Sensitivity and Selectivity Limits of Multiplex NanoSPR Biosensor

Chapter 27

Sensitivity and Selectivity Limits of Multiplex NanoSPR Biosensor Assays

Downloaded by AUBURN UNIV on March 2, 2016 | http://pubs.acs.org Publication Date: September 19, 2008 | doi: 10.1021/bk-2008-0996.ch027

Chenxu Yu and Joseph Irudayaraj Department of Agricultural and Biological Engineering and Bindley Biosciences Center, Purdue University, West Lafayette, IN 47907

Gold nanorods (GNRs) of different aspect ratios were fabricated through seed mediated growth; partial and full functionalization procedures were developed to attach antibodies to the GNRs and yield Gold Nanorods Molecular Probes (GNrMPs). Multiplex sensing was achieved by the distinct response of the plasmon spectra of GNrMPs to binding events of up to three targets. A mathematical model formulated adequately described the ligand binding response of GNrMPs and concentrations of multiple targets were determined from experimental data. The GNrMP sensors were found to be highly specific and sensitive and the dynamic response was found to be in the range between 10 M and 10 M . Comparison of the experimental data with the theoretical model yielded an affinity constant K =1.32x10 M which was in agreement with the IgG-antiIgG binding affinity reported in the literature. The limit of detection (LOD) of GNrMPs was found to be in the low nano-molar range, and is a function of the binding affinity: for a higher probe-target affinity pair the LOD can be expected to reach femto molar levels. This technique can play a key role in developing tunable sensors for sensitive and precise monitoring of biological interactions. -9

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© 2008

American Chemical Society

In Nanoparticles: Synthesis, Stabilization, Passivation, and Functionalization; Nagarajan, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2008.

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Introduction Gold nanoparticles possess optical properties that make them uniquely suitable for biosensing applications. Their optical properties strongly depend on both the particle size and shape and are related to the interaction between the metal conduction electrons and the electric field component of the incident electromagnetic radiation, which leads to strong, characteristic absorption bands in the visible to infrared part of the spectrum (7). Fundamentally, a biosensor is constructed by coupling a ligand to its receptor complement via an appropriate signal transduction element (2). Various signal transduction mechanisms have been explored as biosensing schemes, including optical (3,4), radioactive (5,6), electrochemical (7,8), piezoelectric (9,10), magnetic (11,12), micromechanical (13,14), IR and Raman spectroscopic (15,16), and mass spectrometry (17,18). Although each of these methods has its individual strengths and weakness, optical sensors that utilize the surface plasmon resonance (SPR) phenomenon of planar gold surfaces have shown potential to become methods of choice in many biosensing applications (4,19). Other than macro-scaled SPR sensors using planar gold surface, several research groups have begun to develop micro/nano scaled optical biosensors that utilize the unique optical properties of gold nanostructures (20-29). The optical properties of gold nano structures strongly depend on both the particle size and shape and are related to the interaction between the surface electrons and the incidental electromagnetic radiation, which leads to strong, characteristic absorption in the visible to infrared region of the spectrum (1). For gold nanorods in aqueous solution, two distinct plasmon bands, one associated with the transverse (-520 nm) mode and the other with the longitudinal mode (usually > 600 nm) of the surface electron oscillation could be observed (1). Biosensor applications (20-30) have been designed based on the fact that the wavelengths of these bands are affected by changes in the dielectric properties in the close vicinity of these structures (known as nanoSPR (20), or Localized Surface Plasmon Resonance (LSPR)) (27). These changes are induced by the binding of ligands to the receptor molecules (i.e. antibodies) immobilized onto the nanostructures through chemi- or physisorption. The wavelength shift of the plasmon bands is solely determined by changes in the dielectric properties (i.e., refractive indexes) in the immediate vicinity of the nano structures. In the case of spherical particles, target-binding induced wavelength shift to single particles is small (2-3 nm), a sensor has to be designed based on the controlled aggregation concept (28,31-34). However, aggregation results in significant widening of the plasmon peak, and the resulting spectral resolution is too poor to distinguish multiple targets. When anisotropic particles such as nanorods are used to fabricate Gold Nano-rod Molecular Probes (GNrMPs), single particle sensor could be devised. Recently we have demonstrated that GNrMPs made with gold nanorods of



388 different aspect ratios could be implemented in a multiplex mode to detect presence/absence of multiple targets simultaneously (29). However, in the earlier work the GNrMPs were only partially-functionalized and was prone to non specific binding, and the detection of targets was not quantitatively interpreted. In this research, we studied both partial and full functionalization strategies for making GNrMPs, and developed a scheme to implement the GNrMPs for specific detection of multiple targets. A systematic study of the response of GNrMPs to changes in dielectric properties (refractive indexes) in the vicinity is presented and quantitative analysis of binding events is provided. It is shown that these GNrMPs operate in a manner similar to macro SPR sensors and it is possible to transduce very small changes in refractive index near the surface of the GNrMPs into a measurable wavelength shift. The GNrMPs were found to be extremely sensitive and could measure the targets at low nano-molar level.

Experimental section Materials Hexadecyltrimethylammoniumbromide (CTAB, 99%) and benzyl dimethyl ammoniumchloride hydrate (BDAC, 99%), Sodium borohydride (99%), Lascorbic acid, Gold (III) chloride hydrate (>99%) and Silver nitrate (>99%), gold atomic absorption standard solution, 11-mercaptoundecanoic acid (MUDA), were all purchased from Sigma-Aldrich (St. Louis, MO) and used without further purification. 1-ethyl, 3-(3-dimethylaminopropyl) carbodiimide (EDAC) and 4-(4-Maleimidophenyl) butyric acid JV-succinimidyl ester (NHS), goat anti-human IgG, goat anti-mouse IgG, goat anti-rabbit IgG Fabs and human IgG, mouse IgG, rabbit IgG were all purchased from Pierce Biotechnology (Rockford, IL). Nanopure deionized and distilled water (18.2 MQ) was used for all experiments.

Fabrication and Characterization of Gold Nanorods A seed-mediated growth procedure modified from that suggested by Nikoobakht and El-Sayed (35) was used to fabricate gold nanorods with aspect ratio between 2.5 and 7. The details of the procedure were reported elsewhere (29). The concentration of atomic gold in the solution of gold nanorods was determined by inductively coupled plasmon atomic emission spectroscopy (ICPAES). The gold nanorods were then concentrated to 100 nM by centrifugation. All subsequent characterization, activation, and functionalization were conducted using these nanorods samples.



389 The yield and aspect ratios of gold nanorods was determined using Transmission Electron Microscopy (TEM), acquired with a Philips CM-100 TEM (Philips, Eindhoven, Netherlands). TEM grids were prepared by placing 1 ul of the nanorods solution in a 400-mesh formvar coated copper grid and evaporating the solution at room temperature. Images were then captured using a Tietz F415 slow scan digital camera at 4K resolution. At least 150-200 nanorods could be counted and measured per grid to provide an estimate of the mean aspect ratio of these nanorods after the synthesis step. Absorption spectra of GNrMP samples through each stage of experiments were measured using a Jasco V570 UV-Vis-NIR spectrophotometer (Jasco, Inc., Easton, MD), in the wavelength range between 400 and 1500 nm. The measured spectra were normalized by rescaling the maximum absorbance of the longitudinal plasmon peak to 1.

Functionalization of Gold Nanorods to Synthesize GNrMPs Once synthesized, the nanostructures were fiinctionalized by biologically active agents and deployed as sensors. Biofunctionalization constitutes a two step process: in step 1, termed as the activation step, a chemical anchor layer was formed on the nanorods surface to provide active functional groups to which biological molecules (i.e., antibodies) can be covalently attached; and in step 2, the functionalization step, biomolecules were covalently linked to the anchor layer to produce GNrMPs for target specific sensing. Partial activation offreshly-madegold nanorods was accomplished partially replacement of the CTAB capping, the CTAB capping at the {l,l,0}/{ 1,0,0} side faces of the nanorods is retained and the CTAB capping at the {1,1,1} side faces is replaced with an alkanethiol SAM, to which antibodies can be tethered through NH-CO bonds. Partial activation of gold nanorods was achieved as follows: 0.5 ml of 20mM ethanol solution of MUDA was added to 5 ml of gold nanorods solution and stirred mildly for 24 hours under room temperature. Nanorods were then collected by centrifugation at 5000 rpm for 15 minutes and resuspended in a 0.005 M CTAB solution to yield a final concentration of-100 nM. Full activation of gold nanorods was achieved by the replacement of CTAB capping as in (37). The resulting solution was subjected to two rounds of chloroform extraction to remove the CTAB released from the nanorods. Nanorods were then collected by centrifugation at 5000 rpm for 15 minutes and resuspended in nano pure water (18.2 mQ) to yield a final concentration of-100 nM. Once the MUDA SAM was formed, antibodies (goat anti-human, goat antimouse, goat anti-rabbit IgG Fabs) were then attached to the activated nanorods as follows: to a 5 ml of the activated nanorods (-100 nM), 1 ml of freshly prepared 0.4M EDAC and 0.1M NHS solution was added and sonicated for 25 minutes at 4°C. The resulting structures were then collected by centrifugation at



390 5000 rpm for 5 minutes and resuspended in 5 ml PBS buffer (pH=7.4). Antibodies suspended in PBS was then added to the resulting nanorods suspension (the concentration of antibodies was varied between 200-1000 nM) and then incubated for 1 h under constant sonication at room temperature. The fiinctionalized nanorods were subsequently collected by centrifugation at 5000 rpm for 5 minutes and three rounds of vigorous washing and sonication in PBS solution for 10 mins. The supernatant was collected after each washing step and the cumulative protein content was measured using a Biorad Protein Assay (Biorad Laboratories, Hercules, CA) with bovine serum albumin (BSA) as a protein standard. The amount of antibodies bound to the nanorods was determined by subtracting the antibodies left in the supernatant from the original amount.

Implementation of the Gold Nanorods Molecular Probes 5 ml of the GNrMPs (20 nM) was mixed with 5 ml of targets (respective IgGs) with concentrations spanning from 10" M to 10" M for 30 mins under mild stirring to allow the probe-target binding to reach equilibrium and the sensor response to the probe-target binding depicted by a pronounced shift of longitudinal plasmon peaks, were measured using UV-Vis-NIR spectroscopy. For multiplex analysis, 3 GNrMPs with different aspect ratios were mixed at equal concentrations, and the target solution containing the respective complement (IgGs) at varying concentrations was prepared. Equal amount of GNrMP and target solutions were mixed and kept under mild stirring for 30 mins and the plasmon spectra of the mixture were then measured. The response of GNrMPs to target binding events was then quantitatively evaluated. 6

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Results and Discussion Nanorods Fabrication and Characterization Gold nanorods of aspect ratios in the range between 2.8 and 7 were made following the single and double surfactant protocols discussed earlier. Figure 1 shows the absorption spectra of nanorods with aspect ratio of 2.8, 3, 4.5, 5.5, and 7, respectively. It can be clearly seen that small changes in aspect ratio introduce a significant red-shift in the longitudinal plasmon band of the GNR colloids, implying a significant potential for multiplexing. Within the range of this study, a linear correlation could be established between the aspect ratio of gold nanorods and the absorbance wavelength of the longitudinal plasmon bands; hence the aspect ratio of gold nanorods could be easily deduced from their plasmon spectra.


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Sensitivity and Selectivity Limits of Multiplex NanoSPR Biosensor

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