1.

Introduction

Technologies for clinical diagnoses and monitoring are always needed for discriminating patients from healthy subjects. To identify diseases and to monitor therapy and/or the progression of diseases, most used methods—such as biopsy, computer tomography, magnetic resonance imaging, endoscopy, and blood analysis—are time-consuming, expensive, invasive, painful, stressful, and difficult to perform. Therefore, noninvasive tools are needed to monitor the diseases in their early stages and to holistically measure the physiological status of patients. There are different potential noninvasive techniques, such as breath, nail, hair, mucus, saliva, sweat, and/or tear analysis. Among them, the detection of volatile organic compounds (VOCs) present in breath at trace concentrations with an acceptable accuracy and precision is most promising. This method may provide noninvasive, point-of-care (PoC), easily repeatable, painless, and real-time diagnosis of a number of disease states and therefore is preferable. Breath biomarkers can be analyzed either after administration of a drug or substrate or without any prior administration of a chemical.

A number of breath molecules have been detected and quantified so far using the different methods, such as proton transfer reaction mass spectrometry (PTR-MS), selected ion flow tube mass spectrometry (SIFT-MS), gas chromatography-mass spectrometry (GC-MS), e-noses, and laser-based sensors. Laser spectroscopic detection techniques—such as tunable diode laser absorption spectroscopy (TDLAS), cavity-enhanced absorption spectroscopy (CEAS), integrated cavity output spectroscopy (ICOS), cavity ringdown spectroscopy (CRDS), photoacoustic spectroscopy (PAS), quartz-enhanced photoacoustic spectroscopy (QEPAS), cavity leak-out spectroscopy, and optical frequency comb cavity-enhanced absorption spectroscopy—provide the features of high-sensitivity, high-selectivity, low costs, real-time measurements, and PoC function. Therefore, these methods are candidates for performing accurate breath diagnostics in hospitals.

To identify and quantify the molecules in breath, narrow linewidth ($<1\mathrm{MHz}$), mode-hop-free (1 to $2{\mathrm{cm}}^{-1}$) wavelength-tunable, single-mode light sources are needed. These features can be obtained using external cavity lasers (ECLs). ECL-based spectroscopic techniques may become very suitable for monitoring diseases by analyzing exhaled breath air samples. Many review papers about breath analysis using laser-based techniques have been published. Ciaffoni et al.¹ focused on the current status of volatile sulfur compounds sensing via laser absorption spectroscopy. The review article of Wang and Sahay² is related to breath analysis of almost all of the biomarkers that have been analyzed in actual human breath using high-sensitivity laser spectroscopic techniques. Wojtas et al.³ presented recent advances in human breath analysis using optical methods. Risby and Tittel⁴ reviewed the current status of mid-infrared quantum and interband cascade lasers for clinical breath analysis. The review article of Bielecki et al.⁵ presented an overview on high-sensitivity laser absorption spectroscopic techniques used for trace gas detection. Mid-infrared spectroscopic sensing techniques were reviewed for potential application in breath diagnostics.⁶ In the review paper,⁷ techniques such as mass spectrometry-based (GC-MS, PTR-MS, SIFT-MS), laser absorption spectroscopy-based (CRDS and TDLAS), and other spectroscopic techniques for breath analysis applications were compared in terms of advantages/disadvantages, versatilities and plausibility to be transformed in clinical applications. McCurdy et al.⁸ summarized some of the recent advances in laser absorption spectroscopy based on semiconductor lasers and optical detection techniques for clinically relevant exhaled gas analysis in breath, specifically molecular biomarkers such as nitric oxide, ammonia, carbon monoxide, ethane, carbonyl sulfide, formaldehyde, and acetone.

Regarding the issues above mentioned, the present review article focuses on the latest status of breath analysis using the ECL-based methods. The article is structured as follows: first, we will explain the respiratory system and breath sampling. The absorption of electromagnetic waves by breath molecules is discussed next. The latest developments and functional principle of ECLs are described in detail as well. Finally, the laser-based techniques used for breath analysis and limitations of breath analysis are presented in detail. Regarding this content, the present review article differentiates itself from previous reviews related to the laser-based breath analysis.

2.

Respiratory System

The lung is primarily responsible for gas exchange between the atmosphere and the blood. The molecules present in blood can diffuse into the alveoli and subsequently appear in the exhaled breath. Transport of metabolites from the blood to the exhaled breath air can make it possible to utilize breath analysis as a noninvasive diagnostic tool. Low and high blood-soluble gases present in the breath air:⁹ Gases with low blood-solubility like oxygen (${\lambda }_{b:a}\approx 0.7$) and carbon dioxide (${\lambda }_{b:a}\approx 3$) exchange in the alveoli. For these gases, there is a simple relationship between the breath and blood, which depends predominately on two factors: the blood solubility of the gas (${\lambda }_{b:a}$) and the distribution of ventilation-to-perfusion in all gas exchange units. Gas exchange of high blood-soluble gases like ethanol (${\lambda }_{b:a}\approx 1756$) takes place in the airways, and there is a more complex relationship between end-exhaled breath measurement and blood. Airway gas exchange is related to multiple factors, such as airway temperature, bronchial blood flow, and blood–air partition coefficient. Soluble gases with a blood:air partition coefficient less than 10 (${\lambda }_{b:a}<10$) exchange almost solely with the alveoli, whereas gases with a blood:air partition coefficient greater than 100 (${\lambda }_{b:a}>100$) exchange almost exclusively with the airways. Gases in between these two extremes ($10<{\lambda }_{b:a}<100$) exchange partially with the airways and partially with the alveoli.^9,10

Figure 1 shows expirograms in which gases that exchange in the alveoli and those that exchange in the airways can be seen: phase 1 shows that the anatomic dead space empties. When fresh air is inhaled, low blood-soluble gases in the blood are transfered into the inspired air entering the alveoli. This action continues throughout the entire breathing cycle (inspiration and expiration). When the air is exhaled, molecules present in the conducting airways exit the lung first (phase 1). The airways take part in the gas exchange. Therefore, phase 1 does not exist in the expirogram for gases that exchange completely in the airways (e.g., ethanol). Therefore, an anatomical dead space cannot be given for such gases. For these gases, the end-exhaled breath concentration is always less than that in the alveoli. Phase 2 represents the transition from air residing in the dead space to alveolar air containing high partial pressures of gas. Phase 3 corresponds to air from the alveoli for low soluble gases and airways for high soluble gases. Alveolar air is expired via the conducting airways (phase 3). Molecules with high blood solubility are absorbed from the tissue and mucus lining the airway wall. In contrast to low blood-soluble gases, therefore, these gases present in large concentrations in the airway tissue and mucus for a given partial pressure. The partial pressure of soluble gas in the end-exhaled breath is always less than that in the alveoli due to the absorption–desorption process, which is the major mechanism of pulmonary gas exchange for any gas with a blood–air partition coefficient greater than 100.¹⁰

Fig. 1

Phases of an expirogram from a low blood-soluble gas that exchanges in the alveoli (red line) and from a high blood-soluble gas that exchanges in the airways (blue line).⁹

3.

Breath Sampling

Breath sampling procedures are threefold: alveolar, dead-space, and mixed sampling. Dead space air contains the first 150 mL of expiration air, and here no gas exchange takes place. Alveolar breath is the part of exhaled air coming after the dead-space air, whereas end-tidal air is the last fraction of expired air. Alveolar gas exchange is dependent on ventilation, pulmonary perfusion, and the blood:air partition.¹⁰ The term “end-exhaled breath” should be utilized for highly blood soluble VOCs, whereas the term “alveolar breath” may be used for low blood-soluble compounds.

It is often tacitly assumed that end-tidal air will reflect the alveolar concentration $C_{\mathrm{A}}$, which is related to the concentration of the VOC in mixed venous blood $C_{\mathrm{v}}$, the substance-specific blood:gas partition coefficient ${\lambda }_{b:\text{air}}$, alveolar ventilation ${\dot{V}}_{\mathrm{A}}$, and cardiac output ${\dot{Q}}_{\mathrm{c}}$:^11,12

In mixed sampling, total breath (alveolar + dead space) is collected. Concentrations of endogenous VOCs in alveolar air are two to three times higher than those found in mixed expiratory samples because there is no dilution by dead space gas.¹³

There may be discrepancies between the true alveolar breath and the measured levels of water-soluble VOCs because their concentrations may be diluted on their way (a dilution effect). A single breath or multiple breath cycles can be applied for breath sampling.¹⁴ Breaths may considerably change from each other. The alveolar gas sample may not be represented by the composition of a single breath. Therefore, multiple breaths may be preferable.

4.

Absorption of Electromagnetic Waves by Molecules

Analysis of the breath molecules using laser-based methods is based on the absorption of electromagnetic waves by molecules under investigation. Therefore, we will explain the absorption phenomena in this section. The overall excited energy of a molecule is a sum of the electronic ($E_{\mathrm{el}}$), vibrational ($E_{\mathrm{vib}}$), and rotational transitions ($E_{\mathrm{rot}}$) energy:

Rotational spectra originate from the transitions between rotation levels of a given vibration level in an electronic state [Fig. 2(a)]. In this process, only the rotational quantum number ($j$) changes. These spectra lie in the spectral range from the microwave to the far infrared. The rotational–vibrational spectra consist of the transitions from rotation levels of a vibration level to rotation levels of another vibration level in the same electronic state [Fig. 2(b)]. The rotation and vibration ($\nu$) quantum number change in these transitions. These spectra lie in the infrared spectral region. Electronic spectra consist of the transitions between the rotation levels of different vibration levels of an electronic state and rotation or vibration levels of another electronic level [Fig. 2(c)]. There are changes in all three quantum numbers ($J$, $\nu$, and electronic quantum number). These spectra lie in the near-infrared or visible range or in the UV range.

Fig. 2

Electronic, vibrational, and rotational quantum states and corresponding transitions: (a) rotational transition, (b) rotational–vibrational transition, and (c) electronic transition.

The number $f$ of normal vibrations of a molecule is given as

Fig. 3

Normal vibration modes of a water molecule and its characteristic ground and first excited vibrational energy levels.

The ratio of the transmitted intensity $I$ and initial intensity $I_0$ of laser beam transmitting through an absorbing medium at a particular frequency is given using the Beer–Lambert equation:

The conversion between $S(T)$ and $S_i(T)$ is given as follows:¹⁵

A typical lineshape of an isolated absorption line is centered at ${\nu }_0$ and has a linewidth $\mathrm{\Delta }\nu$. The absorption line can be broadened due to natural broadening, Doppler broadening, and collision broadening. The homogeneous natural and collision broadening result in Lorentzian line shapes, whereas the Doppler broadening gives rise to a Gaussian line profile. The natural linewidth is determined by the lifetimes of the energy states (${\tau }_1$ and ${\tau }_2$):

The linewidth can be broadened due to the Doppler effect. The distribution of random velocities of molecules in gas is described by the Maxwellian velocity distribution function. Each velocity group gives rise to its own Doppler shift. The Doppler broadening is given by¹⁵

The perturbations that shorten energy level lifetimes cause collisional broadening. The linewidth broadening occurs due to the Heisenberg uncertainty principle; shortening the energy level lifetime results in greater uncertainty of the energy of molecule, and thus the absorption lineshape becomes broader. The lineshape for collisional broadening takes the form of a Lorentzian function:¹⁵

At low pressure, the near-Gaussian line shape is obtained because thermal motion is the dominant broadening mechanism. The line shape becomes near-Lorentzian with the increasing pressure. When the Doppler contribution can be neglected ($\mathrm{\Delta }{\nu }_{\mathrm{c}}p\gg \mathrm{\Delta }{\nu }_{\mathrm{D}}$), the line shape can be considered pure Lorentzian. In the case of $\mathrm{\Delta }{\nu }_{\mathrm{c}}p\ll \mathrm{\Delta }{\nu }_{\mathrm{D}}$, a pure Gaussian line shape is received. Molecular ro-vibrational transitions exhibit pressure broadened FWHM of $\le 3\mathrm{MHz}$ ($0.0001{\mathrm{cm}}^{-1}$) at low pressures $<5\mathrm{mbar}$ and $\sim 1.5\mathrm{GHz}$ ($0.05{\mathrm{cm}}^{-1}$) at atmospheric pressure, where Doppler widths of 30 … 90 MHz ($0.001\dots 0.003{\mathrm{cm}}^{-1}$) at room temperature are typically observed for molecular transitions in the infrared.¹⁶ When the condition $\mathrm{\Delta }{\nu }_{\mathrm{c}}p\approx \mathrm{\Delta }{\nu }_{\mathrm{D}}$ is fulfilled (between 5 and 100 Torr for most lightweight gases), the line shape will be a combination of the Gaussian and Lorentzian, which is known as Voigt profile.

5.

External Cavity Diode Lasers

Different methods can be utilized to realize a wavelength-tunable single-mode operation: tunable ${\mathrm{CO}}_2$ lasers, tunable dye lasers, tunable solid-state lasers, tunable excimer lasers, nonlinear optical frequency conversion lasers, tunable free-electron lasers, and tunable external cavity semiconductor lasers. Within the framework of this work, the focus will only be on the tunable external cavity semiconductor lasers (ECLs). Further information on the other tunable lasers can be found in, e.g., Ref. 17.

Grating-coupled ECLs can be realized in Littrow or Littman configurations.¹⁸ In the Litrow configuration [Fig. 4(a)], there are a gain chip, a lens (or a parabolic mirror), and a diffraction grating. The gain chip serves as the gain medium for ECL setup. The lens (or parabolic mirror) is used to collimate the beam emitted from the gain chip. The collimated beam is diffracted by a grating. The first order of the diffracted beam is fed back into the gain medium. The wavelength is tuned by rotating the grating. Because two optical components (mirror or lens and grating) are used, the alignment is easy and the feedback is strong.

Fig. 4

(a) Littrow and (b) Littman configuration.

In the Littman configuration [Fig. 4(b)], a mirror is added to the setup. The diffracted beam is sent back to the grating by a mirror and diffracted again. Finally, the beam reaches to the gain chip and it is amplified. The tuning is usually achieved by rotating the mirror. Because of the double pass of the beam on the grating, the optical feedback strength is reduced. However, the Littman setup has an advantage of the increased wavelength selectivity.

Generally, the Littrow setup is preferable for maximizing the tuning range. To optimize the optical feedback, the incident beam and first order of the diffracted beam have to be colinear. The directions of the various orders of the diffracted beam with respect to the normal are determined by

Finally, the wave, which makes an angle $\mathrm{\Delta }\phi$, is focalized on the facet at a distance:

The reflectivity of the facet inside the external cavity setup is very important for a properly operating ECL. This reflectivity should be as low as possible to effectively suppress the Fabry–Perot modes of the gain chip, so a pure single mode can be selected and amplified. The reflectivity of the outcoupling facet of the gain chip can be reduced by various ways, such as the tilted gain stripes, the buried facets, and the antireflection (AR) coating. The tilting of the waveguide decreases in the internal Fresnel reflection from the facet and therefore increases the losses.^19,20 In the case of buried facets, a semi-insulating material between the end of the guide and the facet, which reduces the coupling of the beam back into the active region, is utilized.²¹ When a facet is AR-coated, a phase shift of $\pi$ and, therefore, an optical path difference of $\lambda /2$ occur after the reflection in the optical thick medium. The reduction of the reflectivity of the AR-coated facet is achieved by the destructive interference between the waves reflected from the front and rear sides of the layer. The reflectivity reduction causes a decrease in the competition between the modes of the small cavity (gain chip) and of the large cavity (ECL) for oscillation. This situation leads to enhancement of the coupling efficiency to the EC, to increased output power of the ECL, and to enlarged coarse/fine tuning range and the range of the stable operating region.

5.1.

Wavelength Selection

Tuning mechanism of an ECL can be explained by considering a sketch for the gain and losses in the ECL setups (Fig. 5).¹⁷ As one can see in Fig. 5, there are three loss mechanisms: (1) Filter loss due to the wavelength selectivity property of the wavelength selective element (e.g., grating), (2) coupling and output loss between the guided waves in the gain chip and vacuum in ECL, and (3) facet etalon loss, which is modulated at the period of the internal Fabry Perot mode spacing $\mathrm{\Delta }{\nu }_{\mathrm{int}}$. The facet etalon loss is caused by the interference between the reflections from facets.

Fig. 5

Gain and losses for ECLs.

The injection current is controlled to tune the gain profile of the gain chip and the internal cavity mode frequency. To vary the external mode frequency, the grating can be rotated and/or the EC length can be changed by applying the voltage to a piezoelectric transducer controlling the grating position. For a perfect operation of the ECLs, the facet etalon loss ripple should be weak and the bandwidth of the wavelength selective element loss should be narrow compared with the period of the ripple. The bandwidth of the grating can be estimated by the following equation:

Eq. (23)

$$\mathrm{\Delta }\lambda =\frac{\lambda d\cos \theta }{a},$$

where $\lambda$ is the wavelength, $a$ is the diameter of the collimated laser beam, and $\theta$ is the incident angle of the beam. Only the EC mode, which is the closest to the loss minimum of the wavelength selective element, can oscillate. The other modes see higher losses and cannot be amplified. Tuning to the next EC mode can be achieved by varying the loss minimum of the wavelength selective element. In the case of the grating-coupled ECLs in Littrow configuration, this variation occurs by rotating the grating [Fig. 6(a)] and/or changing the cavity length [Fig. 6(b)]. A strong facet etalon loss and a wider bandwidth of the wavelength-selective element loss lead to tuning with EC mode jumps approximately equal to $\mathrm{\Delta }{\nu }_{\mathrm{int}}$. This discontinuity is called “tuning gap” [Figs. 6(a) and 6(b)]. The shift in the external mode frequency exceeding half the free spectral range of the external cavity gives rise to mode hops. At this point, the adjacent external mode has moved closer to the peak of the internal mode, and the laser oscillation hops to this new mode.²² To achieve the tuning without any gap (tuning on the EC mode, mode-hop-free tuning) [Fig. 6(c)], there are different methods: (1) the injection current, the grating angle, and/or the external cavity length should be simultaneously controlled;²³ (2) the grating has to be mounted on a pivot arm, which is rotated about an axis lying near the intersection of the grating and laser output facet plane;²⁴ and (3) an electro-optically tunable single-mode ECL has to be built.²⁵

Fig. 6

Possibility of changing the grating position and corresponding wavelength tuning. (a) The grating is rotated around its the center, (b) the grating is moved back and forth, and (c) the grating is rotated around an axis lying near the intersection of the grating and laser output facet plane.

5.3.

Linewidth

The laser linewidth (FWHM of the lineshape) describes the spectral purity of a laser (Fig. 7). The linewidth of a semiconductor laser is given by the Schawlow–Townes formula modified by Henry (Ref. 37 and references therein):

Eq. (26)

$$\mathrm{\Delta }\nu =\frac{\pi h\nu \mathrm{\Delta }{\nu }_c^2{\alpha }_m{n}_{2t}}{P\alpha (n_{2t}-n_{1t})}(1+{\alpha }_e^2),$$

where $\nu$ is the lasing frequency, $\alpha$ is the total losses of the cavity, ${\alpha }_{\mathrm{m}}$ is the mirror losses of the output facet, $P$ is the output power, $\mathrm{\Delta }{\nu }_{\mathrm{c}}=\alpha c/(2\pi n)$ is the FWHM of the cavity resonance, $n_{1t}$ and $n_{2t}$ are the populations of the upper and lower states of the laser transition at threshold, respectively, and ${\alpha }_e=(d{n}_r/dN)/(d{n}_i/dN)$ is the linewidth enhancement factor introduced by Henry. The imaginary ($n_{\mathrm{r}}$) and real parts ($n_{\mathrm{i}}$) of the refractive index change during the relaxation oscillations of the field intensity. Varying of the imaginary part of the index causes the gain to change, whereas the change in the real part of the refractive index gives rise to an additional phase shift. Therefore, the laser linewidth is broadened by a factor ($1+{\alpha }_e^2$) compared with the Schawlow–Townes linewidth. The lower energy level of semiconductor lasers is not empty, which is different from most other lasers. As laser photons in the valence band may reradiate by spontaneous emission, phase fluctuations of the laser field increase. This is another phenomenon that leads to the linewidth broadening of semiconductor lasers.

Fig. 7

Spectral line.

Loh et al.³⁸ investigated experimentally the influence of the grating reflectivity, grating resolution, and AR coating on the intrinsic linewidth of a grating-coupled ECL. They found that the linewidths are little affected by the presence of an AR coating on the diode facet and that the grating resolution has a larger effect on the linewidth than the grating reflectivity.³⁸

The laser linewidth can be determined using several methods, such as optical cavity measurements,²⁹ heterodyne measurements,³⁷ homodyne/heterodyne interferometry using a long interferometer delay,³⁹ measurements from the frequency noise power spectral density,⁴⁰ measurements from root-mean-square frequency deviation,⁴¹ and saturation spectroscopy.⁴²

Tkach and Chraplyvy⁴³ reported that there are five distinct regimes of feedback effects, with well-defined transitions between them as a function of feedback power ratio. At the lowest levels of feedback down to $-90\mathrm{dB}$ (regime I), linewidth narrowing or broadening is observed depending on the phase of the feedback. Regime II is seen at a feedback level (up to around $-45\mathrm{dB}$), which depends on the distance to the external reflector, and frequency splitting of the mode and mode hopping are observed. Regime III is found at a level (about $-39$ to $-45\mathrm{dB}$), which does not depend on the distance to the reflection. In this regime, the mode hopping is suppressed, and the laser is observed to operate on a single narrow line. Regime IV starts at $-39\mathrm{dB}$ and is independent from the distance to the reflection. In this regime, line broadening is observed due to the coherence collapse effect. Relaxation oscillation sidebands that present in each mode in the optical spectrum increase in strength giving rise to the line broadening. Consequently, coherence time decreases. Regime V is independent of distance too and observed at the highest levels of feedback (usually greater than $-10\mathrm{dB}$). AR coating of output facet is needed to achieve this regime. In this regime, the operation of the laser occurs on a single longitudinal mode with narrow linewidth for all phases of the feedback and the laser is relatively insensitive to additional external optical perturbations.⁴³

6.

External Cavity Laser-Based Techniques for Breath Analysis

The ECL-based techniques can be effectively used for the detection and quantification of breath VOCs at concentrations ranging from parts per million by volume (ppmv) to the parts per trillion (pptv) level. The laser sources should have high sensitivity and selectivity for breath molecule sensing. For selectivity, an absorption line that is free of interference from other species in gas mixture should be used. For the best sensitivity, a strong molecular absorption line should be chosen. To achieve a higher signal-to-noise ratio (SNR), either the noise levels should be decreased and/or the signal should be enhanced. For decreasing the noise levels, there are different methods, such as wavelength/frequency modulation spectroscopy. The signal can be further increased by obtaining an effective long pathlength using multipass cells or cavity enhancement methods. ECL-based spectroscopic detection techniques, such as multipass cell absorption method (MCAB), CRDS, CEAS, ICOS, PAS, and QEPAS, provide the features of high-sensitivity, high-selectivity, low costs, real-time measurements, and PoC function. Furthermore, laser-based techniques can be implemented without the need for radioactive labels. Therefore, these methods are candidates for performing accurate breath diagnostics in hospitals. In Table 2, some important breath molecules, detection wavelength, detection limit, and methods are summarized.

Table 2

Some important breath molecules, detection wavelengths, detection limits, and methods.

6.1.

Multipass Absorption Spectroscopy

In the MCAB,⁷⁵ the absorption signal can be enhanced via longer optical pathlengths using optical multipass cells. There are four types of multipass cells most commonly applied: White cells,^76,77 Herriott cells,⁷⁸ Chernin cells,⁷⁹ and astigmatic mirror multipass cells.⁸⁰ Beer–Lambert law of linear absorption is used to determine the absorption coefficient $\alpha (\nu )$:

Eq. (33)

$$\begin{gathered} I(\nu )=I_0\exp [-\alpha (\nu )L_{\mathrm{eff}}] \\ =I_0\exp [-\sigma (\nu )N{L}_{\mathrm{eff}}] \\ =I_0\exp [-S(T)\varphi (\nu -{\nu }_0)N{L}_{\mathrm{eff}}], \end{gathered}$$

where $I(\nu )$ is transmitted intensity of the radiation after a distance $L_{\mathrm{eff}}$ through the medium, $I_0$ is initial intensity of the radiation, $L_{\mathrm{eff}}$ is effective path-length, $\sigma (\nu )$ is absorption cross-section of the absorbing species, $N$ is molecular concentration (the number density of the absorbing species), $S$ is linestrength (i.e., the total absorption per molecule) of the absorbing species at temperature $T$, $\varphi (\nu -{\nu }_0)$ is line shape function for the particular absorption line.

Using Eq. (33), the absolute number density $N$ can be calculated by integrating the absorption coefficient:

Eq. (34)

$${\int }_{-\infty }^{\infty }\alpha (\nu )\mathrm{d}\nu =\frac{1}{L_{\mathrm{eff}}}{\int }_{-\infty }^{\infty }\ln \left[\frac{I_0(\nu )}{I(\nu )}\right]\mathrm{d}\nu =NS.$$

The Herriott cell consists of two mirrors (Fig. 8). The input mirror has a small hole. A laser beam is injected into a Herriott cell through the hole and it is reflected multiple times between the two mirrors. The light path might be lengthened 10 or 100 times inside the cell. The laser beam leaving the cell through the same or the other hole reaches the detector. The sensors employing a multipass gas cell have been reported for many trace gases, such as acetaldehyde,⁵¹ ethane,⁶¹ formaldehyde,⁶⁷ and methane.^71,72

Fig. 8

Herriott multipass cell.

6.2.

Cavity Ringdown Spectroscopy

CRDS⁸¹ is based on the observation of the decay rate of a laser beam stored in an optical cavity formed from ultra-high reflective mirrors (Fig. 9) and is first demonstrated by O’Keefe and Deacon.⁸² In this case, the absorption coefficient is determined using the following equation:

Eq. (35)

$$\alpha (\nu )=N\sigma (\nu )=\frac{1}{c}[\frac{1}{\tau (\nu )}-\frac{1}{{\tau }_0(\nu )}],$$

where ${\tau }_0$ and $\tau$ are the ringdown time without and with the absorbing gas present, $c$ is the light velocity.

Fig. 9

Cavity ringdown absorption cell.

A laser beam, which is coupled into the optical cavity, is reflected back and forth inside the cavity. When beam is reflected every time, a small fraction of this light leaks out of the cavity. Many trace gases, such as acetone,⁵² carbon dioxide,⁵⁷ ethanol,⁶³ isoprene,⁷⁰ and nitric oxide,^73,74 have been analyzed using the CRDS technique.

6.3.

Integrated Cavity Absorption Spectroscopy and Cavity-Enhanced Absorption Spectroscopy

ICOS⁸³ and CEAS⁸⁴ are the same method. ICOS is used in the context of pulsed excitation, and CEAS is used in the context of continuous-wave cavity excitation. This method is based on analyzing the intensity of light transmitted through a high finesse optical cavity as a function of wavelength (Fig. 10). To reduce the noise levels and therefore to improve the detection limit, the laser beam can be coupled into the optical cavity at a small angle with respect to the cavity axis or away from the central axis of the optical cavity. This is known as “off-axis alignment,” which has advantage over on-axis alignment: it is almost insensitive to vibrations. In the case of ICOS and CEAS, Eq. (36) is applied to extract the absorption coefficient:

Eq. (36)

$$\alpha (\nu )=N\sigma (\nu )=[\frac{I_0(\nu )}{I(\nu )}-1]\frac{1-R}{d},$$

where $R$ is mirror reflectivity and $d$ is the distance between mirrors.

Fig. 10

High finesse optical cavity for ICOS or CEAS.

The gases—such as acetone,⁵³ ammonia,⁵⁴ carbon monoxide,⁵⁴ carbonyl sulfide,⁶⁰ and ethane⁶⁰—have been analyzed using the ICOS or CEAS techniques.

6.4.

Photoacoustic Spectroscopy and Quartz-Enhanced Photoacoustic Spectroscopy

In the PAS,⁸⁵ the acoustic waves resulting from the absorption of radiation (photoacoustic effect) are detected and analyzed using a sensitive microphone (Fig. 11). The PAS response $S$ to optical absorption is given by Eq. (37):

Eq. (37)

$$S=k\frac{\alpha CPQ}{fA},$$

where $\alpha$ is the absorption coefficient per unit concentration of the target species, $C$ is the concentration of the target species, $P$ is the optical power, $f$ is the PA sound frequency, $A$ is the resonator cross-section area, and $k$ is a constant describing the microphone transfer function and other system parameters. Usually, PA resonators are designed for $f$ values in the 500 to 4000 Hz range and have $Q$ factors of $\sim 20$ to 200.⁸⁶

Fig. 11

Photoacoustic cell.

Another approach to photoacoustic detection of trace gases is QEPAS,^86,87 which utilizes a quartz tuning fork (QTF) as a sharply resonant acoustic transducer. The QTF is a piezoelectric element. To further increase the QEPAS signal, a microresonator can be added to the QEPAS sensor system.⁸⁸ Also, to achieve the best QEPAS detection sensitivity and selectivity, it is important to choose the optimum operating pressure for a QEPAS sensor system. The PAS- or QEPAS-based sensors have been used for the detection of many gases, such as ammonia,^55,56 carbonyl disulfide,⁵⁸ carbon monoxide,⁵⁹ ethanol,⁶² ethylene,^64–66 and hydrogen sulfide.^68,69

7.

Breath Biomarkers

Owing to its noninvasiveness, ease of use, easy repeatability, and painlessness, breath analysis has attracted considerable attention for scientific and clinical studies. Until now, many studies have demonstrated that the human breath air contains more than 1000 different molecules.^2,89 The matrix of exhaled breath air is much less complicated compared to serum or urine. A few atmospheric molecules in % concentration (${\mathrm{N}}_2$, ${\mathrm{O}}_2$, ${\mathrm{H}}_2\mathrm{O}$, ${\mathrm{CO}}_2$), inorganic gases (e.g., NO, ${\mathrm{NH}}_3$, and CO), VOCs at the levels of parts per million (ppm), parts per billion (ppb), or parts per trillion (ppt) (e.g., ${\mathrm{CH}}_4$, ${\mathrm{HCO}}_2$, ${\mathrm{N}}_2\mathrm{O}$), and nonvolatile substances measured in breath condensate (e.g., isoprostanes, cytokines, leukotrienes) have been found to be present in exhaled human breath (Table 3). Spectral fingerprints of these molecules span from the UV to the IR spectral regions. The mixture is exhaled at temperatures between 34°C and 37°C while relative humidity may range from 91% to 96% in oral exhalations and from 82% to 85% in nasal exhalations.⁹⁰ The mixture of exhaled breath air varies from person to person both quantitatively and qualitatively.⁹¹ Analysis of some VOCs, which present in everyone, can provide a safe method for studying human metabolism, detecting diseases, clinical diagnosis, therapeutic monitoring, and investigation of potential adverse effects of new drug therapy. In Table 4, some important breath molecules, concentration, physiological basis, and related diseases are summarized. The 872 breath VOCs from apparently healthy humans have been reported in the review article of de Lacy Costello et al.⁸⁹

Table 3

Main breath molecules.3

Table 4

Some important breath molecules, concentration, physiological basis, and related diseases.

8.

Limitations of Breath Analysis

It has been known for a long time that the smell of exhaled breath is an indicator for diseases.¹²² The work of Pauling et al., in which hundreds of VOCs were determined in normal human breath at the levels of parts per billion (ppb) or lower, has been accepted as the beginning of the modern breath analysis. In spite of the long investigation time, this method has not yet been introduced as a standard tool into clinical diagnosis. Very few diseases are actually diagnosed using exhaled breath air analysis; for example, breath urea analysis. There are several potential confounders and issues blocking the introduction of breath analysis into clinical applications. To widely use the breath test for more specific diseases in hospitals, the following issues should be addressed:^123–125

To address all these issues and to move breath analysis forward, collaboration among optical engineers, experts in breath analysis, clinicians, and spectroscopists is required.

9.

Conclusion and Outlook

Several issues should be addressed for advances in the field of breath monitoring and for widespread usage of this method as a routine clinical tool. Breath analysis has not yet been used in hospitals as a clinical tool due to lack of generally accepted evaluation criteria for data. Multiple potential confounders, such as interference from other molecules, smoking and alcohol consumption, dermal absorption, inhalation from the ambient air, and so on, should be carefully considered when performing measurements of breath biomarkers.

Breath research may be performed as (1) exploratory research (the discovery of new breath molecules, e.g., breath markers for chronic kidney disease), (2) focused research (the in-depth evaluation of a molecule whose biochemistry is already well-understood, e.g., breath acetone), and (3) breath condensate (the analysis of nonvolatile molecules).¹²³

Breath analyses conducted by the techniques, such as GC-MS, SIFT-MS, and PTR-MS, are still in laboratory research. The development of inexpensive, real-time, portable breath monitors is a key factor in the advancement of breath tests. Such devices should be moved from laboratory research to commercial reality. Laser-based spectroscopic instruments are a good candidate for this field. The current laser-based methods are not satisfactory for medical applications as routine diagnostics tool. Technologies in laser physics and optical engineering should be further improved. The development of absorption spectroscopy instruments with lasers may provide certain advantages for sensitive examination of not only small molecules but also heavy molecules with relatively broad spectral structures. The issue of spectral interferences can be eliminated using single-mode tunable laser. Another type of interference, which should be considered, is biointerferences; for instance, ammonia is a biomarker for both asthma and chronic kidney diseases. This challenging field requires the extensive interdisciplinary collaborations.

The availability of a cheap breath analyzer can even enable patients to measure their own biomarker levels at home. This situation can result in performing more large cross-sectional and longitudinal in-depth studies with many data points, instead of making just small pilot studies limiting the gain of widespread usage of breath analysis. One may expect that the issues mentioned in the present manuscript may be soon addressed and breath analysis may be introduced into clinical practice.