bioplasmonic paper as a platform for detection

Bioplasmonic Paper as a Platform for Detection of Kidney CancerBiomarkersLimei Tian, Jeremiah J. Morrissey,, Ramesh Kattumenu, Naveen Gandra, Evan D. Kharasch,,,

and Srikanth Singamaneni,,*Department of Mechanical Engineering and Materials Science, Washington University in St. Louis, St. Louis, Missouri 63130, UnitedStatesDepartment of Anesthesiology, Division of Clinical and Translational Research, Washington University in St. Louis, St. Louis,Missouri 63110, United StatesSiteman Cancer Center, Department of Biochemistry and Molecular Biophysics, Washington University in St. Louis, St. Louis,Missouri 63110, United States

*S Supporting Information

ABSTRACT: We demonstrate that a common laboratory lter paper uniformly adsorbedwith biofunctionalized plasmonic nanostructures can serve as a highly sensitivetransduction platform for rapid detection of trace bioanalytes in physiological uids. Inparticular, we demonstrate that bioplasmonic paper enables rapid urinalysis for thedetection of kidney cancer biomarkers in articial urine down to a concentration of 10 ng/mL. Compared to conventional rigid substrates, bioplasmonic paper oers numerousadvantages such as high specic surface area (resulting in large dynamic range), excellentwicking properties (naturally microuidic), mechanical exibility, compatibility withconventional printing approaches (enabling multiplexed detection and multimarkerbiochips), and signicant cost reduction.

Renal cancer is generally silent, frequently fatal andaccounts for 3% of adult malignancies. It is estimatedthat in 2012 alone 64 770 new cases will be diagnosed with and13 570 deaths will occur of cancer of the kidney and renalpelvis.1 Altogether, this disease represents the sixth leadingcause of death due to cancer. In a recent study, it has beendemonstrated that the proteins aquaporin-1 (AQP1) andadipophilin (ADFP) in urine form excellent candidates for thenoninvasive and early detection of renal cancer carcinoma(RCC).2 While this study clearly demonstrated the possibilityof using these proteins as potential biomarkers for early andnoninvasive detection of RCC, an inexpensive and highthroughput biodetection platform is required to translatethese biomarkers to clinical setting and incorporation intoroutine health physical. Conventional labeled assays such asenzyme-linked immunosorbent assay (ELISA) are time-consuming and expensive and require tedious labelingprocedures making them unsuitable for rapid and routineurinalysis. The above considerations clearly suggest the needfor a label-free approach, for rapid and quantitative detection ofthe proteins in urine at physiologically relevant concentrations(ng/mL).Localized surface plasmon resonance (LSPR) of metal

nanostructures, which involves collective coherent oscillationof dielectrically conned conduction electrons, is sensitive tonumerous factors such as composition, size, shape, surrounding

dielectric medium, and proximity to other nanostructures.36

The sensitivity of LSPR to local changes in dielectricenvironment renders it an attractive transduction platform forchemical and biological sensing.712 LSPR of metal nanostruc-tures has been shown to be sensitive enough to dierentiateinert gases with a refractive index contrast (n) on the order of3 104, probe the conformational changes of biomacromo-lecules, detect single biomolecule binding events, monitor thekinetics of catalytic activity of single nanoparticles, and evenoptically detect single electron.1317 Detection of variousbiomolecules such as proteins1822 and DNA,2325 have beendemonstrated in the past few years making the transductionplatform promising for the development of simple, highlysensitive, label-free, and cost-eective diagnostics.Most of the LSPR sensors demonstrated so far rely on rigid

planar substrates (e.g., glass, silicon) owing to theircompatibility with various well-established lithographic ap-proaches (e.g., photolithography, e-beam lithography andnanosphere lithography), which are routinely employed foreither fabrication or assembly of plasmonic nanotrans-ducers.2629 Compared to these conventional substrates,paper has numerous advantages such as high specic surface

Received: August 13, 2012Accepted: October 25, 2012Published: October 25, 2012

Article

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2012 American Chemical Society 9928 dx.doi.org/10.1021/ac302332g | Anal. Chem. 2012, 84, 99289934

area, excellent wicking properties, compatibility with conven-tional printing approaches, signicant cost reduction and easydisposability. For all these reasons, paper is gaining increasedattention as a potential substrate for various applicationsincluding biodiagnostics, food quality testing as well asenvironmental monitoring.3036 Paper substrates functionalizedwith inorganic nanoparticles such as gold, silver, and titaniahave been investigated for a wide range of applicationsincluding self-cleaning,37 disinfection,38 colorimetric sensing,39

surface enhanced Raman scattering (SERS) based trace analytedetection,4042 and antimicrobial activity.43

Here, we report for the rst time a bioplasmonic paper thatcomprises a common laboratory lter paper uniformly adsorbedwith biofunctionalized plasmonic nanostructures for thedetection of target bioanalytes from model physiological uids.The sensitivity and detection limit of the paper-based LSPRsubstrate is on par or better compared to the conventional rigidsubstrates using similar plasmonic nanostructures. Furthermore,we demonstrate the deployment of such bioplasmonic paper asa exible swab to collect and detect trace quantities (few gspread over cm2 area) of model bioanalytes on real-worldsurfaces.

EXPERIMENTAL SECTIONSynthesis of Gold Nanorods (AuNRs). Gold nanorods

were synthesized using a seed-mediated approach.42,43 Seedsolution was prepared by adding 0.6 mL of an ice-cold sodiumborohydride solution (10 mM) into 10 mL of 0.1 Mcetyltrimethylammonium bromide (CTAB) and 2.5 104

M chloroauric acid (HAuCl4) solution under magnetic stirringat room temperature. The color of the seed solution changedfrom yellow to brown. Growth solution was prepared by mixing95 mL of CTAB (0.1 M), 0.5 mL of silver nitrate (10 mM), 5mL of HAuCl4 (10 mM), and 0.55 mL of ascorbic acid (0.1 M)in the same order. The solution was homogenized by gentlestirring. To the resulting colorless solution, 0.12 mL of freshlyprepared seed solution was added and set aside in dark for 14 h.Prior to use, the AuNR solution was centrifuged at 10 000 rpmfor 10 min to remove excess CTAB and redispersed innanopure water (18.2 M-cm). The centrifugation procedurewas repeated twice.AuNR-IgG Conjugates Preparation. To a solution of

heterobifunctional polyethylene glycol (SH-PEG-COOH) inwater (37.5 L, 20 M, Mw = 5000 g/mol, JenkemTechnology), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide(EDC, Thermo Scientic), and N-hydroxy succinimide (NHS,Thermo Scientic) with the same molar ratio as SH-PEG-COOH were added followed by shaking for 1 h. The pH of theabove reaction mixture was adjusted to 7.4 by adding 10concentrated phosphate buered saline (PBS), followed by theaddition of rabbit immunoglobulin G (IgG) (10 L, 75 M, Mw= 150 kDa, Thermo Scientic). The reaction mixture wasincubated for 2 h, and then ltered to remove any byproductduring the reaction using a 50 kDa lter. The nal SH-PEG-IgG conjugates solution (0.75 M) was obtained after washingwith PBS buer (pH 7.4) twice. AuNR-IgG conjugates solutionwas prepared by adding 50 L SH-PEG-IgG conjugatessolution to 1 mL twice centrifuged AuNR solution withincubation for 1 h. The amount of SH-PEG-IgG was optimizedto obtain maximum coverage of IgG on AuNR surface (FigureS4 of the Supporting Information).AuNR and AuNR-IgG Conjugates on Rigid Planar

Substrates. Glass substrates were cut into approximately 1 2

cm rectangular slides and cleaned in piranha solution (3:1 (v/v)mixture of H2SO4 and 30% H2O2) followed by extensive rinsingwith nanopure water. AuNR or AuNR-IgG conjugates wereadsorbed onto glass substrates following the modication of thesurface with (3-mercaptopropyl)-trimethoxy-silane (MPTMS)by exposing the glass surface to 5% MPTMS in ethanol for 15min followed by ultrasonication in ethanol for 30 min andrinsing with water. Subsequently, the glass surface was exposedto AuNR or AuNR-IgG conjugates solution for 1 h, followed byrinsing with water to remove the loosely bound nanorods.Similar approach was employed for biofunctionalization ofAuNR with anti-AQP1 (Sigma), which were employed for thedetection of AQP1 (Origene) from articial urine (Surine,Cerilliant).Plasmonic Paper Substrates Preparation. Adsorption of

AuNR-IgG conjugates on paper substrate was achieved byimmersing a 1 1 cm2 paper in AuNR-IgG conjugates solution(2 mL) for 12 h. After removing from the solution, the paperwas thoroughly rinsed with water, and then exposed to SH-PEG solution to block nonspecic binding. LSPR measure-ments were performed by exposing the plasmonic paper tovarious concentrations of anti-Rabbit IgG (Thermo Scientic)in PBS for 1 h, followed by thorough rinsing with PBS andwater and drying with a stream of nitrogen. Six UVvisextinction spectra were collected for each substrate before andafter anti-IgG exposure. Each spectrum represented a dierentspot within the same substrate.Characterization Techniques. Transmission electron

microscopy (TEM) micrographs were recorded on a JEM-2100F (JEOL) eld emission instrument. Samples wereprepared by drying a drop of the solution on a carbon-coatedgrid, which had been previously made hydrophilic by glowdischarge. Scanning electron microscope (SEM) images wereobtained using a FEI Nova 2300 Field Emission SEM at anaccelerating voltage of 10 kV. Atomic force microscopy (AFM)images were obtained using Dimension 3000 (Digital instru-ments) AFM in light tapping mode.44 DLS measurements wereperformed using Malvern Zetasizer (Nano ZS).Extinction Spectra and Raman Spectra Measure-

ments. Extinction spectra from paper substrates were collectedusing a CRAIC microspectrophotometer (QDI 302) coupled toa Leica optical microscope (DM 4000M) with 100 objectivein the range of 450800 nm with 10 accumulations and 0.8 sexposure time in reection mode. The spectral resolution of thespectrophotometer is 0.2 nm. Shimadzu UV-1800 spectropho-tometer was employed for collecting UVvis extinction spectrafrom solution and glass samples. Raman spectra were obtainedusing a Renishaw inVia confocal Raman spectrometer mountedon a Leica microscope with 20x objective (NA = 0.4) and 785nm wavelength diode laser (0.5 mW). The spectra wereobtained in the range of 4001800 cm1 with threeaccumulations and 10 s exposure time.

RESULTS AND DISCUSSIONLaboratory lter paper (Whatman # 1) employed as aplasmonic substrate in this study is characterized by cellulosebers with a diameter of 10 m (Figure S1 of the SupportingInformation). AFM images revealed the hierarchical brousmorphology of the lter paper with cellulose nanobers braidedinto microbers (average diameter of 400 nm) (Figure S1 ofthe Supporting Information). The root-mean-square (RMS)surface roughness of the paper was found to be 72 nm over 5 5 m2 area, which indicates the large surface area of the paper

Analytical Chemistry Article

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substrate available for adsorption of biofunctionalized plas-monic nanostructures.We employed Goat Rabbit IgG (IgG henceforth) as model

capture biomolecule and Goat anti-Rabbit IgG (anti-IgG) asmodel target bioanalyte to demonstrate the paper-based LSPRbiosensor. Bioplasmonic paper, which serves as a LSPRbiosensor, is comprised of biofunctionalized gold nanorods(AuNRs) adsorbed on the lter paper. Figure 1 illustrates thesteps involved in the fabrication and deployment ofbioplasmonic paper as a LSPR sensor for the detection oftarget bioanalytes. Thiol-terminated polyethylene glycol (SH-PEG), a hydrophilic polymer, serves as a exible linker toincrease the accessibility of IgG to target biomolecules andforms a stable protective layer around AuNRs to reducenonspecic binding. AuNR-IgG conjugates were prepared byfunctionalizing AuNRs with SH-PEG-IgG molecules (exper-imental section for details). Subsequently, AuNR-IgG con-jugates were adsorbed onto paper substrate by exposing thepaper substrates to AuNR-IgG conjugate solution followed bythorough rinsing with water to remove the weakly adsorbednanorods. Following the adsorption of the AuNR-IgGconjugates, the paper substrates were exposed to SH-PEGsolution to further block nonspecic binding. Exposure to anti-IgG solution resulted in its specic binding to IgG, which canbe detected as a spectral shift of the LSPR wavelength ofAuNRs.AuNRs are particularly attractive as plasmonic nano-

transducers considering the large refractive index sensitivity oflongitudinal LSPR, facile and large tunability of the LSPRwavelength of nanorods with aspect ratio and the electro-magnetic (EM) hot-spots at the edges.45 AuNRs, synthesizedusing a seed-mediated approach,46,47 are positively charged witha length of 55.8 3.6 nm and a diameter of 22.0 2.1 nm(TEM image in part A of Figure 2). UVvis extinction spectraof the AuNRs are characterized by two distinct bandscorresponding to the transverse (lower wavelength) andlongitudinal (higher wavelength) oscillation of electrons withincident EM eld. The longitudinal plasmon band of AuNRs,which is known to be more sensitive to the refractive index

change of the surrounding medium compared to the transverseband, exhibited a red shift of 8.5 nm and an increase inextinction intensity upon SH-PEG-IgG appendage (part B ofFigure 2). The red shift and increase in intensity correspond tothe increase in the refractive index around the AuNRs with SH-PEG-IgG binding. SERS spectra collected from AuNRs andAuNR-IgG conjugates also conrm the biofunctionalization ofAuNRs (part C of Figure 2). The successful bioconjugation ofIgG is evidenced by the Raman bands at 831 cm1 and 852cm1, 1004 cm1 and 1031 cm1, 1230 cm1, and 1685 cm1

corresponding to tyrosine, phenylalanine, amide III, and amideI, respectively.The average hydrodynamic diameter of AuNR and AuNR-

IgG measured using dynamic light scattering (DLS) was foundto be 25.0 and 35.0 nm, respectively (Figure 2D). Theincrease in hydrodynamic diameter of AuNR can be primarilyattributed to the successful appendage of IgG (Mw = 150 KDa)to AuNR surface. Only a small increase of 0.2 nm wasobserved for the conjugation of SH-PEG (Mw = 5 KDa)(Figure S2 of the Supporting Information). Part E of Figure 2shows the AFM images of the AuNR and AuNR-IgG adsorbedon silicon surfaces. Apart from indicating the stability of AuNR-IgG in solution (absence of any large scale aggregates), a smallincrease in the average diameter ( 3 nm) of nanorods wasobserved following SH-PEG-IgG functionalization, whichclosely corresponds to the size of IgG (part F of Figure2).48,49 The discrepancy between the DLS and AFM measure-ments can be attributed to two factors: (i) AFM imaging wasperformed in dry state as opposed to DLS measurements,which reveals the hydrodynamic radius of AuNR, (ii) possibledeformation (compression under AFM tip) of the biomoleculesand polymer during AFM imaging although the imaging wasperformed in light tapping mode.An important consideration in the design of bioplasmonic

paper substrates is the optical homogeneity of the substrate.The homogeneity determines the noise oor of the spectralshift, which in turn determines the resolution of thebioplasmonic paper. SEM images revealed highly uniformdistribution of AuNR-IgG conjugates on paper surface with no

Figure 1. Schematic illustration representing the steps involved in the fabrication of bioplasmonic paper for anti-IgG detection.



signs of aggregation or patchiness on the substrate (part A ofFigure 3). Higher magnication image reveals the preferentialalignment of AuNR-IgG conjugates along cellulose bers. Thesurface density of the AuNR-IgG conjugates adsorbed on thepaper substrate was found to be 351 11/m2, which can becontrolled by incubation time (part B of Figure 3). SERSspectra were collected from AuNRs and AuNR-IgG conjugatespaper to conrm the preservation of IgG conjugation duringincubation process (Figure S5 of the Supporting Information).Longitudinal LSPR wavelength of AuNR-IgG conjugates

adsorbed on paper substrate exhibited a signicant blue shift(17 nm) compared to that in solution due to the change inthe refractive index of the surrounding medium (aqueous to air+substrate) (part C of Figure 3).50 The extinction spectrumfrom the paper substrate was collected from a 2 2 m2 areausing a microspectrometer mounted on an optical microscope,which corresponds to 1400 nanorods. The narrow andsymmetric extinction band (fwhm of 115 nm) corresponding tothe longitudinal LSPR of AuNR indicates the absence of AuNRaggregates on paper surface, which is in agreement with theSEM images discussed above. Extinction spectra collectedacross 0.5 0.5 cm2 area revealed excellent optical uniformity

of the bioplasmonic paper substrate. LSPR wavelengthmeasured from six dierent spots over such large area exhibiteda small standard deviation of 1 nm (part D of Figure 3). Theexcellent spectral homogeneity is due to the highly uniformadsorption of AuNR-IgG conjugates as evidenced by the SEMimages. The spectral homogeneity observed here is quiteremarkable considering the simplicity of the fabrication processand the inherent heterogeneity of the paper substrates (largesurface roughness and hierarchical nature of the brous mat).The biosensing capability of the paper substrates is

demonstrated by using anti-IgG as a model bioanalyte, whichis known to exhibit strong and specic binding to IgG.51

Extinction spectra were obtained from paper substratesadsorbed with bare AuNRs, AuNR-IgG conjugates blockedwith SH-PEG (for blocking nonspecic binding) andsubsequently exposed to 24 g/mL of anti-IgG (part A ofFigure 4). LSPR wavelength exhibited a 17 nm red shift withthe partial replacement of CTAB layer with AuNR-IgGconjugates and SH-PEG molecules, and a further red shift of23 nm upon specic binding of anti-IgG (24 g/mL) to IgG.On the other hand, a small red shift of 2 nm was observedupon exposure to bovine serum albumin (BSA) (24 g/mL),

Figure 2. (A) TEM image of AuNRs. (B) UVvis extinction spectra conrming AuNR and SH-PEG-IgG conjugation. UVvis extinction spectrumof 56 22 nm AuNRs shows a longitudinal LSPR wavelength of 658 nm (AuNRs, red). After incubation with SH-PEG-IgG, the max red shifts by 8.5nm (AuNR-IgG, black). (C) Hydrodynamic diameters obtained from DLS show that the average hydrodynamic diameter increased by 10 nmfollowing SH-PEG-IgG conjugation. (D) Surface enhanced Raman spectra before and after the conjugation of antibody on gold nanorods reveal theRaman bands corresponding to IgG. (E, F) AFM images of AuNRs and AuNR + IgG conjugates (Z scale for both images is 100 nm), revealing theincrease in the average diameter of the nanorods following the bioconjugation.



which indicates small nonspecic binding of the biofunction-alized nanorods (part B of Figure 4).The extinction spectrum was deconvoluted by tting the

extinction spectrum with two Gaussian peaks, from which thelongitudinal LSPR wavelength of AuNRs was employed to

monitor the biomolecule binding (part C of Figure 4). Asemilog plot of the longitudinal LSPR wavelength shift fordierent concentrations of anti-IgG revealed that LSPR shiftmonotonically increases with increase in the concentration ofanti-IgG. The plot also reveals extremely small nonspecic

Figure 3. (A, B) SEM images of paper adsorbed with AuNR-IgG conjugates. (C) Extinction spectra of AuNR-IgG conjugates in solution and onpaper (insert, shows photographs of the bare lter paper (left) and lter paper after adsorption of AuNR IgG conjugates (right)). (D) Sixextinction spectra collected from dierent spots on a plasmonic paper of 0.5 0.5 cm2 area showing the remarkable spectral homogeneity ofbioplasmonic paper.

Figure 4. (A) Extinction spectra of AuNR paper substrate (black), AuNR-IgG conjugates on the paper substrate before (red) and after binding ofanti-IgG (blue). (B) Control experiment showing the small nonspecic binding of BSA on bioplasmonic paper substrate. (C) LSPR spectradeconvoluted using two Gaussian peaks. (D) Plot showing the LSPR peak shift of bioplasmonic paper for various concentrations of anti-IgG andBSA.



binding of BSA at various concentrations (part D of Figure 4).Conventional glass substrates exhibited smaller shift (20 nm)compared to the bioplasmonic paper (24 nm) under similarconditions (Figure S3 of the Supporting Information). Thelarger shift in the case of paper substrate compared to glasssubstrate is possibly due to the 3D porous structure of papersubstrate, which enables better uptake and transport of thebioanalytes to the plasmonic nanostructures. A detection limitof 24 pg/mL (0.16 pM) was noted in the case ofbioplasmonic paper, which is on par with that observed inthe case of other rigid substrates.52

Similar approach was employed to detect AQP1, a biomarkerfor RCC in urine. We employed AuNR appended with anti-AQP1 as biofunctionalized transducers for the detection ofAQP1 in articial urine. Part A of Figure 5 shows the shift inthe longitudinal LSPR of AuNR upon binding of AQP1 (4.67g/mL) from articial urine sample (part A of Figure 5). Amonotonic increase in the LSPR shift is observed withincreasing the concentration of AQP1 in articial urine (partB of Figure 5). The measured LSPR shift for 10 ng/mL (0.35nM) of AQP1 is 1.1 nm. This shift is signicantly higher thanthe noise oor of the bioplasmonic paper (0.6 nm). Thedetection limit matches with the lower limit of the range ofAQP1 in patients with RCC.53 The detection limit of AQP1 ishigher than anti-IgG, which is possibly due to: (i) the molecularweight of AQP1 is 28.3 kDa, much smaller than modelbioanalyte anti-IgG 150 kDa, causing a smaller change inrefractive index; and (ii) limited binding sites accessible toAQP1 compared to that available for anti-IgG, which binds tothe heavy chain of IgG. The detection limit can be lowered byoptimizing the ratio of SH-IgG and AuNRs, and selectingnanostructures or their organized assemblies with higher LSPRsensitivity.One of the distinct advantages of the bioplasmonic paper is

the ability to collect trace amount of bioanalytes from real-world surfaces by simply swabbing across the surface. Todemonstrate such unique ability, 12 g of anti-IgG wasdeposited on a tomato as purchased from a grocery store. Aslightly wet (in PBS buer) bioplasmonic paper substrate wasswabbed on the surface of the tomato to collect the bioanalytes(part A of Figure S6 of the Supporting Information). Thelongitudinal LSPR wavelength exhibited a red shift (21 nm)upon specic binding of anti-IgG, which was collected from sixdierent spots in the center of the plasmonic paper (part B ofFigure S6 of the Supporting Information). The insertphotographs of part B of Figure 6 shows that the color ofthe bioplasmonic paper turned from blue to gray upon bindingof the target biomolecules to the biofunctionalized nanorods. A

control experiment was performed by swabbing a slightlywetted plasmonic paper on the surface of as purchased tomato(i.e., no prior cleaning). Longitudinal LSPR wavelengthexhibited a 3 nm red shift, which can be attributed to thesurface contaminants nonspecically adsorbing to AuNR on theunwashed tomato (part C of Figure S6 of the SupportingInformation). Considering that the swabbing of the surfaceresults in only a fraction of the biomolecules being absorbedonto the paper, the much higher LSPR shift upon specicbinding of anti-IgG on the surface clearly indicates thebioplasmonic paper substrate to be excellent candidate forcollection and detection of trace amount of bioanalytes fromreal-world surfaces.

CONCLUSIONSIn conclusion, we have demonstrated a bioplasmonic paper forthe rapid and label-free detection of AQP1, a biomarker forearly and non-invasive detection of RCC. Bioplasmonic paperexhibited excellent spectral homogeneity, sensitivity, dynamicrange and selectivity, comparable to or better than theconventional rigid LSPR substrates. Furthermore, owing to itsunique exible and conformal nature, bioplasmonic paperenables simultaneous collection and detection of trace amountof bioanalytes from real-world surfaces. Paper-based LSPRsubstrates oer numerous advantages such as low cost, easystorage, transport and disposability, which lead to simple,inexpensive plasmonic biochips for point-of-care diagnostics.Besides being highly cost-eective and environmental-friendly,the use of paper-based LSPR substrates when transformed intoprintable microuidic devices will enable the detection ofmultiple bioanalytes in complex physiological uids. Thesynergism of paper-based microuidics and LSPR-basedbiosensing is expected to be truly transformative by openingup novel avenues in multianalyte biological detection.

ASSOCIATED CONTENT*S Supporting InformationAdditional information as noted in the text. This material isavailable free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATIONCorresponding Author*E-mail: [email protected].

NotesThe authors declare no competing nancial interest.

Figure 5. (A) Extinction spectra of AuNR appended with anti-AQP1 adsorbed on a paper substrate before (black) and after exposing to 4.7 g/mLAQP1 in articial urine (red), (B) plot showing the LSPR peak shift of bioplasmonic paper for various concentrations of AQP1.



ACKNOWLEDGMENTSThis work was supported by Oce of Congressionally DirectedMedical Research Programs under contract No. W81XWH-11-1-0439 to SS, and National Institutes of Health under grant No.R01CA14152 and the Barnes-Jewish Hospital Foundation toJJM.

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