicity,RSA has previously provided good insight into the mechanisms of protein adsorption(Tie et al2003).In its simplest form,the RSA model assumes that the adsorbing particles are hard spheres that impinge sequentially all over the surface at randomly chosen positions. Employing RSA modeling further implies that the maximum attainable bBSA coverage would be about55%(Adamczyk et al1994).As a consequence,around250bBSA molecules can potentially adsorb on the surface of one nanodisk,which gives a mass of127ng cm?2of gold surface or25ng cm?2of the sensor surface.Since bBSA has an ellipsoid shape with two axes of4and8nm,an average diameter can be used as an approximation in the model.Therefore,we presume that440bBSA molecules can potentially adsorb per nanodisk, which corresponds to a mass of226ng cm?2of gold surface or45ng cm?2of the sensor surface.For the experiment with the lowest injected bBSA concentration,i.e.,1ng ml?1 corresponding to14.3pM,a peak shift of0.05nm was detected,which gives a corresponding mass of3.8ng cm?2 (i.e.,about7.4bBSA molecules per nanodisk),calculated assuming an8nm protein layer thickness.
With the extremely low noise level of the present system (10?4nm),the interpolated detection limit would be about 4bBSA molecules per100nanodisks,which effectively corresponds to20pg cm?2of gold surface and,since the total gold area of the disks is20%of the total sensor area,the potentially attainable LOD would be as low as4pg cm?2of protein binding(57attomoles cm?2,or,considering the size of the illumination spot,7.5attomoles of bBSA).Notably, with the above-mentioned SPR-based biosensors the
ultimate Figure4.EAP assay.(a)Complete kinetics series—starting with16 MHDA SAM-functionalized nanodisk surface,followed by the EDC/NHS activation,binding of anti-EAP,surface deactivation and ?nally the injection of the EAP analyte solution at three different concentrations,marked on the right.(b)Mapping the
limits-of-detection for EAP with various injected concentrations and following the eventual LSPR peak shift upon complete adsorption. achievable LOD,considering operation at a wavelength of 760nm and a typical analyte such as DNA or BSA,is 0.91pg mm?2(Homola2008).It is worth mentioning that earlier developed LSPR-based biodetection schemes, which did not involve single-particle sensing and were based on surface-con?ned bottom-up nanoplasmonic particle arrays(similarly to the present case),demonstrated detection limits in the low picomolar–high femtomolar range(solution concentration),corresponding to100–1000pg mm?2of areal mass sensitivity(Haes and Van Duyne2002,Riboh et al 2003).LSPR-based colorimetric detection with solution-phase oligonucleotide-modi?ed plasmonic nanoparticles equally yields a low-picomolar–mid femtomolar LOD(Rosi and Mirkin2005).
3.3.EAP binding assay
As discussed in section1,there is a high demand for ultra-sensitive and compact biomedical sensing schemes. Armed with the exceptional sensing characteristics of the biochemosensing setup discussed in the present study,we show that it is indeed feasible to consider the LSPR sensing scheme for the development of a fully functional diagnostics tool for point-of-care usage.We exemplify the potential of the devised setup with the detection of a S.aureus-related protein—the EAP,mentioned earlier.Figure4presents the complete kinetics sequence of the LSPR-detected functionalization of the sensing chip with the eventual detection of three injected concentrations of EAP,i.e.1,5and50μg ml?1(see ?gure4(a)).Notably,for this case we have chosen a low-aspect ratio nanodisk system(100nm×70nm)to ensure the
positioning of the LSP resonance in the middle of the visible spectral range.The latter feature would eventually facilitate the construction of a hand-held LSPR biochemosensor as the current availability,cost,dimensions and the integration capabilities for the optical components in the visible range are extremely promising.As evidenced in?gure4(a),reliable detection of EAP is readily achieved.Interestingly,the antibody functionalization step(at the21st minute as in ?gure4(a))resulted in a2.2nm LSPR peak shift.Assuming that the16MHDA SAM provides a1.9nm spacer between the antibody and the surface of a nanodisk and the size of vertically conformed anti-EAP(about15nm),the amount of antibody coupled to the surface is estimated as645ng cm?2 on Au or about770molecules per nanodisk.This number can help to estimate the number of EAP molecules per nanodisk for the areal mass sensitivity extraction,though one needs to take into account that EAP is prone to oligomerization(Flock and Flock2001),which is a probable reason for the observation of a roughly three times larger signal from EAP adsorption as compared to the antibody adsorption.Again,for the estimation of the LOD the saturation coverage of EAP was achieved at the2μM injected concentration(?gure4(b)),which relates to a LSPR peak shift of1.26nm.Correspondingly,the lowest injected EAP amount was8.3nM with a LSPR response of 0.03nm(?gure4(b)).Considering the noise level extracted from?gure4(a),the estimated detection limit gets down to an EAP concentration of8pM.Importantly,Flock et al have shown that only about30%of the excreted EAP molecules re-bind to the surface of S.aureus,resulting in less than 50×103molecules per bacterium that are available for binding with the present assay(Flock and Flock2001).In a typical whole blood sample,the amount of bacteria is less than 30ml?1(Wellinghausen et al2009),which indicates that a high femtomolar sensitivity would assure a reliable working tool for S.aureus diagnostics
3.4.PSA binding assay
Another biomedical application for LSPR sensing is the detection of PSA.As mentioned earlier,continuous monitoring of PSA levels is needed for timely cancer diagnostics purposes, so the development of a portable and sensitive detection solution would be largely appreciated by the biomedical community.In our PSA assay,presented in?gure5, 20μg ml?1of the injected antibody gives rise to a LSPR peak shift of3.962nm.The size of the antibody can be considered as15nm(in the vertical dimension).When those values are used in equations(1)–(5),the bound protein mass is calculated as441ng cm?2on the nanodisk surface or207 antibody molecules per nanodisk.Notably,this is comparable to the estimated number of bBSA molecules per nanodisk, discussed earlier.
When1ng ml?1of PSA solution is injected onto the anti-PSA functionalized surface after the ethanolamine deactivation,a noticeable peak shift of0.037nm is detected (inset of?gure5).The PSA molecules are relatively small (33kDa);the size of PSA,derived from its crystallographic structure,is3.8nm×3.8nm,so plugging these numbers
into Figure5.PSA assay.The adsorption series for binding PSA starts with the activation of16MHDA SAM by EDC/NHS,and is followed by the binding of anti-PSA,surface deactivation and PSA injection at 1ng ml?1.Inset—zoom(on the vertical scale)of the kinetics curve region directly adjacent to the inset box.
equations(1)–(5)one could relate the detected LSPR peak shift to32.7ng cm?2of Au nanodisk area or,on average,to 15bound PSA molecules per nanodisk.With the extremely low noise level of the system,we could tentatively project the LOD to0.3ng cm?2of the total sensor surface(if we consider the5min before the PSA1ng ml?1injection,the standard deviation is4.5×10?4nm which gives a signal-to-noise ratio of82and thus the LOD32.7/82×3= 1.2ng cm?2on the nanodisk surface(1PSA molecule per two nanodisks),corresponding to1.2×0.20=0.3ng cm?2 on the total sensor area).With such reasoning,it appears possible to be able to potentially observe about36.5pg ml?1 (1pM)(note that using the noise level extracted from the bBSA binding experiments here,this value is pushed further down to8.1pg ml?1(250fM)—LOD of10pg cm?2).It is worth mentioning that the total amount of PSA that binds to the surface is greatly diffusion limited,and an enhanced micro?uidic system with a constant?ow could provide yet lower detection limits.Previous studies of PSA detection using an immunochromatographic assay,where Au plasmonic nanoparticles were used as labels,conjugated to primal antibodies,demonstrated LOD of0.2ng ml?1(Tanaka et al 2006).Further comparison to similarly low levels of PSA detection(1ng ml?1)that used aggregation of colloidal Au plasmonic nanoparticles with a sandwich immunoassay(Cao and Sim2007)demonstrates how the biodetection method presented here,while keeping ultrahigh sensitivity,provides a straightforward possibility for multiplexing due to the presence of the supporting surface.In another study,by using secondary antibodies to enhance the performance of a SPR-based biosensor,the LOD of PSA detection with SPR was shown to be10ng ml?1(300pM)(Cao et al2006). Huang et al utilized a secondary antibody and colloidal Au nanoparticles to demonstrate the sensitivities at1ng ml?1of PSA(30pM)(Huang et al2005),whereas Choi et al used the same concept to further push down the LOD to300fM(Choi et al2008).This number compares well to the potential LOD of the biosensing scheme presented here(250fM),with the
latter obviously being free from extra labeling with secondary antibody and nanoplasmonic colloidal particles.
4.Conclusions
We demonstrated a simple scheme for label-free biomolecular optical detection that nonetheless presents a possibility for biomolecular sensing with an ultrahigh sensitivity of several pg cm?2(or several tens of attomoles cm?2).The distinct advantages of the present scheme are its cost-effective constituent optical components,large-scale bottom-up nanofabrication of nanoplasmonic sensing surfaces,and the above-mentioned ultrahigh areal mass sensitivity that, to the best of our knowledge,not only surpasses by more than one order of magnitude the performance of SPR biosensing instruments,but also delivers so far the best areal mass sensitivity among comparable LSPR-based biodetection schemes.In the example EAP and PSA assays,the present scheme also demonstrates state-of-the-art sensitivities, required in clinical settings.As the presented scheme does not utilize any temperature and/or mechanical stabilization,we believe that with the further introduction of a slightly more sophisticated and ef?cient micro?uidic liquid-handling system and integration of the used optical components on a single board,the presented biodetection platform might provide a viable route towards the construction of a simple and broadly accessible point-of-care diagnostics tool kit for use in clinical settings or in personalized health care systems. Acknowledgments
We acknowledge the?nancial support of the Swedish Research Council.Magnus Jonsson(Applied Physics,Chalmers) is greatly acknowledged for the assistance with QCMD measurements and helpful discussions.Professor Jan-Ingmar Flock,Karolinska Hospital University,Stockholm,Sweden is acknowledged for providing the anti-EAP and EAP for the biosensing assay.
References
Acimovic S S,Kreuzer M P,Gonz′a lez M U and Quidant R2009 Plasmon near-?eld coupling in metal dimers as a step toward
single-molecule sensing ACS Nano31231
Adamczyk Z,Siwek B,Zembala M and Belouschek P1994Kinetics of localized adsorption of colloid particles Adv.Colloid
Interface Sci.48151
Anker J N,Hall W P,Lyandres O,Shah N C,Zhao J and
Duyne R P V2008Biosensing with plasmonic nanosensors Nat.
Mater.7442
Blow N2009Proteins and proteomics:life on the surface Nat.
Methods6389
Cao C,Kim P J,Kim B W,Chae H,Yoon C H,Yang S S and Sim S J2006A strategy for sensitivity and speci?cify
enhancements in prostate speci?c antigen-α1-antichymotrypsin detection based on surface plasmon resonsnace Biosens.
Bioelectr.212106
Cao C and Sim S J2007Preparation of highly stable oligo(ethylene glycol)derivatives functionalized gold nanoparticles and their
application in LSPR-based detection of PSA/ACT complex
J.Nanosci.Nanotechnol.73754Choi J W,Kang Da Yeon,Jang Y H,Kim H K,Min J and Oh B K2008Ultra-sensitive surface plasmon resonance based immunosensor for prostate-speci?c antigen using gold
nanoparticles–antibody complex Colloids Surf.A313655 Cooper M A2006Optical biosensors:where next and how soon?
Drug Discov.Today111061
Dahlin A,Tegenfeldt J O and Hook F2006Improving the instrumental resolution of sensors based on localized surface
plasmon resonance Anal.Chem.784416
Dahlin A,Z¨a ch M,Rindzevicius T,K¨a ll M,Sutherland D S and H¨o¨o k F2005Localized surface plasmon resonance sensing of
lipid-membrane-mediated biorecognition events J.Am.Chem.
Soc.1275043
Dmitriev A,H¨a gglund C,Chen S,Fredriksson H,Pakizeh T, K¨a ll M and Sutherland D S2008Enhanced nanoplasmonic
optical sensors with reduced substrate effect Nano Lett.83893 Dmitriev A,Pakizeh T,K¨a ll M and Sutherland D S2007
Gold–silica–gold nanosandwiches:tunable bimodal plasmonic resonators Small3294
Esteban R,V ogelgesang R,Dorfmueller J,Dmitriev A,Rockstuhl C, Etrich C and Kern K2008Direct near-?eld optical imaging of higher order plasmonic resonances Nano Lett.83155
Flock M and Flock J-I2001Rebinding of extracellular adherence protein Eap to Staphylococcus aureus can occur through a
surface-bound neutral phosphatase J.Bacteriol.1833999 Fredriksson H,Alaverdyan Y,Dmitriev A,Langhammer C, Sutherland D S,Z¨a ch M and Kasemo B2007Hole-mask
colloidal lithography Adv.Mater.194297
Haes A J and Van Duyne R P2002A nanoscale optical biosensor: sensitivity and selectivity of an approach based on the localized surface plasmon resonance spectroscopy of triangular silver
nanoparticles J.Am.Chem.Soc.12410596
Hanarp P,K¨a ll M and Sutherland D S2003Optical properties of short range ordered arrays of nanometer gold disks prepared by colloidal lithography J.Phys.Chem.B1075768
Harraghy N,Hussain M,Haggar A,Chavakis T,Sinha B, Herrmann M and Flock J-I2003The adhesive and
immunomodulating properties of the multifunctional
Staphylococcus aureus protein Eap Microbiology1492701 Homola J2008Surface plasmon resonance sensors for detection of chemical and biological species Chem.Rev.108462
Homola J and Piliarik M2006Surface plasmon resonance(SPR) sensors Surface Plasmon Resonance Based Sensors(Springer
Ser Chem Sens Biosens)vol4ed J Homola(Berlin:Springer) Huang L,Reekmans G,Saerens D,Friedt J M,Frederix F,Francis L, Muyldermans S,Campitelli A and van Hoof C2005
Prostate-speci?c antigen immunosensig based on mixed
self-assaembled monolayers,camel antibodies and colloidal
gold enhanced sandwich assays Biosens.Bioelectron.21483 Lal S,Link S and Halas N J2008Nano-optics from sensing to wavelguiding Nat.Photon.1641
Loeb S and Catalona W J2008What to do with an abnormal PSA test?Oncologist13299
Lowy F D1998Staphylococcus aureus infections New Eng.J.Med.
339520
McFarland A D and Van Dyune R P2003Single silver nanoparticles as a real-time optical sensors with zeptomole sensitivity Nano
Lett.31057
Miller M M and Lazarides A A2005Sensitivity of metal nanoparticle surface plasmon resonance to the dielectric
environment J.Phys.Chem.B10921556
Nehl C L,Liao H and Hafner J H2006Optical properties of star-shaped gold nanoparticles Nano Lett.6683
Neuzil P and Reboud J2008Palm-sized biodetection system based on localized surface plasmon resonance Anal.Chem.806100 Nicu L and Le¨?chl′e T2008Biosensors and tools for surface functionalization from the macro-to the nanoscale:the way
forward J.Appl.Phys.104111101
Nusz G J,Curry A C,Marinakos S M,Wax A and Chilkoti A2009 Rational selection of gold nanorod geometry for label-free
plasmonic biosensors ACS Nano3795
Pakizeh T,Dmitriev A,Abrishamian M S,Granpayeh N and K¨a ll M2008Structural asymmetry and induced optical
magnetism in plasmonic nanosandwiches J.Am.Opt.Soc.B
25659
Riboh J,Haes A J,McFarland A,Yonzon C R and
Van Duyne R P2003A nanoscale optical biosensor:real-time immunoassay in physiological buffer enabled by improved
nanoparticle adhesion J.Phys.Chem.B1071772
Rosi N L and Mirkin C A2005Nanostructures in biodiagnostics Chem.Rev.1051547
Stewart M E,Anderton C R,Thompson L B,Maria J,Gray S K, Rogers J A and Nuzzo R G2008Nanostructured plasmonic
sensors Chem.Rev.108494
Svedhem S,Pfeiffer I,Larsson C,Wingren C,Borrebaeck C and H¨o¨o k F2003Patterns of DNA-labeled and
scFv-antibody-carrying lipid vesicles directed by
material-speci?c immobilization of DNA and supported lipid
bilayer formation on an Au/SiO2template ChemBioChem4339 Tanaka R,Yuhi T,Nagatani N,Endo T,Kerman K,Takamura Y and Tamiya E2006A novel enhancement assay for
immunochromatographic test strips using gold nanoparticles
Anal.Bioanal.Chem.3851414
Tie Y,Calonder C and Tassel P R V2003Protein adsorption: kinetics and history dependence J.Colloid Interface Sci.2681Toftegaard R,Arnbjerg J,Daasbjerg K,Ogilby P R,Dmitriev A, Sutherland D S and Poulsen L2008Metal-enhanced1270nm singlet oxygen phosphorescence Angew.Chem.Int.Edn
476025
Unger A and Kreiter M2009Analyzing the performance of plasmonic resonators for dielectric sensing J.Phys.Chem.C
11312243
Unger A,Rietzler U,Berger R and Kreiter M2009Sensitivity of crescent-shaped metal nanoparticles to attachment of dielectric colloids Nano Lett.92311
V¨o ros J2004The density and refractive index of adsorbing protein layers Biophys.J.87553
Wang H,Brandl D W,Le F,Nordlander P and Halas N J2006 Nanorice:a hybrid plasmonic nanostructure Nano Lett.6827 Wellinghausen N,Siegel D,Gebert S and Winter J2009Rapid detection of Staphylococcus aureus bacteremia and methicillin resistance by real-time PCR in whole blood samples Eur.J.
Clin.Microbiol.Infect.Dis.81001
Whitney A V,Elam J W,Zou S,Zinovev A V,Stair P C, Schatz G C and Van Duyne R P2005Localized surface
plasmon resonance nanosensor:a high-resolution
distance-dependence study using atomic layer deposition
J.Phys.Chem.B10920522
Willets K A and Van Duyne R P2007Localized surface plasmon resonance spectroscopy and sensing Annu.Rev.Phys.Chem.
58267