Online Data Supplement for:

1. EXTENDED METHODS
1.1 Breath Collection
Alveolar exhaled breath was collected in chemically inert Mylar bags (Eco Medics) in a
controlled way, following a 3 minute procedure of lung wash as described elsewhere. 1,2 The
procedure was designed to avoid ambient contaminants and nasal entrainment of gas from
entering the sampling bags. Each subject provided at least one Mylar bag (750 ml). The content
of each bag was transferred through an off-line procedure to reusable tenax sorbent tubes
(SKC). Room-air samples were collected by pumping ambient air at the collection site through a
similar sorbent tube. The tubes were kept at 4ºC in a clean environment. The sorbent tubes
were then shipped to the Technion (Haifa, Israel) for analysis by the gas-chromatography/mass-
spectrometry (GC-MS) and by the chemical nanoarray.

1.2 Chemical analysis of the breath samples by GC-MS

Gas-chromatography/mass-spectrometry (GC-MS; QP2010; Shimadzu Corporations) with a
SLB-5ms capillary column (with 5% phenyl methyl siloxane; 30 m length; 0.25 mm internal
diameter; 0.5 μm thicknesses; from Sigma-Aldrich), combined with a thermal desorption (TD)
system (TD20; Shimadzu Corporation) was used for the chemical analysis of the breath samples.
At first the VOCs were released from the Tenax sorbent material of each storage tube by
heating the sorbent material for 10 min. at 270°C in a pre-heated stainless steel thermal
desorption chamber (350 ml); then the air with the breath VOCs from the chamber was
pumped through a TD tube (flow rate: 100 ml/min). The TD tubes were injected into the GC-
system in splitless mode at 30 cm/sec constant linear speed and under 0.70 ml/min column
flow. The following oven temperature profile was set: (a) 10 min at 35°C; (b) 4°C/min ramp until
150°C; (c) 10°C/min ramp until 300°C; and (d)15 min at 300°C. The GC-MS chromatograms were
analyzed using the GCMS post-run analysis program (GCMS solutions version 2.53SU1,
Shimadzu Corporation), and the compounds were tentatively identified through spectral library

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match (Compounds library of the National Institute of Standards and Technology, Gaithersburg,
MD 20899-1070 USA). Statistical analysis was carried out using SAS JMP, Version 10.0 (SAS
Institute Inc., Cary, NC, USA, 1989-2005) for Wilcoxon/Kruskal-Wallis tests.

1.3 Breath Analysis using the Chemical Nanoarray

The nanomaterial-based sensor array used in this study to analyze the breath samples
contained cross-reactive, chemically diverse chemiresistors that were based on two types of
nanomaterials: (i) organically stabilized spherical gold nanoparticles (GNPs, core diameter: 3-4
nm), and (ii) Single wall carbon nanotubes (SWCNTs) capped with polycyclic aromatic
hydrobarbon (PAH). The chemical diversity of the sensors was achieved through different
organic functionalities. The GNP and SWCNT/PAH or SWCNT/HBC sensors used in this study
responded rapidly and reversibly when exposed to typical VOCs in the breath. 3-5 Additionally,
we have confirmed that they have a very low response to water. 2,4-7 The effect of the high and
varying room air humidity and the high and varying humidity levels in exhaled breath is
insignificant, since we used Tenax TA sorbet material to trap the breath samples, which shows
low water trapping of generally less than ∼2-3 mg of water/g of adsorbent even at 100%
relative humidity at room temperature.8
1.3.1 Organically stabilized spherical gold nanoparticles (GNPs)
The Monolayer capped Au NPs were synthesized by the two-phase Brust method with
different modifications as described elsewhere.2,9-12 The cross-reactive chemical sensors that
were used are based on spherical gold nanoparticles (GNPs; 3–4 nm core diameter) coated
with: hexanethiol, 2-ethylhexanethiol, 3-methyl-1-butanethiol, octadecylamine, decanethiol,
dodecanethiol, 2-mercaptobenzoazole, 4-methoxy-toluenethiol, tert-dodecanethiol, 2-amino-4-
chlorobenzenethiol, 2-mercaptobenzimidazole, benzylmercaptan, 2-nitro-4-trifluoro-
methylbenzenethiol, 2-naphthalenethiol, 2-nitro-4-trifluoro-methylbenzenethiol and 2-
mercaptobenzoazole, where the organic ligands provided the broadly cross-selective
adsorption sites for the breath VOCs. 2,9-11,13,14 The chemiresistive layers were formed by drop-
casting the solution onto semi-circular microelectronic transducers, until the baseline resistance
of the devices has reached the range of 1kΩ-1MΩ. The device was dried for 2 hours at ambient

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temperature and then baked overnight at 50°C in a vacuum oven. The microelectronic
transducers consisted of ten pairs of circular interdigitated (ID) gold electrodes on silicon with
300 nm thermal oxide (Silicon Quest International, Nevada, US). The outer diameter of the
circular electrode area was 3mm, and the gap between two adjacent electrodes and the width
of each electrode both 20 m.

1.3.2 Single wall carbon nanotubes (SWCNTs) capped with polycyclic aromatic
hydrobarbon (PAH)
The SWCNT/PAH sensors were based on electrically continuous random networks of
SWCNTs that were formed by drop-casting a solution of SWCNTs (supplied from ARRY
International LTD, Germany; ∼30% metallic, ∼70% semiconducting, average diameter = 1.5 nm,
length = 7 mm) in dimethylformamide (DMF, from Sigma Aldrich Ltd., >98% purity) using
sonication for 15 min, resulting in a 0.02 wt% dispersion, which was then left for half an hour in
a 50 ml vial for sedimentation of large aggregates. Ultracentrifugation for 25 min of the
resulting homogeneous dispersion above the precipitate was done for further purification. This
procedure was performed twice, to ensure that the majority of the aggregates and impurities
are removed. The device was let left to dry overnight under ambient conditions to enhance the
self-assembly of the SWCNTs and to allow the solvent evaporation. This procedure was
repeated until the device reached a resistance of 100 KΩ - 10 MΩ. The microelectronic
transducers for the SWCNT sensors consisted of ten pairs of 4.5 mm wide, interdigitated Ti/Pd
electrodes on silicon with two microns of thermal oxide (Silicon Quest International, Nevada,
US). The gap between two adjacent electrodes was 100 m. The SWCNT sensors were
organically functionalized with cap-layers that were composed of two Polycyclic Aromatic
Hydrocarbon (PAH) derivatives and two Hexabenzocoronene (HBC) derivatives. PAH-5 and PHA-
6 contain hydrophobic mesogens that are terminated with alkyl chains and functionalized with
alcohol and carboxylic acid, respectively.4 HBC-C12 and HBC-C6,2 have discotic aromatic cores and
a corona composed of straight C12 and branched C6,2 alkyl chains.5,7
Table S1 The organic functionalities of the nanomaterial-based sensors in the array. The sensors
that were used in the different DFA models.
Organic layer Sensing feature

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 HBC-1 Normalized change of sensor’s
resistance at the area under curve
1,6 hexanedithiol Normalized change of sensor’s
resistance at the end of the exposure
HBC-1 Normalized change of sensor’s
resistance at the end of the exposure
HBC-3 Normalized change of sensor’s
resistance at the middle of the
exposure
Dodecanethiol Normalized change of sensor’s
resistance at the middle of the
exposure
Decanethiol Normalized change of sensor’s
resistance at the peak of the exposure
Octadecanethiol Normalized change of sensor’s
resistance at the peak of the exposure

1.3.3 Exposure of the Chemical Nanoarray to Breath Samples

The fabricated sensors described in sections 1.3.1-2 were mounted on a custom
polytetrafluoroethylene PTFE circuit board inside a stainless steel test chamber with a volume of
100 cm3. The sampling system delivered pulses of the breath samples from the thermal
desorption device to the chemical nanoarray. The chamber was evacuated between exposures.
An Agilent multifunction switch 34980 was used to measure the resistance of all sensors as a
function of time. Typically, the sensors’ responses were recorded for 5 min in (40 mtorr)
vacuum, followed by 5 min under breath sample exposure, followed by another 5 min in
vacuum. In order to supervise the sensor’s functionality, during the experiment, and also to

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overcome sensors’ response drift, a fixed calibration gas mixture, containing 23.8 ppm isopropyl
alcohol, 6.3 ppm Trimethylbenzene and 1.2 ppm 2-ethylhexanol was exposed to the sensors on
a daily bases, then the raw signals of the breath samples response, was normalized by its
parallel response to the calibration gas, form the same day. All samples received from the
clinical collaborators were tagged with and patient's number. The samples were blindly run and
then analyzed in the Technion-IIT. The results were then conjugated with the relevant clinical
data.

1.4 Pattern Recognition Analysis:

During exposure of the sensors to the breath samples or the calibration gas, the
interaction between the volatile compounds and the organic sensing layer results in a change in
the sensors resistance, however, it recovers to baseline resistance, almost immediately with
evacuating the sample from the chamber. From each response, four sensing features were read
out; the normalized change of sensor’s resistance at the peak, middle and end of the exposure,
and the area under curve of whole measurement. Then, discriminate function analysis (DFA)
was used to classify the data.15 DFA is a linear, supervised pattern recognition method that is
supplied with the classification information regarding every measurement in the training set.
Using a linear combination of the input variables, DFA finds new orthogonal axes (canonical
values), in order to minimize the variance within each given class and maximize the variance
between two classes. In order to prevent over fitting of the data, we used minimal number of
sensing features to build discriminative model, stressing a ratio lower than 1:6 of features to
samples, moreover, Leave-one-out cross-validation method was then employed to calculate
the prediction accuracy. Given n measurements, the model was computed n times using n-1
training vectors, each time, and classifying the vector left out, accordingly, then we determined
whether the result last turned out to be, True Positive (TP), False Positive (FP), True Negative
(TN) or False Negative (FN). All possibilities of leave-one-sample-out were considered, and the
classification accuracy was identified by calculating the sensitivity and specificity of the built
model, while:

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1.5 Wilcoxon/Kruskal-Wallis test:
The Wilcoxon/Kruskal-Wallis test is a nonparametric test that compares two data sets and is
based solely on the order in which the observation from the two groups fall. This statistical test
is useful when the data are not necessarily normally distributed; if the variances for the two
groups are markedly different; or if the data are measurements on ordinal scale. This analysis
serves as alternative for the paired student's t-test. Moreover, this test is suitable when dealing
with small sizes data sets.

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