Non-Invasive Disease Specific Biomarker Detection Using Infrared Spectroscopy: A Review.

Many life-threatening diseases remain obscure in their early disease stages. Symptoms appear only at the advanced stage when the survival rate is poor. A non-invasive diagnostic tool may be able to identify disease even at the asymptotic stage and save lives. Volatile metabolites-based diagnostics hold a lot of promise to fulfil this demand. Many experimental techniques are being developed to establish a reliable non-invasive diagnostic tool; however, none of them are yet able to fulfil clinicians' demands. Infrared spectroscopy-based gaseous biofluid analysis demonstrated promising results to fulfil clinicians' expectations. The recent development of the standard operating procedure (SOP), sample measurement, and data analysis techniques for infrared spectroscopy are summarized in this review article. It has also outlined the applicability of infrared spectroscopy to identify the specific biomarkers for diseases such as diabetes, acute gastritis caused by bacterial infection, cerebral palsy, and prostate cancer.

Towards a standard operating procedure for revealing hidden volatile organic compounds in breath: the Fourier-transform IR spectroscopy case.

Human breath contains a large amount of small volatile organic compounds (VOCs) and could therefore be used as a carrier of metabolic information for medical diagnostics. Still, in spite of several promising techniques that have been applied during the last decades to study breath content, there is a lack of breath-based diagnostic tools available for physicians. Among several promising techniques, infrared (IR) spectroscopy has already proved its potential for reliable detection of VOCs in the breath. However, due to the large dynamic range of molecular concentrations and overlapping absorption spectra of different VOCs, many low-absorption molecules stay hidden in spectroscopic measurements. To overcome this obstacle, we propose the Matryoshka method for removing masking effects and revealing the buried spectral structures in any bio-fluid in the gas phase. By exploiting both physical and digital removal steps, we demonstrate how the method reveals methane, acetone, aldehyde, and methyl butyrate in a real breath.

Two-dimensional Infrared Spectroscopy Reveals Better Insights of Structure and Dynamics of Protein.

Proteins play an important role in biological and biochemical processes taking place in the living system. To uncover these fundamental processes of the living system, it is an absolutely necessary task to understand the structure and dynamics of the protein. Vibrational spectroscopy is an established tool to explore protein structure and dynamics. In particular, two-dimensional infrared (2DIR) spectroscopy has already proven its versatility to explore the protein structure and its ultrafast dynamics, and it has essentially unprecedented time resolutions to observe the vibrational dynamics of the protein. Providing several examples from our theoretical and experimental efforts, it is established here that two-dimensional vibrational spectroscopy provides exceptionally more information than one-dimensional vibrational spectroscopy. The structural information of the protein is encoded in the position, shape, and strength of the peak in 2DIR spectra. The time evolution of the 2DIR spectra allows for the visualisation of molecular motions.

Monitoring the Reaction of the Body State to Antibiotic Treatment against Helicobacter pylori via Infrared Spectroscopy: A Case Study.

The current understanding of deviations of human microbiota caused by antibiotic treatment is poor. In an attempt to improve it, a proof-of-principle spectroscopic study of the breath of one volunteer affected by a course of antibiotics for Helicobacter pylori eradication was performed. Fourier transform spectroscopy enabled searching for the absorption spectral structures sensitive to the treatment in the entire mid-infrared region. Two spectral ranges were found where the corresponding structures strongly correlated with the beginning and end of the treatment. The structures were identified as methyl ester of butyric acid and ethyl ester of pyruvic acid. Both acids generated by bacteria in the gut are involved in fundamental processes of human metabolism. Being confirmed by other studies, measurement of the methyl butyrate deviation could be a promising way for monitoring acute gastritis and anti-Helicobacter pylori antibiotic treatment.

Efficient Isotope Editing of Proteins for Site-Directed Vibrational Spectroscopy.

Vibrational spectra contain unique information on protein structure and dynamics. However, this information is often obscured by spectral congestion, and site-selective information is not available. In principle, sites of interest can be spectrally identified by isotope shifts, but site-specific isotope labeling of proteins is today possible only for favorable amino acids or with prohibitively low yields. Here we present an efficient cell-free expression system for the site-specific incorporation of any isotope-labeled amino acid into proteins. We synthesized 1.6 mg of green fluorescent protein with an isotope-labeled tyrosine from 100 mL of cell-free reaction extract. We unambiguously identified spectral features of the tyrosine in the fingerprint region of the time-resolved infrared absorption spectra. Kinetic analysis confirmed the existence of an intermediate state between photoexcitation and proton transfer that lives for 3 ps. Our method lifts vibrational spectroscopy of proteins to a higher level of structural specificity.

Vibrational spectroscopy of Methyl benzoate.

Methyl benzoate is studied as a model compound for the development of new IR pulse schemes with possible applicability to biomolecules. Anharmonic vibrational modes of Methyl benzoate are calculated on different level (MP2, SCS, CCSD(T) with varying basis sets) ab initio PESs using the vibrational self-consistent field (VSCF) method and its correlation corrected extensions. Dual level schemes, combining different quantum chemical methods for diagonal and coupling potentials, are systematically studied and applied successfully to reduce the computational cost. Isotopic substitution of ß-hydrogen by deuterium is studied to obtain a better understanding of the molecular vibrational coupling topology.

Broadband two dimensional infrared spectroscopy of cyclic amide 2-Pyrrolidinone.

In the past one-and-a-half decade there has been a significant methodological and technological development of two dimensional infrared (2DIR) spectroscopy, which unfolds many underlying physical and chemical processes of complex molecules, especially for biological molecules. Due to the extreme technical difficulties and non-uniform performance of ultrafast laser, so far, the method has been mostly applied to a small spectral region. A rather simple experimental methodology is presented here which is able to cover a broad spectral range from 1500 cm(-1) to 3500 cm(-1) to explore the molecular structure of cyclic amide, 2-Pyrrolidinone, via the time-resolved coupling of CO, CH and NH stretch vibrations. The signature of the coherent as well as incoherent coupling has been found. The amide-I band is incoherently coupled to CH and NH stretch vibrations and acts as an acceptor mode for vibrational energy relaxation from CH and NH stretch vibrations.

Baseline correction for the infrared spectra of exhaled breath.

Infrared spectroscopy appears to be a promising analytical method for the metabolic analysis of breath. However, due to the presence of trace amounts in exhaled breath, the absorption strength of the metabolites remains extremely low. In such low detection limits, the nonlinear detection sensitivity of the infrared detector and electronic noise strongly modify the baseline of the acquired infrared spectra of breath. Fitting the reference molecular spectra with the baseline-modified spectral features of breath metabolites does not provide accurate identification. Therefore, baseline correction of the acquired infrared spectra of breath is the primary requirement for the success of breath-based infrared diagnosis. A selective spectral region-based, simple baseline correction method is proposed for the infrared spectroscopy of breath.

Neonatal Exhaled Breath Sampling for Infrared Spectroscopy: Biomarker Analysis.

Monitoring health conditions in neonates for early therapeutic intervention in case deviations from physiological conditions is crucial for their long-term development. Due to their immaturity preterm born neonates are dependent on particularly careful physical and neurological diagnostic methods. Ideally, these should be noninvasive, noncontact, and radiation free. Infrared spectroscopy was used to analyze exhaled breath from 71 neonates with a special emphasis on preterm infants, as a noninvasive, noncontact, and radiation-free diagnostic tool. Passive sample collection was performed by skilled clinicians. Depending on the mode of respiratory support of infants, four different sampling procedures were adapted to collect exhaled breath. With the aid of appropriate reference samples, infrared spectroscopy has successfully demonstrated its effectiveness in the analysis of breath samples of neonates. The discernible increase in concentrations of carbon dioxide, carbon monoxide, and methane in collected samples compared to reference samples served as compelling evidence of the presence of exhaled breath. With regard to technical hurdles and sample analysis, samples collected from neonates without respiratory support proved to be more advantageous compared to those obtained from intubated infants and those with CPAP (continuous positive airway pressure). The main obstacle lies in the significant dilution of exhaled breath in the case of neonates receiving respiratory support. Metabolic analysis of breath samples holds promise for the development of noninvasive biomarker-based diagnostics for both preterm and sick neonates provided an adequate amount of breath is collected.

Hydrogen bonding characteristics of 2-pyrrolidinone: a joint experimental and theoretical study.

The mid-IR spectrum of the hydrogen bonding lactam 2-pyrrolidinone in CCl(4) was studied using FT-IR spectroscopy accompanied by a quantum chemical anharmonic normal mode analysis combined with a Monte Carlo approach based on semi-empirical harmonic frequencies. We characterized and assigned the spectroscopic features in the range from 1500 cm(-1) to 3600 cm(-1) covering the respective amide bands related to hydrogen bonding CO and NH groups as well as the backbone CH vibrational band. By comparing theory and experiment we are able to assign all mid-IR features to a variety of distinct structures of 2-pyrrolidinone, ranging from monomers, singly and doubly hydrogen bonded dimers to longer hydrogen-bonded chains. These chains account for the most prominent features in the IR spectrum. Furthermore, two peaks in the CH band have been identified as highly localized axial and equatorial C-H stretch vibrations qualified for probes of conformational dynamics. The results provide a detailed microscopic picture of 2-pyrrolidinone in solution, helpful for further dynamical structure studies of this amide.

Theory helps experiment to reveal VOCs in human breath.

Volatile organic compounds (VOCs) present in human breath not only provide information about the internal chemistry of the body but can also be specific to diseases. Therefore, detection and analysis of specific VOCs can be used for medical diagnostics. However, up until today in spite of several existing VOC-based detection techniques and significant efforts, breath analysis is not a diagnostic method available for clinicians. Infrared absorption spectroscopy is a promising technique to fill this gap, with tens of identified VOCs in breath. Currently, a lack of digital spectral databases and several masking effects make difficult reliable molecular identification of observed absorption features. We demonstrate that calculations of rotational bands of vibrational spectra could serve as a basic method for molecular identification of spectral features observed in experiment. Results of comparison of several known VOCs spectra with the predictions of the theoretical model are presented.

Ultrafast vibrational coupling between C H and C O band of cyclic amide 2-Pyrrolidinone revealed by 2DIR spectroscopy.

Coupling between C H and C O vibrational modes play an essential role on determination of biological structure and dynamics. However, due to the weakness of the C H absorption and strong absorption of the C O vibrational band make such experiments less straightforward than those with transitions of nearly the same strength. In this communication the characteristics of the C H and C O coupling has been studied using dual frequency two dimensional infrared spectroscopy. 2-Pyrrolidinone has been used as a model molecule of biological system. The coherent and incoherent couplings between C H and C O vibrational bands have been observed. The cross peaks dynamics have been discussed and time constant of the cross peak intensity has been calculated.

Breath indeed carries significant information about a disease: Potential biomarkers of cerebral palsy.

Objective and reliable noninvasive medical diagnostics of a large variety of diseases is still a dream. As a step in the direction of realization, a spectroscopic breath study of cerebral palsy (CP) was performed. Principal component analysis revealed data clustering for a healthy group and CP individuals was observed, with a P-value below 10-5 . Learning algorithms resulted in 91% accuracy in distinguishing the groups. With the help of manual analysis of absorption spectral features of breath samples, two volatile organic compounds were identified that demonstrate significant deviations in the groups. These represent two esters of propionic acid (PPAE). A transportation scheme was hypothesized that links the gut where propionic acid (PPA) and PPAE are produced, the brain of CP patients, through which PPA and PPAE transmit, and the lungs where PPAE releases. The results show a possibility to detect one more brain-related disorder via breath, in this case CP.

Human beings as islands of stability: Monitoring body states using breath profiles.

By checking the reproducibility of conventional mid-infrared Fourier spectroscopy of human breath in a small test study (15 individuals), we found that a set of volatile organic compounds (VOC) of the individual breath samples remains reproducible at least for 18 months. This set forms a unique individual's "island of stability" (IOS) in a multidimensional VOC concentration space. The IOS stability can simultaneously be affected by various life effects as well as the onset of a disease. Reflecting the body state, they both should have different characteristics. Namely, they could be distinguished by different temporal profiles: In the case of life effects (beverage intake, physical or mental exercises, smoking etc.), there is a non-monotonic shift of the IOS position with the return to the steady state, whereas a progressing disease corresponds to a monotonic IOS shift. As a first step of proving these dependencies, we studied various life effects with the focus on the strength and characteristic time of the IOS shift. In general, our results support homeostasis on a long time scale of months, allostasis on scales of hours to weeks or until smoke quitting for smokers, as well as resilience in the case of recovery from a disease.

Structural sensitivity of CH vibrational band in methyl benzoate.

The CH vibrational bands of methyl benzoate are studied to understand its coupling pattern with other vibrational bands of the biological molecule. This will facilitate to understand the biological structure and dynamics in spectroscopic as well as in microscopic study. Due to the congested spectroscopic pattern, near degeneracy, and strong anharmonicity of the CH stretch vibrations, assignment of the CH vibrational frequencies are often misleading. Anharmonic vibrational frequency calculation with multidimensional potential energy surface interprets the CH vibrational spectra more accurately. In this article we have presented the importance of multidimensional potential energy surface in anharmonic vibrational frequency calculation and discuss the unexpected red shift of asymmetric CH stretch vibration of methyl group. The CD stretch vibrational band which is splitted to double peaks due to the Fermi resonance is also discussed here.

Sensitive spectroscopic breath analysis by water condensation.

Breath analysis has great potential for becoming an important clinical diagnosis method due to its friendly and non-invasive way of sample collection. Hundreds of endogenous trace gases (volatile organic compounds (VOCs)) are present in breath, representing different metabolic processes of the body. They are not only characteristic for a person, their age, sex, habit etc, but also specific to different kinds of diseases. VOCs, related to diseases could serve as biomarkers for clinical diagnostics and disease monitoring. However, due to the large amount of water contained in breath, an identification of specific VOCs is a real challenge. In this work we present a technique of water suppression from breath samples, that enables us to identify several trace gases in breath, e.g., methane, isoprene, acetone, aldehyde, carbon monoxide, etc, using Fourier-transform infrared spectroscopy. In the current state, the technique reduces the water concentration by a factor of 2500. Sample preparation and data acquisition take about 25 min, which is clinically relevant. In this article we demonstrate the working principle of the water reduction technique. Further, with specific examples we demonstrate that water elimination from breath samples does not hamper the concentration of trace gases in breath. Preliminary experiments with real breath also indicate that the concentrations of methane, acetone and isoprene remain the same during the sample preparation.