Dynamic Nanoparticle-Based Flexible Sensors: Diagnosis of Ovarian Carcinoma from Exhaled Breath

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1 Supporting Information for: Dynamic Nanoparticle-Based Flexible Sensors: Diagnosis of Ovarian Carcinoma from Exhaled Breath Nicole Kahn (1), Ofer Lavie (2), Moran Paz (2),Yakir Segev (2) and Hossam Haick *(1) (1) Department of Chemical Engineering and Russell Berrie Nanotechnology Institute, Technion Israel Institute of Technology, Haifa , Israel. (2) Gynecological Oncology and Surgery Unit, Carmel Medical Center, Haifa , Israel. * Corresponding Author (H.H.): hhossam@technion.ac.il 1. Methods 1.1 Synthesis of Nanoparticles Biligand nanoparticles were synthesized using a modified 2-phase Brust synthesis 1, 2. In short, mol HAuCl 4 H 2 O (Sigma Aldrich) was dissolved in 8 ml distilled water and combined with mol tetraoctylammonium bromide (Sigma Aldrich) in 27 ml toluene. The mixture was extracted and the organic phase divided in half mol dodecylamine (Sigma Aldrich) in 4 ml toluene was added to one half, and mol of a thiol ligand in 4 ml toluene was added to the other. The 2 halves were combined, and mol sodium borohydride (Sigma Aldrich) in 8 ml distilled water was added. After reduction, the solution was mixed for 3 h, and after extraction the organic solvent was evaporated. The nanoparticles precipitated in ice-cold ethanol were re-suspended in toluene for analysis or thin film deposition. Synthesized nanoparticles were analyzed with TEM to verify size and shape. The thiol ligands used were 2-nitro-4-flourmethylbenzenethiol (Santa Cruz), 2-napthalenethiol (Sigma Aldrich), 4- chlorobenzenemethanethiol (Sigma Aldrich), 3-ethoxythiopenol (Sigma Aldrich), and 4-tertbutylbenzenethiol (Sigma Aldrich). 1

2 1.2 Sensor Preparation and Characterization Electrodes were printed with silver on 50 micron Kapton, using 2 mil copper as a base metal. Electrodes were interdigitated with 0.5 mm spacing and the entire active electrode area was 1cm x 1cm. Layer-by-layer (LbL) film was deposited by immersing the electrodes alternately in a GNP solution with an absorbance of 2 and a linker solution containing mol hexadecanedithiol (Sigma Aldrich) in toluene each for 15 min. The electrodes were washed by immersion in toluene for 2 min after each submersion. Film thickness was measured indirectly by UV-VIS absorbance with a double-beam spectrophotometer, and surface analyzed by HR- SEM. 1.3 Exposure to High Concentrations of VOCs High concentration experiments involved analytes that had were either statistically significant or close to statistically significant in the separation of ovarian cancer breath from control breath. 3 VOC concentration was controlled using a bubbler system with nitrogen as a carrier gas. Bending protocols included a period of 60 seconds in which the sensors were stabilized in nitrogen, after which the chamber was exposed to the relevant analyte while the sensors remained unbent for two min. Each exposure was repeated 3 times, and an identical bending procedure was carried out under nitrogen flow before each exposure. Triplicates of each sensor type were utilized. The array for DFA separation experiments contained a single sensor from each ligand (NTMBT, CBMT, ETP, tbbt, NT) at 0.3 OD, a single ligand (CBMT) at 2 additional thicknesses (0.2 OD and 0.4 OD), and 2 sensors of single ligand at a single thickness (CBMT at 0.3 OD) sintered at C for 5 and 30 min. The sintered sensors were added additional features to the sensing array, as sintering significantly affected electrical characteristics. 1.4 Exposure to Low Concentrations of VOCs Low concentration experiments used only the 2 statistically significant analytes, as well as 2 biomarkers for potential confounding factors. Analyte concentration here was controlled using a gas generator, and exposure was done by transferring the gas to the system in bags. Exposures in this way gave flow rates of 100 ml/min through the chamber. The bending took 6 steps the sensors were deflected by 0.8 mm in each step at 2.4 mm/min in 3 increments for a total deflection of 2.4 mm, and then the crosshead was raised to the initial position in 3 increments each of 0.8 mm. In between each bend, the sensors were steady under exposure for 20 sec. Low concentration exposures were run at slightly higher velocities and shorter waiting times between bending steps, due to the limited volume of analyte gas. The various thicknesses 2

3 and ligand types were maintained in arrays for all exposures in this study. However, the sintered sensors used varied due to their significant lack of stability. Low concentration analysis and the clinical study used CBMT sensors at multiple thicknesses (OD 0.3 and 0.4) sintered at C for 5 min. Sintered sensors of OD 0.2 were quickly affected by multiple bending cycles, and were therefore not included in the analysis. 1.5 Collection of Breath Samples Breath samples were collected from female volunteers at Carmel Medical Center, Haifa. The subjects were instructed not to ingest food or liquids (other than water) for 2 h before sample collection. Samples were collected in controlled manner, in which inhaled ambient air was filtered and dead space volume excluded. These clinical samples were adsorbed on 2-bed ORBOTM420 TenaxVR TA sorption tubes (Sigma Aldrich) and stored in a cool environment until analysis. Immediately before exposure, the tubes were thermally desorbed at 190ºC under a nitrogen flow of 60 ml/min for 10 min, and collected in a bag for further analysis. 2. Sensing Mechanism of GNP-based Chemiresistive Sensors Generally speaking, molecularly capped gold nanoparticle (GNP) thin films conduct via a thermally activated tunneling mechanism according to the following equations 4, 5 : σ(t, δ) e δβ e E A RT (1) E A = 1 2 e2 (1 r 1 r+δ ) 4πε 0 ε (2) in which σ is electronic conductivity, δ is the interparticle distance, β is the electron transfer coupling coefficient, E A is the tunneling activation energy, R is the gas constant and T is the absolute temperature, r is the particle radius, ε is the dielectric constant of the interparticle medium and δ is the surface-to-surface interparticle spacing. When VOCs are adsorbed into the thin film, swelling increases interparticle distance as well as the permittivity of the interparticle space, affecting the electrical characteristics of the film according to equation (1). Multiple parameters affect analyte response swelling can increase resistance by distancing adjacent nanoparticles, or can decrease resistance by reducing the distance between adjacent island growths, increasing the number of percolation pathways 6. Analyte response can be positive or negative, generally correlating with analyte dielectric constant, but this can change with analyte concentration, indicating response mechanism that cannot always be easily explained. 6 When 3

4 GNP films are prepared on flexible substrates, the spatial organizational changes that result from bending or stretching allow them to be used as strain sensors. Substrate type 7, 8 and thickness 8, as well as nanoparticle size 9, capping ligand chemistry 10, and linker type 7 all regulate film strain sensitivity. Strain response can be described using the following equations 11 : ΔR R b = e gε 1 (3) g = β(d + δ) (4) where R/R b is the resistance change normalized to a baseline resistance, g is the gauge factor, and D is the nanoparticle diameter. Strain response therefore can either be due to increased distance between adjacent nanoparticles 9, 12 or fewer percolation pathways caused by film cracking 7,13. Micro-cracks and plastic grain deformation caused by significant strain permanently affect electrical characteristics Signal Analysis Data was extracted and processed using Matlab. Bending features were calculated as follows: the static feature of normalized bending response (overall change in resistance caused by bending), and the dynamic features of gauge factor, normalized resistance change between bending steps, and normalized rate of bending response, as illustrated in Figure S1. The gauge factor was determined by simple geometric calculations using the deflection (sagitta) to measure the radius of curvature, which was then used for the following equation: gf= e e+d (5) where e is the substrate thickness and d is the diameter of curvature. The rate of bending response was calculated as the bending response slope normalized by the pre-bending resistance, and the resistance change at rest was calculated as the difference between the resistance at start and end of rest normalized to the initial resistance. The unbent response to analyte exposure was calculated as the normalized rise to 95% response. Repeated measurements were averaged and standard error was calculated. For multivariate data analysis, discriminant factor analysis (DFA) was run using JMP software, and blind tests were carried out using k-fold analysis and leave-one-out calculations when noted. DFA is a linear supervised pattern recognition method that effectively reduces multidimensional 4

5 Resistance [Ohm] experimental data, in which the classes to be discriminated are defined before the analysis is performed. DFA was also used as a heuristic to select the most relevant sensors and bending features by filtering out non-contributing features. DFA determines the linear combinations of the input variables (features being extracted from each sensor) so that the variance in each class is minimized and the variance between classes maximized. The DFA output variables (i.e. canonical variables - CV) are achieved in mutually orthogonal dimensions; the first CV is the most powerful discriminating dimension. 4. Bending-Related Feature Extraction The following figure illustrates the bending-related features extracted from the sensors, as well as the unbent VOC response. Gauge factor calculation is not illustrated in the figure, and was calculated as described in the previous section. Nitrogen VOC exposure Unstrained Exerted strain Constant strain R(3) R(4) R3 - R overall bending response = R change at rest = R 4 - R 3 R R(2) normalized response rate = R 3 - R 2 t 3 - t 2 R 2 R(1) 0.95( R - R ) 2 1 unbent VOC response = R 1 Time [s] Figure S1 Features extracted from sensor strain and VOC response. Purple arrows indicate VOC exposure times, and orange arrows describe the progression of strain exertion. 5

6 5. Effect of Film Thickness on Film Morphology For the sensing array used in the following section, a variety of sensors were prepared using different ligands, film thicknesses and sintering times. While both NTMBT and CBMT gave good selectivity (Figure 2), CBMT was used for further examination in film thickness experiments due to excellent reproducibility and repeatability. Naked Kapton OD 0.2 OD 0.3 OD 0.4 Figure S2 HR-SEM images of naked Kapton, and 3 CBMT films deposited on Kapton at different thicknesses. CBMT sensors were prepared at varying film thicknesses (as characterized by UV absorption) and film morphology was examined using high resolution SEM (Figure S2). Thin films (OD 0.2) gave a porous coverage that was not complete over the substrate. GNP substrate surface coverage increases with cycle number, following the island growth process previously observed. 6 This correlates with the thin film growths visualized on smooth (PET) substrates seen previously - as film thickness increases and pores are filled with nanoparticles, additional deposits cluster on the surface. Sensors were curved to a bending radius of 6 mm, a relatively small radius that provides significant curvature allowing visualization of morphological distortions. Multiple cracks were seen in the 0.3 OD film, but none were seen in the 0.2 OD and 0.4 OD films. It is possible that the non-continuous GNP film did not undergo the same strain in thinner film as the substrate below; in thicker films, the film was too robust to crack. 6

7 6. Effect of Thickness of VOC and Strain Response Figure S3(B) shows the effect of film thickness on baseline resistance, which followed the previously observed trend of decreasing resistance with increased thickness 6. The unbent response to VOC exposure is also shown, decanal being provided as a representative example, but the trend of increased response with increased thickness was observed for many VOCs used in these exposure experiments (data not shown). When bending under nitrogen, no clear trend was visualized, OD 0.3 films giving the smallest bending response. Two main mechanisms are responsible for bending response; film swelling due to analyte adsorption increases resistance, whereas changes in the dielectric constant of inter-particle space decrease resistance. In non-continuous films, the pores may fill with adsorbed VOC, replacing air with a medium that has a greater dielectric constant opposing the resistance increase caused by film swelling and leading to a weaker overall bending response. Therefore, the results in Figure S3(C) can be explained by the morphological findings shown in Figure S3(A); non-continuous films exhibit greater responses due to relatively few percolation pathways that can be disconnected with small changes in island distance. However, when bending-induced cracks appear, they bear the strain, leaving the majority of the percolation pathways unaffected; this prompts a relatively minor bending response. It has been suggested that, in addition to the interparticle rearrangement described above, the entire film can restructure to accommodate the strain, inducing smaller changes along the individual percolation pathways. 14 7

8 Baseline resistance [MOhm] Normalized response to 0.15% strain [%] 95% rise normalized to baseline A OD 0.2 OD 0.3 OD Optical density [OD] B Optical density [OD] C OD 0.2 OD 0.3 OD 0.4 ' VOC concentration [P/P0] D Figure S3 - (A) Morphological differences visualized by HR-SEM in films of varying thicknesses bent to a radius of 6 mm. Nano-cracks are highlighted with red squares. (B) The effect of film thickness on baseline resistance. (C) The bending response under nitrogen. (D) Example of the effect of film thickness on unbent VOC (decanal) response. The correlation between VOC and bending response in relation to film thickness was measured using 7 compounds related to ovarian cancer of varying properties (Figure S4). Bending responses under VOC exposure were normalized to bending response under nitrogen, a response found to vary with VOC type and concentration. These specific analytes were chosen as they provide a group with a variety of functional groups that are all statistically significant or almost significant in differentiating between the breath of ovarian cancer patients and control groups. 3 8

9 Normalized strain r esponse under VOC A B OD 0.2 OD 0.3 OD ethylhexanol 3-heptanone Water Decanal VOC Hexadecane No nanal Styrene Figure S4 - Film thickness dependence of VOC response under flexural strain exertion for CBMT sensors. Response was defined as the overall (static) normalized resistance change accompanying a flexural strain of 0.15% under VOC exposure, normalized to the response to an identical strain under nitrogen. (A) Hotplot of thickness-dependent VOC/strain response. Film thicknesses are defined by optical density. The VOCs are 2-ethylhexanol (VOC1), 3-heptanone (VOC2), water (VOC3), decanal (VOC4), hexadecane (VOC5), nonanal (VOC6), and styrene (VOC7). Concentration is given in units of P/P 0 after VOC type on the x-axis. (B) VOC/strain response under exposure to a single concentration of P/P 0 =0.04. As seen from Figure S4, concentration-dependent bending response varied with the analyte. No significant concentration effect was visualized for the more polar analytes (2- ethylhexanol, 3-heptanone, water), whereas the relative response increased with concentration under exposure to decanal, hexadecane, and styrene, and decreased with concentration under 9

10 nonanal. 3-heptanone can be grouped with polar molecules when compared to nonanal and decanal due to the more polar nature of the ketone carbonyl. 3-heptanone also has relatively large errors, making it possible that the response follows a trend that cannot be understood from this experiment. It is possible that the polar VOCs either adsorbed with more difficulty into the aromatic ligand-capped GNP film, leading to a maximum response independent of concentration. The smallest relative responses have been visualized for water and hexadecane, both unable to adsorb easily into the film because of polarity and length, respectively. Therefore the response to bending under these VOCs is similar to that under nitrogen. This is particularly significant for sensors used for breath analysis up to 90% relative humidity is found in exhaled breath, leading to potential difficulty in obtaining significant responses to the desired VOCs. A low response to water indicates that this barrier can be overcome with these specific sensors. In general, there is a positive relationship between film thickness and relative response to bending. While the thicker and less porous films have a slightly decreased bending response, they have a significantly increased analyte response, leading to a greater overall response. Swelling leads to a situation in which there are fewer nanoparticles in every unit of volume, therefore greater interparticle distance (and therefore greater bending response) is induced by bending the substrate to a constant strain Effect of Ligand Type on Bending and VOC Response Adjacent ligand-ligand interactions, as well as ligand-analyte interactions, define the response mechanisms. To understand more fully these interactions, sensors were prepared of the allligand types at a single film thickness (0.3 OD) and exposed to the 7 analytes used as ovarian cancer biomarkers (Figure 2). The ETP ligand contains a strongly activating ether substituent, and therefore pi stacking between adjacent ligands can be considered significant. Bending response therefore has a negative relative value (when normalized to the bending response under nitrogen) because styrene adsorbed into the increased distances between adjacent nanoparticles increases film conduction due to the additional delocalized electrons now being involved in conductance. Water and 2-ethylhexanol have significant positive relative bending responses in ETP sensors any polar analyte succeeding in penetrating the film (perhaps helped by interaction with the electronegative ether) will disrupt the pi stacking that so significantly increases conduction. In this sensory type, as in most others examined, decanal and nonanal have similar responses, whereas nonanal induces a greater relative response, possibly due to a size effect. NT ligands 10

11 also have significant pi stacking due to the increased likelihood of aromatic ligands from adjacent nanoparticles overlapping, and also their purely aromatic structure. Therefore, styrene has a negative relative effect on the bending response described above, and water has the most strongly positive response. This may be explained by naphthalene being less aromatic than benzene and therefore polar molecules partially penetrate the film, thereby disrupting pi stacked interactions. NTMBT contains 2 highly electronegative substituents, with strong dipoledipole interactions. This limits pi stacking and therefore styrene has a positive (albeit minor) relative bending response due to limited adsorption. 2-ethylhexanol and 3-heptanone, the VOCs with the strongest dipoles, have the greatest positive relative bending response due to significant adsorption. Water has a negative relative response due to significant adsorption and hydrogen bonding with the electronegative substituents that can increase conductance. tbbt contains a branched substituent that prevents the structural alignment necessary for successful pi stacking. Styrene therefore induces a positive relative response due to only film swelling. Hexadecane is the largest VOC examined, and tbbt is the most non-conjugated sterically impaired ligand. It is therefore highly likely that swelling was limited, as described earlier 15, and the change in permittivity decreased the resistance. These same factors could decrease the response to bending due to limited particle distancing as a function of flexural strain. The CBMT ligand contains a single electronegative deactivating group, and therefore pi stacking is less significant than in other ligands; this leads to styrene causing a positive relative bending response. All the other analytes used in this ligand induced relatively strong positive relative bending responses, possibly due to the fact that this ligand can interact with both polar and nonpolar group due its weakly aromatic and weakly electronegative aspects. 11

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