Recent advances of laser-spectroscopy-based techniques for applications in breath analysis

Abstract

Laser absorption spectroscopy (LAS) in the mid-infrared region offers a promising new effective technique for the quantitative analysis of trace gases in human breath. LAS enables sensitive, selective detection, quantification and monitoring in real time, of gases present in breath. This review summarizes some of the recent advances in LAS based on semiconductor lasers and optical detection techniques for clinically relevant exhaled gas analysis in breath, specifically such molecular biomarkers as nitric oxide, ammonia, carbon monoxide, ethane, carbonyl sulfide, formaldehyde and acetone.

Full text

IOP PUBLISHING JOURNAL OF BREATH RESEARCH
J. Breath Res. 1(2007) 014001 (12pp) doi:10.1088/1752-7155/1/1/014001
TOPICAL REVIEW
Recent advances of laser-spectroscopy-
based techniques for applications in
breath analysis
Matthew R McCurdy1,2, Yury Bakhirkin1, Gerard Wysocki1,
Rafal Lewicki1andFrankKTittel
1
1Rice Quantum Institute, Rice University, 6100 Main St., Houston, TX 77005, USA
2Medical Scientist Training Program, Baylor College of Medicine, 1 Baylor Plaza, Houston, TX 77030,
USA
E-mail: fkt@rice.edu
Received 27 March 2007
Accepted for publication 18 May 2007
Published 11 July 2007
Online at stacks.iop.org/JBR/1/014001
Abstract
Laser absorption spectroscopy (LAS) in the mid-infrared region offers a promising new
effective technique for the quantitative analysis of trace gases in human breath. LAS enables
sensitive, selective detection, quantification and monitoring in real time, of gases present in
breath. This review summarizes some of the recent advances in LAS based on semiconductor
lasers and optical detection techniques for clinically relevant exhaled gas analysis in breath,
specifically such molecular biomarkers as nitric oxide, ammonia, carbon monoxide, ethane,
carbonyl sulfide, formaldehyde and acetone.
(Some figures in this article are in colour only in the electronic version)
Introduction
Analyzing compounds in breath for clinical diagnosis and
therapeutic management is a burgeoning field with great
potential. Breath analysis may in time be incorporated into
routine clinical care as blood tests are used today—for disease
screening, diagnosis, prognosis, activity (inflammatory status)
and response to therapy.
Development of trace gas sensor technologies is a
key factor in the advancement of breath analysis. Small,
affordable, easy-to-use, sensitive and accurate techniques are
needed for detection of breath compounds. Ideally, exhaled
molecules can be quantified in real-time using a hand-held
device [1].
Analytical instrumentation involving mass spectrometry
with or without prior separation by gas chromatography
is the most commonly used method to quantify exhaled
molecules. Currently, atmospheric pressure ionization mass
spectrometry (API-MS) and selected ion flow tube mass
spectrometry (SIFT-MS) are the most frequently used methods
for direct breath analysis [1]. New infrared semiconductor
laser based trace gas sensor technology offers the feasibility
of compact (hand held), reliable, non-aliasing, user friendly,
autonomous and low-cost devices without sacrificing high
detection ranges from ppmv, to sub-ppbv levels depending on
the specific biomarker gas species and the detection method
employed [2,3]. LAS is particularly suited for applications
where continuous monitoring of targeted exhaled gases with
sensitivity, selectivity and fast response are required, such as in
critical care and operating room settings. LAS does not require
consumable products or an operator. Single-molecule exhaled
breath LAS sensors are already commercially available. For
example, an exhaled nitric oxide sensor (Breathmeter) is
available from Ekips Technologies, Inc., and an exhaled
ammonia sensor (Nephrolux) is available from Pranalytica,
Inc. At present, LAS performs best with small molecules
and is not amenable to characterization of a large number of
molecules, such as in screening for bio-markers of diseases
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J. Breath Res. 1(2007) 014001 MRMcCurdyet al
and cancers. However, recent advances in broadly tunable
laser sources using quantum cascade lasers suggest that LAS
can be an effective tool for breath profiling (quantification
of multiple exhaled molecules including broadband absorbing
species) in the future [4].
Laser absorption spectroscopy
The mid-infrared spectral range is ideal for tunable
laser absorption spectroscopy (LAS) since most molecular
gases possess strong, characteristic fundamental rotational–
vibrational lines. High-resolution LAS can resolve absorption
features of targeted molecules and selectively access optimal
spectral lines at low (100 Torr) pressure without interference
from CO2and H2O to achieve high levels of trace gas
detection sensitivity and specificity. Avoiding CO2and H2O
interferences is particularly important in the development of
biomedical gas sensors for breath analysis.
Laser absorption spectroscopy operates on the principle
that the amount of light absorbed by a sample is related
to the concentration of the target species in the sample.
Light of known intensity is directed through a gas sample
cell and the amount of light transmitted through the sample
cell is measured by a detector. If we assume incident
light intensity, I0(x =0,λ), and transmitted light intensity,
I(x,λ), the Beer’s law relates the transmitted light to the
incident light and the absorption coefficient of the sample,
α(λ), as
I(x,λ) =I0e−α(λ)x (1.1)
where λis the wavelength and xis the path length.
Concentration is determined from the absorption coefficient.
Each gas has an absorption line at a unique wavelength,
preferably free of interference of other gases in the sample cell.
Equation (1.1) shows that the ability of a LAS-based sensor
to detect a specific concentration depends on the path length
through the absorbing medium.
The strongest molecular rotational–vibrational
transitions, which are desired to perform ultra-sensitive
concentration measurement, are in the mid-infrared (mid-IR)
spectral region. The usefulness of laser spectroscopy in
this region is limited by the availability of reliable, tunable,
continuous wave, infrared laser sources. The most practical
sources include lead salt diode lasers, coherent sources based
on difference frequency generation (DFG), optical parametric
oscillators (OPOs), tunable solid-state lasers, quantum and
interband cascade lasers. Sensors which utilize lead salt
diode lasers are typically large in size and require cryogenic
cooling because such lasers operate at temperatures of <90 K.
DFG-based spectroscopic sources (especially bulk and
waveguide periodically poled lithium niobate [PPLN] based)
have recently been shown to be reliable and compact [5].
The recent advances of quantum cascade (QC) and
interband cascade (IC) lasers fabricated by band structure
engineering offer an attractive new source option for mid-
infrared absorption spectroscopy with ultra-high resolution
and sensitivity [6]. The most technologically developed mid-
infrared QC laser source to date is based on type-I intersubband
transitions in InGaAs/InAlAs heterostructures [7–9]. More
recently, interband cascade lasers (ICLs) based on type-II
interband transition have been reported in the 3–5 µmregion
[10–13].
LAS-based techniques
Obtaining detection sensitivities at ppb or sub-ppbv
levels either requires long effective optical pathlengths or
suppression of laser and optical noise. Long optical path
lengths (30 m), typically realized in multipass absorption
cells, are used [14–17]. As an example, for nitric oxide
detection, a sensitivity of 0.7 ppb Hz−1/2 can be achieved using
a multipass cell with a 36 m path and a 0.3 l volume (Aerodyne
Research, Inc.—Model AMAC 36) [17].
Another detection technique is based on the absorption
that occurs in a high finesse cavity, which can be determined
from the rate of decay of light intensity that occurs inside
the cavity and is known as cavity ringdown spectroscopy
(CRDS). CRDS has been successfully applied to measure NO
concentration at ppbv levels [6,18,19] (for a review, see [20]).
The advantage over direct absorption spectroscopy results
from (i) the intrinsic insensitivity of the CRDS technique to
light source intensity fluctuations, and (ii) the extremely long
effective path lengths (many kilometers) that can be realized
in stable high finesse optical cavities. In CRDS, laser light is
resonantly coupled into a high-Qoptical cavity that consists
of two ultra-low-loss dielectric mirrors (∼250 ppm or better).
After the incident laser light is interrupted, the intensity of
light in the cavity decays exponentially. The ringdown time τ
for a two-mirror cavity is given by
τ=1
c
1
αl −ln(R1R2)1/2(1.2)
where lis the cavity length, cis the speed of light, R1
and R2are the cavity mirror reflectivities, and αis the
absorption coefficient of the medium inside the cavity. Thus,
by measuring the decay constant, the absorption by a sample
present in the cavity can be determined.
Another technique, which employs a high-finesse optical
cavity and is simpler to implement than CRDS, is integrated
cavity output spectroscopy (ICOS). This technique is based
upon the excitation of a dense spectrum of transverse
cavity modes and time averaging the cavity output. This
scheme relies on the accidental coincidence of the frequencies
associated with excitation laser source and the ICOS cavity.
By external dithering of one of the ICOS mirrors the number
of excited ICOS cavity modes can be increased [21,22].
Less critical alignment of the exciting laser beam with
respect to the ICOS cavity can be realized with an off-
axis ICOS optical geometry, which provides an increased
spectral density of cavity modes, and thereby minimizing
noise in the resulting absorption spectra [23–25]. The off-axis
ICOS geometry preserves the cavity path length enhancement
F/π (where Fis the cavity finesse) as well as producing a
dense mode spectrum. This interaction between the exciting
laser and the ICOS cavity reduces the associated amplitude
noise. Moreover, all transverse TEMmn modes contribute
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to detection of the intracavity trace gas absorber, and the
off-axis ICOS measurement technique is more insensitive to
vibrations and misalignments than CRDS and on-axis ICOS.
All three forms of cavity-enhanced absorption spectroscopy
require sufficient excitation power at levels >1 mW because
of reduced transmission through a high finesse optical
cavity.
Nitric oxide
The presence of endogenous nitric oxide (NO) in exhaled
breath of humans and animals was first reported in 1991
[26]. Since then it has become increasingly apparent that
measurements of exhaled NO constitute a new way to monitor
the inflammatory status in respiratory disorders, such as
asthma and other pulmonary conditions. Exhaled nitric oxide
as a measure of inflammation is suggested as providing
the best combination of disease evaluation and practical
implementation for improved asthma outcomes [27]. Exhaled
nitric oxide has been successfully employed in chronic
asthma treatment monitoring to reduce the dose of inhaled
corticosteroids, which have serious side effects, without
compromising asthma control [28]. In treating asthma, which
affects 19 million Americans, exhaled NO may soon be
incorporated into routine clinical care [29].
Nitric oxide is the only exhaled biomarker for which
breath collection guidelines have been published. The
American Thoracic Society and the European Respiratory
Society published an updated Joint Statement [30] in 2005 with
recommendations for standardized procedures for the online
and offline measurement of exhaled lower airway and nasal
NO. Exhaled NO concentrations from the lower respiratory
tract exhibit significant expiratory flow rate dependence [31].
Because of this, exhaled NO is commonly collected using
a single breath maneuver at a constant exhalation flow rate.
Exhaled NO sharply rises and reaches a plateau, which can
have a positive, negative or near-zero slope. The plateau
concentration is defined as the average concentration overa 3 s
window in the plateau region [30]. The plateau level is reported
as the exhaled NO value. Measurements of the NO plateau at
multiple flow rates allow for determination of the origin of NO
(proximal or distal lung) by estimating the flow-independent
exchange parameters [32]. The American Thoracic Society
recommends that an NO detection system has a sensitivity of
1 ppb, and response time of <0.5 s. Exhaled NO
analyzers based on the chemiluminescent technique [33]are
commercially available. Competing current technologies
such as LAS, electrochemical [34], and resonance-enhanced
multiphoton ionization coupled with time-of-flight mass
spectrometry [35] may provide lower initial and operating
costs, smaller size and less frequent calibration.
Several groups have reported NO analyzers using LAS.
Nelson et al [36] reported measurements of nitric oxide in air
with a detection limit of less than 1 nmol mol−1(<1 ppbv)
using a thermoelectrically cooled quantum cascade laser
operated in a pulsed mode at 5.26 µm (1897 cm−1) and coupled
to a 210 m path length multiple-pass absorption cell at a
reduced pressure (50 Torr). The sensitivity of the system was
enhanced by normalizing pulse-to-pulse intensity variations
with temporal gating on a single HgCdTe detector. A detection
precision of 0.12 ppb Hz−1/2was achieved with a liquid-
nitrogen (LN2)-cooled detector. This detection precision
corresponds to an absorbance precision of 1 ×10−5Hz−1/2
or an absorbance precision per unit path length of 5 ×
10−10 cm−1Hz−1/2. More recently, a sensitivity of
0.7 ppb Hz−1/2 was reported using a cw TEC-cooled QCL
and multipass cell with a 36 m path and a 0.3 l volume [17].
A spectroscopic gas sensor for nitric oxide detection
based on a CRDS technique achieved a 0.7 ppb standard
error for NO in N2for a data collection time of 8 s [18]. In
this experiment, a cw LN2-cooled distributed-feedback (DFB)
QC laser operating at 5.2 µm was used as a tunable single-
frequency mid-infrared light source. Both laser-frequency
tuning and abrupt interruptions of the laser radiation were
performed by manipulation of the laser current. A single
ringdown event sensitivity was achieved to an absorption of
2.2 ×10−8cm−1.
A gas analyzer based on off-axis integrated cavity output
spectroscopy (ICOS) and a cw LN2-cooled QCL also operating
at ∼5.2 µm was developed by our group to measure NO
concentrations in exhaled human breath [37]. A compact
ICOS sample cell (length =5.3 cm, volume ∼80 cm3),
which was suitable for on-line and off-line measurements
during a single breath cycle, was designed and evaluated.
Using a combination of wavelength modulation technique and
ICOS resulted in a noise-equivalent (signal-to-noise ratio of 1)
sensitivity of 2 ppbv of NO for a 15 s data acquisition and
integration time.
Subsequently, a nitric oxide sensor based on off-axis
ICOS and a thermoelectrically cooled, cw DFB QCL
laser operating at 5.45 µm (1835 cm−1) combined with a
wavelength-modulation technique was developed to determine
NO concentrations at the sub-ppbv levels [38,39]. The
sensor as shown in figure 1employed a 50 cm long high-
finesse optical cavity that provided an effective path length of
700 m. A noise equivalent minimum detection limit
(MDL) of 0.7 ppbv with a 1 s integration time was
achieved. A wavelength-modulated signal for a calibrated
NO concentration of 23.7 ppbv was fitted by a pre-acquired
reference spectrum using a general linear fit procedure [40]
(shown in figure 2).
In [41] a thermoelectrically cooled, continuous-wave,
QC laser operating between 1847 and 1854 cm−1was used
in combination with wavelength modulation spectroscopy to
achieve sub-ppbv level for NO. The P7.5 doublet of NO
centered around 1850.18 cm−1was used for concentration
measurements. Using an astigmatic multiple-pass absorption
cell with an optical path length of 76 m and a total acquisition
time of 30 s, a detection limit of 0.2 ppbv was achieved.
The corresponding minimal detectable absorption was
8.8 ×10−9cm−1Hz−1/2.
Recently, the optical performance of a NO/CO2sensor
employing integrated cavity output spectroscopy (ICOS) with
a5.45µm TEC cw DFB QCL [39] and a LN2-cooled
cw DFB QCL operating at 5.22 µm capable of real-time
simultaneous NO and CO2measurements in a single breath
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Figure 1. CW-TEC-DFB QC laser based nitric oxide off axis-integrated cavity output spectroscopy [39].
Figure 2. 2f OA-ICOS based NO absorption signal [39].
cycle was reported by our group [42]. A NO noise-
equivalent concentration of 0.4 ppb within a 1 s integration
time was achieved. The off-axis ICOS sensor performance
was compared to a chemiluminescent NO analyzer and a non-
dispersive infrared (NDIR) CO2absorption capnograph for
offline and online human exhaled breath measurements. The
off axis ICOS sensor measurements were in good agreement
with the data acquired with the two commercial gas analyzers.
Figure 3depicts online nitric oxide concentrations at 3 l min−1
exhalation as a function of time for the ICOS sensor and
chemiluminescent analyzer. Fifteen exhaled breaths were
measured online with (a) ICOS and (b) chemiluminescence
each with 2.4 s averaging/computation time. The NO
plateau regions for ICOS and chemiluminescence were in good
agreement, with all data points within 1 standard deviation of
the 15 measurements. The ICOS measurements had a larger
standard deviation for each averaged concentration.
Figure 3. Online nitric oxide concentrations at 3 l min−1exhalation
as a function of time. Fifteen exhaled breaths were measured online
with (a)ICOSand(b) chemiluminescence each with 2.4 s
averaging/computation time. Solid line represents Sievers data
(16 samples s−1) smoothed with a 50 point first-order
Savitzky–Golay routine; squares with large cap error bars represent
Sievers data after 2.4 s averaging; circles with dashed line and small
cap error bars represent ICOS data after 2.4 s averaging and
computation; error bars are ±1 standard deviation [42].
In 2005 concentration measurements of NO for two N
isotopes (14N and 15N) simultaneously using a CO-laser at
λ=5µm and continuous-wave ring-down detection scheme
have been reported [43]. A linear ring-down cavity (length =
0.5 m) with high reflective mirrors (R=99.99%) was used
to achieve a noise-equivalent absorption coefficient of 3 ×
10−10 cm−1Hz−1/2. This corresponds to a noise-equivalent
concentration of 800 parts per trillion (ppt) for 14NO and
40 ppt for 15NO in 1 s averaging time. The detection of
the 15NO is unique to this system and allows 15NO to be used
as a tracer molecule in biochemical processes. A precursor
molecule containing the 15N tracer can be introduced into
a physiological system, such as the gastrointestinal tract or
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1935 1940 1945 1950 1955 1960
0.0
0.2
0.4
0.6
0.8
1.0
1947.16 1947.17 1947.18
0.0
0.2
0.4
0.6
Absorption
Wavenumber [cm
-1
]
R
3/2
(20.5) R
1/2
(23.5)
HITRAN 2000 simulation
Measured spectra
NO @ 4.9torr
9 cm pathlength
Absorption
Wavenumber [cm
-1
]
Figure 4. Nitric oxide absorption spectra measured at different
EC-QCL grating angles. The narrow laser linewidth allows a
spectral resolution of <0.001 cm−1[4].
lungs, and the elimination of 15NO in exhaled breath can be
measured.
Better selection of an optimum absorption line for
sensitive NO measurement as well as simultaneous multi-
species detection become feasible with widely tunable
external cavity (EC) QCLs. Our first EC-QCL with a
thermoelectrically cooled QCL gain medium fabricated using a
bound-to-continuum design and operating in continuous wave
at ∼5.2 µm was reported in 2005 [4]. This EC-QCL exhibited
a coarse single mode tuning over 35 cm−1and a continuous
mode-hop free fine tuning range of ∼1.2 cm−1. A direct
absorption NO spectrum measured with this EC-QCL is shown
in figure 4. The EC-QCL employs a piezo-activated cavity
mode tracking system for mode-hop free operation suitable
for high-resolution spectroscopic applications (see the inset in
figure 4) and multiple species trace-gas detection. Recently
a redesigned EC-QCL architecture as depicted in figure 5
was developed. Using a new gain chip it was possible to
(a)(b)
Figure 5. (a) Schematic diagram of the EC-QCL spectroscopic source and associated measurement system. (QCL—quantum cascade laser;
TEC—thermoelectric cooler; CL—collimating lens; LB—laser beam; GR—diffraction grating; PP—pivot point of the rotational
movement; M— mirror (mounted on the same platform with GR). (b) Photograph of EC-QCL assembly [44].
extend the EC-QCL tuning range to 155 cm−1from ∼1825 to
1980 cm−1, which covers almost the entire P, Q and R branches
of the fundamental NO vibrational band at 5.2 µm with a
maximum available optical power of ∼11 mW as depicted in
figure 6[44].
Ammonia
Ammonia (NH3), a product of urease hydrolysis of urea
to ammonia and carbamate, is one of the key steps in
the nitrogen cycle. In a recent longitudinal study of
30 healthy volunteers, exhaled ammonia concentrations
averaged 0.83 ppm and ranged from 0.25 to 2.9 ppm [45].
Exhaled NH3has been shown to increase steadily in the fasting
state [46]. Exhaled NH3is a potential non-invasive marker of
liver [47] and kidney function as well as peptic ulcer disease,
and these clinical applications need to be assessed in human
clinical studies.
A compact, transportable ammonia sensor based on a
thermoelectrically cooled pulsed QC-DFB laser operating at
∼10 µm was reported to have a sensitivity of better than
0.3 ppmv was achieved witha1mopticalpathlength [48].
The laser was scanned over two absorption lines of the NH3
fundamental ν2 band. This sensor was successfully applied to
continuous long-term monitoring of NH3concentration levels
in the range of 1 to 10 ppmv in bioreactor vent gases at the
NASA Johnson Space Center, Houston, TX.
More recently, a gas analyzer based on a pulsed,
thermoelectrically-cooled mid-IR quantum cascade laser
operating near 970 cm−1using CRDS was developed for the
detection of exhaled NH3[49]. A sensitivity of ∼50 parts
per billion with a 20 s time resolution was achieved for NH3
detection from human exhaled breath.
Quartz-enhanced photoacoustic spectroscopy (QEPAS)
first introduced in 2002 [50] has a high potential for
development of a sensitive miniature breath gas analyzers.
Ammonia detection with a MDL of 0.65 ppm (with 1 s
lock-in time constant) was demonstrated with a QEPAS
sensor employing a 1.53 µm telecommunication diode laser
delivering 38 mW of optical power [51]. The sensitivity of
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5000 5100 5200 5300 5400 5500
0.0
0.5
1.0
TOTAL TUNING RANGE: 155 cm
-1
Normalized signal
Wavelength [nm]
2000 1980 1960 1940 1920 1900 1880 1860 1840 1820
Wavenumber [cm-1]
0
2
4
6
8
10
12
Optical power [mW]
P
max
= 11.1 mW @ 950mA
Laser operation conditions:
CW, I=900mA, T=-30
o
C
CW, I=950mA, T=-30
o
C
Figure 6. Laser frequency tuning range and corresponding output power of a 5.2 µmEC-QCL[44].
the sensor can be significantly improved (up to ∼200 times)
by targeting the strongest ammonia absorption lines from the
fundamental vibrational band in mid-IR (∼10 µm) as well as
by using a redesigned absorption detection module. Recently
we have evaluated a thermoelectrically-cooled, distributed
feedback QCL that can deliver up to 150 mW of optical power
at 9.53 µm, which will be applied to both a LAS and QEPAS
[52] based sensor technique. Preliminary calculations indicate
that QEPAS detection employing such a QCL will result in a
MDL of ammonia concentration at the level of ∼3 ppb for 1s
lock-in time constant.
Carbon monoxide
Carbon monoxide (CO) is a gas that is both formed
endogenously and inhaled from the environment. Exhaled
CO as a biomarker to assess different diseases (cardiovascular,
diabetes, and nephritis) was first reported by Nikberg et al in
1972 [53]. Over the last 20 years exhaled CO has been used in
smoking cessation, to monitor bilirubin production, including
hyperbilirubinemia in newborns, and in the assessment of the
lung diffusion capacity. Exhaled CO levels, which are of
interest for breath analysis, range from 1 to 5 ppm in healthy
subjects.
A pulsed, thermoelectrically cooled distributed feedback
QC laser operating at 4.6 µm[54] achieved a noise-equivalent
detection limit of 12 ppb with a 2.5 min integration time
using a pathlength of 102 cm and the selected CO absorption
line at 2158.3 cm−1. The laser frequency could be tuned
over a 0.41 cm−1region encompassing the R(3) absorption
line at 2158.3 cm−1with appropriate settings of the QC
laser temperature and current. This transition is free from
interference of H2O and CO2, which are abundant in exhaled
breath. Absolute frequency assignment was performed by
comparison of experimental absorption spectra of CO and N2O
with the HITRAN 2000 database [55].
Recently, a thermoelectrically cooled, distributed-
feedback pulsed QC laser operating between 2176 and
2183 cm−1was used to measure CO in human breath [56].
The QC laser emission overlaps with the strong R(8)1 CO
ro-vibrational transition at 2176.2835 cm−1. A minimal
detectable absorption of 1.2 ×10−5cm−1was achieved
at an acquisition rate of 3 Hz by utilizing the frequency
chirp associated with a QCL with long laser pulses. With
short laser pulses and slow frequency scanning a minimal
detectable absorption 8.2 ×10−7cm−1was achieved with a
data acquisition time of 60 s. The breath measurements were
performed without frequency scanning, yielding a minimum
detectable absorption of 7.2 ×10−6cm−1, corresponding to a
detection limit of 175 ppbv using a 0.2 s integration time.
Ethane
Saturated hydrocarbons, such as ethane and pentane, are
generated from ω-3 and ω-6 fatty acids, respectively. In
contrast to the predominantly airway source of exhaled NO,
hydrocarbons are representative of blood-borne concentrations
through gas exchange in the blood/breath interface in the
lungs. Exhaled concentrations of ethane may be used to
monitor the degree of oxidative damage in the body [57].
Initial clinical studies have suggested the use of exhaled
ethane in inflammatory lung diseases such as asthma and
chronic obstructive pulmonary disease (for a review, see [58]).
Exhaled ethane may also correlate with disease status of non-
lung inflammatory diseases such as rheumatoid arthritis and
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J. Breath Res. 1(2007) 014001 MRMcCurdyet al
inflammatory bowel disease. Trace-gas sensors suitable for
breath ethane analysis should be capable of concentration
measurements at 100 ppt levels.
A detection limit of the order of 100 ppt ethane in
human breath was achieved by Dahnke et al usinga5s
integration time using a high-finesse ring-down cavity and a
LN2-cooled, tunable CO-overtone sideband laser in the 3 µm
region [59]. Quantitative time-resolved breath ethane
measurements were made during single exhalations [60].
The ethane alveolar plateau was characterized in three
subjects. Additionally, the ethane washout process was
described after inhalation of ethane. This work was the
first to demonstrate ppb-level time-resolved exhaled ethane
measurements. Although the number of subjects was small
(n=3), the work showed that an ethane sensor with sufficient
detection sensitivity and time resolution for real-time exhaled
ethane measurements can be realized. Subsequently the
same group reported an optical parametric oscillator combined
with cavity leak-out spectroscopy to achieve a minimum
detectable absorption coefficient of 1.6 ×10−10 cm−1Hz−1/2,
corresponding to an ethane detection limit of 6 parts per
trillion/Hz1/2 [61]. Further, frequency-tuning capabilities
enabled multi-gas analysis with simultaneous monitoring of
ethane, methane and water vapor in human breath.
ALN
2-cooled mid-IR lead-salt laser and Herriott cell were
employed by Skeldon et al to achieve an ethane detection
sensitivity of 70 ppt with a 0.7 s response time [62]. The
sensor was used to measure exhaled ethane from animals and
humans in several pilot studies [63] including a small lung
cancer study of 52 randomly selected patients at a respiratory
clinic [64]. Twelve patients were subsequently diagnosed with
lung cancer. Twelve control subjects age-matched to the lung
cancer group were taken from a larger control group of healthy
adults. After correcting for the ambient background, ethane
in the control group ranged from 0 to 10.54 ppb (median of
1.9 ppb), lung cancer patients ranged from 0 to 7.6 ppb (median
of 0.7 ppb), and non-lung cancer patients presenting for an
investigation of respiratory disease ranged from 0 to 25 ppb
(median 1.45 ppb). The laser-based ethane sensor proved to
be an effective tool for accurate and rapid sample analysis,
although there was no significant difference in exhaled ethane
among any of the subject groups.
Recently an optical sensor designed to target two trace
gases using two separate LN2cooled interband cascade
lasers (ICLs) in the spectral regions of 3.3 µm (ethane)
and 3.6 µm (formaldehyde) was demonstrated by our group
(see figure 7). The sensor based on 100 m astigmatic
Herriott multi-pass cell utilized a wavelength modulation
technique at two different modulation frequencies for ethane
and formaldehyde concentration measurements. The sensor
exhibited a minimum detection limit of 3.6 ×10−5Hz−1/2,
which corresponds to an ethane concentration of 150 pptv
measured with a 1 s integration time. The sensor was
evaluated for dual gas sensing using a custom mixture of
79 ppbv ethane and 330 ppbv formaldehyde (balance N2).
During the simultaneous dual trace-gas detection presented in
figure 8, the sensor showed a linear response to progressive
dilution of both gases and no cross-talk between the channels
[65,66].
Figure 7. Schematic diagram of a dual ICL based trace gas sensor
for simultaneous ethane and formaldehyde concentration
measurements [65,66].
Formaldehyde
Exhaled formaldehyde detection may be useful in monitoring
exposure to formaldehyde in the environment, which
according to American Conference of Governmental Industrial
Hygienists (ACGIH) should not exceed 300 ppb at any time.
Exhaled formaldehyde may also be used as a screening test
for primary or metastatic cancer. Exhaled formaldehyde has
been studied in a mouse model of breast cancer and in six
human subjects [67]. Exhaled formaldehyde concentrations
from three subjects without breast cancer were 0.3–0.6 ppm
and from three subjects with breast cancer were 0.45–1.2 ppm.
The sensor depicted in figure 7provided a minimum
detection limit for formaldehyde (1σ)of∼3.5ppbvwitha1s
integration time. By appropriate selection of the laser sources
in both optical channels, the sensor can be reconfigured to
target a number of trace-gas species, which possess absorption
features within the tuning range of two ICLs or QCLs.
Simultaneous multispecies detection of molecules which are
involved in closely related bio-chemical processes enables
studies of a complex process dynamics.
Formaldehyde detection was also demonstrated using off-
axis ICOS with an ICL at 3.53 µm[68]. A 12 mW continuous-
wave, mid-infrared, distributed feedback ICL was used to
quantify H2CO in gas mixtures containing ≈1–25 ppmv of
H2CO. Analysis of the spectral measurements indicated that a
H2CO concentration of 150 ppbv produces a spectrum with a
signal-to-noise ratio of 3 for a data acquisition time of 3 s.
Carbonyl sulfide
Carbonyl sulfide is a relatively unstudied exhaled gas. The
physiological origins of exhaled OCS can include the oxidative
metabolism of carbon disulfide [69] and the incomplete
metabolism of sulfur-containing essential systems, including
methionine [70]. Exhaled OCS has been measured in
normal subjects [71,72], lung transplant recipients [73,74],
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J. Breath Res. 1(2007) 014001 MRMcCurdyet al
Figure 8. Response of the two-channel optical sensor to a custom mixture of 79 ppbv ethane and 330 ppbv formaldehyde (balance N2)
successively diluted by an ultra high purity (UHP) N2(left panel) and by certified mixture of 100 ppb of ethane in N2(right panel)
[65,66].
and patients with cystic fibrosis [72]. In a recent study
[71], exhaled OCS in normal subjects ranged from ∼100–to
300 pptv.
A transportable system employing a thermoelectrically
cooled pulsed QCL operating at 4.85 µm coupled to a
compact 36 m multipass absorption cell measured OCS
with a MDL (1σ) of 1.2 ppb (for 100 averaged 400 point
frequency scans acquired within ∼0.4 s) [74]. The availability
of a neighboring CO2line within the tuning range of the
QCL allowed ventilation monitoring simultaneously with
an OCS measurement and could be used to normalize the
resulting OCS concentrations. Application of a pulsed QCL
allows utilization of a single HgCdTe detector followed by
a time resolved data acquisition system sensor system for
simultaneous measurement of the reference and sample beam
signals, which is used to minimize pulse-to-pulse fluctuations
of the laser radiation and thus significantly improves a signal-
to-noise ratio of the OCS sensor.
Ultra-high sensitivity measurements of an exhaled OCS
using laboratory setup based on CRDS and a continuous wave,
CO laser at 5 µm were also reported by Halmer et al [72].
A Fabry–Perot ringdown cavity (0.5 m in length) with R
99.99% mirrors was used to achieve a noise-equivalent
absorption coefficient of 7 ×10−11 cm−1Hz−1/2. The system
was used for real-time OCS monitoring in ambient air and
breath samples at ppt levels.
Acetone
Acetone (propylketone) is formed by decarboxylation
of acetoacetate, which derives from lipolysis or lipid
peroxidation. Acetone is a ketone body and is oxidized
via the Krebs cycle in peripheral tissue. Ketone bodies
in blood (including acetoacetate and β-hydroxybutyrate) are
increased in subjects who are in a fasting state. A very
significant application of exhaled acetone is in monitoring
blood glucose concentration in patients with diabetes mellitus.
Figure 9. Schematic diagram for a QEPAS sensor platform with
CW EC-QCL operating at 8.6 µm as a spectroscopic source
[80].
The prevalence of diabetes for all age-groups worldwide
was estimated to be 3% and is a major cost of healthcare
[75]. Exhaled acetone concentrations increase in patients with
uncontrolled diabetes mellitus (levels of 300 ppb to 1 ppm)
[76] and have been shown to correlate well with blood glucose
concentration in these patients [77].
To detect and monitor diabetes requires acetone detection
sensitivities of ∼100 ppb. The acetone is a relatively complex
molecule and its absorption spectrum contains only broadband
ro-vibrationally unresolved features. Measurements of such
compounds using laser spectroscopic methods are usually
limited due to limited wavelength tunability of available
laser sources. To address this requirement, we have used
the same EC-QCL architecture developed for a NO sensor
operating at 5.2 µm as described above in the NO section
after replacing the grating with one blazed for the longer
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J. Breath Res. 1(2007) 014001 MRMcCurdyet al
Tabl e 1. Representative human breath biomarkers.
Compound Concentration Physiological basis/pathology indication
Acetaldehyde ppb Ethanol metabolism
Acetone ppm Decarboxylation of acetoacetate, diabetes
Ammonia ppb protein metabolism, liver and renal disease
Carbon dioxide % Product of respiration, Heliobacter pylori
Carbon disulfide ppb Gut bacteria, schizophrenia
Carbon monoxide ppm Production catalyzed by heme oxygenase
Carbonyl sulfide ppb Gut bacteria, liver disease
Ethane ppb Lipid peroxidation and oxidative stress
Ethanol ppb Gut bacteria
Ethylene ppb Lipid peroxidation, oxidative stress, cancer
Hydrocarbons ppb Lipid peroxidation/metabolism
Hydrogen ppm Gut bacteria
Isoprene ppb Cholesterol biosynthesis
Methane ppm Gut bacteria
Methanethiol ppb Methionine metabolism
Methanol ppb Metabolism of fruit
Methylamine ppb Protein metabolism
Nitric oxide ppb Production catalyzed by nitric oxide synthase
Oxygen % Required for normal respiration
Pentane ppb Lipid peroxidation, oxidative stress
Water % Product of respiration
wavelengths in order to optimize EC QCL performance. A
MOCVD grown buried heterostructure Fabry–Perot QCL gain
chip operating at 8.4 µm in cw mode with thermoelectric
cooling was used [9]. The resulting EC-QCL source has a
single mode laser frequency tuning range of ∼180 cm−1from
1110 to 1288 cm−1with a maximum cw output power of
∼50 mW [44].
The 8.4 µm EC-QCL spectroscopic source was integrated
with a quartz-enhanced photoacoustic spectroscopic (QEPAS)
[52,78,79] sensor platform (see figure 9) to detect and quantify
broadband absorbers possessing strong absorption features in
the accessible spectral region, such as acetone at ∼1210 cm−1
and Freon 125 (C2HF5, a convenient safe simulant for toxic
chemical and biological agents). In the case of broadband
absorbers, the QEPAS signal is generated by modulating the
amplitude of the laser radiation with 100% modulation depth
and a 50% duty cycle at the resonance frequency of the quartz
tuning fork (QTF) used as the acoustic transducer in QEPAS.
To demonstrate the capability of this EC-QCL based AM-
QEPAS sensor to perform multi-species detection, a spectrum
of a mixture of acetone and Freon 125 in N2as a buffer gas
was acquired. Measured concentrations of acetone at the level
of 47.2 ppm and Freon 125 at the level of 4.4 ppm could
be retrieved in the data post-processing. The corresponding
spectra are shown in figure 10. The system response to the
acetone absorption was determined for a single spectral point
at the laser frequency (1217.7 cm−1) corresponding to the
maximum absorption coefficient of acetone within the tuning
range using a calibration mixture of 8 ppm acetone in N2.For
a 1 s lock-in time constant and an effective optical power of
6.8 mW a MDL (1σ)of∼520 ppb was obtained. At this
stage this sensor can be applied to offline breath analysis, in
which high measurement speeds are not required. With a
1 min. lock-in time constant the system is capable of acetone
detection with a MDL of ∼70 ppb [80].
Figure 10. Optical power normalized photoacoustic signal of Freon
125 and acetone mixture (blue line) plotted together with a retrieved
component spectra of Freon 125 (red line) and acetone (green line).
The calculated acetone spectrum was fitted by a reference spectrum
of acetone from the PNNL spectroscopic database shown as a black
line [80].
Detection of large organic molecules
The feasibility for sensing small concentrations of large
organic molecules in the vapor phase was examined by Fisher
et al [81] who investigated the potential of mid-infrared
photoacoustic spectroscopy for the detection of volatile doping
agents (banned substances used by some athletes). Spectra
of different doping classes (stimulants, anabolic agents,
diuretics and beta blockers) were obtained with an optical
parametric oscillator based photoacoustic spectrometer. The
sample preparation time will determine whether or not laser
spectroscopy is faster than gas chromatography or liquid
chromatography with mass spectrometry. Further analysis
using urine-like samples containing any doping substances
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J. Breath Res. 1(2007) 014001 MRMcCurdyet al
should provide further insight into the sample preparation
needed. The advantage of photoacoustic technique described
is that only a single scan over the entire wavelength
range is needed for the detection whereas for conventional
investigations the samples need to be analyzed separately for
each class of substances. Since only pure doping substances
were studied, the issue of selectivity needs to be investigated
in the presence of interfering gases present in urine or exhaled
breath. Furthermore the sensitivity of the photoacoustic sensor
for each molecule needs to be characterized.
Summary
Laser absorption spectroscopy (LAS) and quartz enhanced
spectroscopy (QEPAS) have been demonstrated to play an
important role in the future of exhaled breath analysis. The
key attributes of these techniques are sensitivity, selectivity,
fast response, ease-of-use, size and low operating cost which
make LAS and QEPAS competitive technologies for a number
of exhaled bio-markers such as nitric oxide, ammonia, ethane,
carbon monoxide, carbon dioxide, acetone and formaldehyde.
As many as 400 different molecules in breath, many with
well-defined biochemical pathways have been reported in
the literature. Table 1depicts a representative list of these
biomarkers. Several investigators have reported sensors with
adequate sensitivity for these molecules in ambient air or in
cursory exhaled breath samples as an important first step. In
many cases, further studies are needed to characterize the
ability of these sensors to adequately determine real-time
breath concentrations in terms of time resolution (brief data
acquisition time and sample cell volume small enough to allow
for changes in concentration on the 0.5 s time scale) and
selectivity (interfering gases in exhaled breath in normal and
disease states). After this characterization, sensors are ready
for animal and human clinical investigation, such as the
study of exhaled ethane in lung cancer patients reported by
Skeldon and colleagues [64]. The use of laser-based sensors
in clinical applications is a critical development in the field and
demonstrates the utility and relevance of LAS in exhaled breath
analysis. Further advances in efficient mid-infrared sources
and related technologies will improve detection sensitivities
and enable multiple trace gas species detection. At this time
commercially available laser-based sensors are limited to the
detection of exhaled nitric oxide and ammonia.
Acknowledgments
Financial support of the work performed was provided by a
National Science Foundation ERC MIRTHE grant, DARPA
via a sub-award from Pacific Northwest National Laboratory
(PNNL), Richfield, WA, Department of Energy via a sub-
award from Aerodyne, Inc., National Aeronautics and Space
Administration (NASA) and the Robert Welch Foundation.
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