Breath biosensing: using electrochemical enzymatic sensors for detection of biomarkers in human breath

Detection of biomarkers for disease by noninvasive methods is critical for the early diagnosis and screening of disease, enabling prompt treatment. Breath biosensors are a viable option as the exhaled breath contains several biomarkers linked to lung cancer, oxidative stress, diabetes, and other diseases. Breath analysis has been achieved by advanced analytical techniques such as gas chromatography and infrared spectroscopy. However, electrochemical enzymatic breath biosensors offer a cost-effective, sensitive platform for biomarker detection without complex analysis and interpretation by trained laboratory personnel. This review aims to summarize recent advances in the field of electrochemical enzymatic breath biosensors and offer future opportunities from other applications of nonelectrochemical enzymatic breath biosensors.     

Breath analysis by optical fiber sensor for the determination of exhaled organic compounds with a view to diagnostics

Breath analysis constitutes a promising tool in clinical and analytical fields due to its high potential for non-invasive diagnostics of metabolic disorders and monitoring of disease status. An optical fiber (OF) sensor has been developed for determination of volatile organic compounds (ethane, pentane, heptane, octane, decane, benzene, toluene and styrene) in human breath for clinical diagnosis.The analytical system developed showed a high performance for breath analysis, inferred for the analytical signal intensity and stability, linear range, and detection limits ranging from 0.8 pmol L⁻¹, for heptane, and to 9.5 pmol L⁻¹, for decane. The OF sensor also showed advantageous features of near real-time response and low instrumentation costs, besides showing an analytical performance equivalent to the breath analysis by gas chromatography-mass spectrometry (GC-MS), used as the reference method.     

Analysis of human breath with micro extraction techniques and continuous monitoring of carbon dioxide concentration

The detection of volatile organic compounds (VOCs) in human breath can be useful for the clinical routine diagnosis of several diseases in a non-invasive manner. Traditional methods of breath analysis have some major technical problems and limitations. Membrane extraction with a sorbent interface (MESI), however, has many advantages over current methods, including good selectivity and sensitivity, and is well suited for breath analysis. The aim of this project was to develop a simple and reproducible sampling device and method based on the MESI system for breath analysis. The feasibility and validity of the MESI system was tested with real human breath samples. Internal standard calibration methods were used for the quantitative analysis of various breath samples. Calibration curves for some main components (target analytes such as acetone and pentane) were determined in the research. The optimized stripping-side and feeding-side gas velocities were determined. The use of breath CO₂ as an internal standard for the analysis of breath VOCs is an effective method to solve the difficulties associated with variations in the target analyte concentrations in a sample, which are attributed to mass losses and different breathing patterns of different subjects. In this study, the concentration of breath acetone was successfully expressed normalized to CO₂ as in the alveolar air. Breath acetone of healthy males and females profiled at different times of the day was plotted using the MESI system, and results were consistent with the literature. This technique can be used for monitoring breath acetone concentrations of diabetic patients and for applications with other biomarker monitoring.     

Estimating the measurement uncertainty in forensic breath-alcohol analysis

The evidentiary weight attributed to forensic breath alcohol results in drunk-driving prosecutions requires that measurement uncertainty be established and shown to be fit-for-purpose. The principal components contributing to breath alcohol measurement uncertainty include: (1) biological/sampling, (2) instrumental, (3) traceability and (4) the water/air partition coefficient for control standards. Employing duplicate breath results from over 92,000 subjects to estimate the biological/sampling component and assuming reasonable forensic values for the other components, the combined and expanded uncertainty is determined for a practical example. The combined uncertainty for an unbiased single determination breath alcohol measurement was: . Employing the expanded uncertainty (k = 2.58), the 99% confidence interval for a mean breath alcohol concentration of 0.0935 g/210 L was 0.0866 to 0.1004 g/210 L. The proportion of combined uncertainty associated with each component was determined to be: biological/sampling 73%, analytical 10%, traceability 13% and water/air partition coefficient 4%. These are forensically acceptable estimates and demonstrate fitness-for-purpose of breath alcohol measurement when employing appropriate elements of quality control.     

Determination of fentanyl in human breath by solid-phase microextraction and gas chromatography–mass spectrometry

Fentanyl, a kind of intravenous narcotic analgesic, is widely used in clinical anesthesia. As a potential pollution, it was detected in both the air of the cardiothoracic operating room and patients' expiratory circuit. However, whether the fentanyl in patients' expiratory circuit is exhaled by patients is unknown. In this study, breath samples were taken from the expiratory circuits of anesthetic machine linked to the patients who received intravenous fentanyl, a solid-phase microextraction (SPME) coupled with gas chromatography–mass spectrometry (GC–MS) method was developed to detect and quantify fentanyl in breath samples. The parameters influencing adsorption (extraction time, temperature,) and desorption (desorption time) of the analyte on the fiber were investigated and validated for method development. The developed method was proved to be simple, easy, and inexpensive and offer high sensitivity and reproducibility. Linear range was obtained from 0.05 ng/mL to 0.8 ng/mL. The limit of detection was 0.01 ng/mL while an interday precision of less than 12.13% (n = 5) could be achieved. Six patients were involved in this study; results showed presence of fentanyl in the breath of patients who received intravenous fentanyl, and fentanyl concentrations in breath varied from 6.00 to 20.89 pg/mL. In conclusion, fentanyl can be exhaled by patients who received intravenous fentanyl.     

Influence of weakly bound adduct ions on breath trace gas analysis by selected ion flow tube mass spectrometry (SIFT-MS)

This paper describes how weakly bound adduct ions form when the precursor ions used in selected ion flow mass spectrometry, SIFT-MS, analyses, viz. H₃O⁺, NO⁺ and O₂⁺, associate with the major components of air and exhaled breath, N₂, O₂ and CO₂. These adduct ions, which include H₃O⁺N₂, H₃O⁺CO₂, NO⁺CO₂, O₂⁺O₂ and O₂⁺CO₂, are clearly seen when dry air containing 5% CO₂ (typical of that in exhaled breath) is analysed using SIFT-MS. These adduct ions must not be misinterpreted as characteristic product ions of trace gases; if so, serious analytical errors can result. However, when exhaled breath is analysed these adduct ions are partly removed by ligand switching reactions with the abundant water molecules and the problems they represent are alleviated. But the small fractions of the adduct ions that remain in the SIFT-MS spectra, and especially when they are isobaric with genuine characteristic product ion of breath trace gases, can result in erroneous quantifications; such is the case for H₃O⁺N₂ interfering with breath ethanol analysis and H₃O⁺CO₂ with breath acetaldehyde analysis. However, these difficulties can be overcome when the isobaric adduct ions are properly recognised and excluded from the analyses; then these two important compounds can be properly quantified in breath. The presence of O₂⁺CO₂ in the product ion spectra interferes with the analysis of CS₂ present at low levels in exhaled breath. It is likely that similar problems will occur as other trace compounds are detected in exhaled breath when consideration will have to be given to the possibility of overlapping between their characteristic product ions and ions produced by hitherto unknown reactions. Similar problems are evident in other systems; for example, H₃O⁺CH₄ adduct ions are observed in both SIFT-MS analyses of methane rich mixtures like biologically generated waste gases and in model planetary atmospheres.     

呼气中痕量挥发性有机物的质子转移反应质谱在线检测研究

通过对自主研制的大气成分在线检测质子转移反应质谱的进样管路系统进行改造,建立了可在线检测呼气中痕量挥发性有机物的质子转移反应质谱装置。通过对呼气进样系统的旁路流量控制,实现对进样速度的调控,既可提高进样速度,以满足实时监测呼气中指定成分浓度变化;也可适时关闭旁路,以降低进样速度,从而对呼气成分进行全谱分析,避免采样袋采样和浓缩的复杂程序和潜在干扰。以作者呼出气体作为研究对象,对装置性能进行测试,结果表明:装置最快响应时间可达1s,对呼气中丙酮的探测灵敏度高达每10-9(V/V)浓度的信号强度为14.6counts/s,多次呼气测量重复性好,有望广泛应用于呼气疾病诊断研究。     

Design-of-experiment optimization of exhaled breath condensate analysis using a miniature differential mobility spectrometer (DMS)

Analytical instruments that can measure small amounts of chemicals in complicated biological samples are often useful as diagnostic tools. However, it can be challenging to optimize these sensors using actual clinical samples, given the heterogeneous background and composition of the test materials. Here we use gas chromatography-differential mobility spectrometry (GC/DMS) to analyze the chemical content of human exhaled breath condensate (EBC). Ultimately, this system can be used for non-invasive disease diagnostics. Many parameters can be adjusted within this instrument system, and we implemented a factorial design-of-experiments to systematically test several combinations of parameter settings while concurrently analyzing effects and interactions.We examined four parameters that affect sensitivity and detection for our instrument, requiring a 2⁴ factorial design. We optimized sensor function using EBC samples spiked with acetone, a known clinical biomarker in breath. Two outputs were recorded for each experiment combination: number of chemicals detected, and the amplitude of acetone signal. Our goal is to find the best parameter combination that yields the highest acetone peak while also preserving the largest number of other chemical peaks in the spectra. By optimizing the system, we can conduct further clinical experiments with our sensor more efficiently and accurately.     

Direct measurement of ammonia in simulated human breath using an inkjet-printed polyaniline nanoparticle sensor

A sensor fabricated from the inkjet-printed deposition of polyaniline nanoparticles onto a screen-printed silver interdigitated electrode was developed for the detection of ammonia in simulated human breath samples. Impedance analysis showed that exposure to ammonia gas could be measured at 962 Hz at which changes in resistance dominate due to the deprotonation of the polymer film. Sensors required minimal calibration and demonstrated excellent intra-electrode baseline drift (≤1.67%). Gases typically present in breath did not interfere with the sensor. Temperature and humidity were shown to have characteristic impedimetric and temporal effects on the sensor that could be distinguished from the response to ammonia. While impedance responses to ammonia could be detected from a single simulated breath, quantification was improved after the cumulative measurement of multiple breaths. The measurement of ammonia after 16 simulated breaths was linear in the range of 40–2175 ppbv (27–1514 μg m⁻³) (r² = 0.9963) with a theoretical limit of detection of 6.2 ppbv (4.1 μg m⁻³) (SN⁻¹ = 3).     

Chemotherapy control by breath profile with application of SPME‐GC/MS method

Chemotherapy used as a treatment against lung cancer has influence on metabolic processes occurring in healthy cells. The changes of biochemical pathways proceeded inside cells might be observed in expired air. In the experiment, breath analysis was carried out before and after anticancer therapy. Expired air samples were collected from 22 patients with a biopsy confirmed lung cancer. Volatile organic compounds present in breath were analyzed by gas chromatography/mass spectrometry. For enrichment of analytes solid‐phase microextraction technique was applied. Eight fibers covered by different sorbents were tested. Carboxen‐polydimethylsiloxane fiber revealed the highest extraction efficiency in relation to analytes in breath. The data showed that cytostatic drugs increase the concentration of acetone and isoprene in the breath collected after chemotherapy. Volatile metabolites of administrated drugs were not identified in expired air.     

Determination of breath alcohol using a differential-type amperometric biosensor based on alcohol dehydrogenase

A differential-type amperometric biosensor based on conventional thick-film technology has been developed for breath alcohol measurement. The amperometric breath alcohol biosensor utilizes the alcohol dehydrogenase (ADH) and nicotinamide adenine dinucleotide (NAD⁺) cofactor which produce reduced NADH as a product of the oxidation of alcohol. The biosensor was designed in a differential format consisting of a common Ag/AgCl reference electrode, an active working electrode containing the ADH, and the inactive working electrode containing only bovine serum albumin instead of the ADH. The differential signal between the active working electrode and the inactive working electrode minimized the interference from a large number of oxidizable species present in a person's breath. Prior to the amperometric measurement the biosensor was hydrated simply by dipping it into a phosphate buffer solution at pH 7.4. The NADH produced from the enzymatic reaction was oxidized at the working electrode biased at a potential of 470 mV vs. an on-board Ag/AgCl reference electrode. The biosensor can measure a person's breath alcohol over the concentration range 20–800 ppm routinely required in a test of drunken driving.     

Effects of carbon dioxide in breath gas on proton transfer reaction-mass spectrometry (PTR-MS) measurements

PTR-MS is becoming a common method for the analysis of volatile organic compounds (VOCs) in human breath. Breath gas contains substantial and, particularly for bag samples, highly variable concentrations of water vapour (up to 6.3%) and carbon dioxide (up to 6.5%). The goal of this study was to investigate the effects of carbon dioxide on PTR-MS measurements; such effects can be expected in view of the already well known effects of water vapour. Carbon dioxide caused an increase of the pressure in the PTR-MS drift tube (1% increase for 5% CO₂), and this effect was used to assess the CO₂ concentration of breath gas samples along the way with the analysis of VOCs. Carbon dioxide enhanced the concentration ratio of protonated water clusters (H₃O⁺H₂O) to protonated water (H₃O⁺) in the drift tube. Using the observed increase, being 60% for 5% CO₂, it is estimated that the mobility of water cluster ions in pure CO₂ is almost 65% lower than in air. Carbon dioxide had a significant effect on the mass spectra of the main breath gas components methanol, ethanol, 1-propanol, 2-propanol, acetone, and isoprene. Carbon dioxide caused a small increase (<10% for 5% CO₂) of the normalised main signals for the non-fragmenting molecules methanol and acetone. The increase can be much higher for the fragmenting VOCs (ethanol, propanol, and isoprene) and was, for 5% CO₂, up to 60% for ethanol. This effect of CO₂ on fragment patterns is mainly a consequence of the increased abundance of protonated water clusters, which undergo softer reactions with VOCs than the hydronium ions. Breath gas samples stored in Teflon bags lost 80% of CO₂ during 3 days, the decrease of VOC signals, however, is mainly attributed to decreasing VOC concentrations and to the loss of humidity from the bags.     

Breath Biomarkers in Diagnostic Applications

The detection of chemical compounds in exhaled human breath presents an opportunity to determine physiological state, diagnose disease or assess environmental exposure. Recent advancements in metabolomics research have led to improved capabilities to explore human metabolic profiles in breath. Despite some notable challenges in sampling and analysis, exhaled breath represents a desirable medium for metabolomics applications, foremost due to its non-invasive, convenient and practically limitless availability. Several breath-based tests that target either endogenous or exogenous gas-phase compounds are currently established and are in practical and/or clinical use. This review outlines the concept of breath analysis in the context of these unique tests and their applications. The respective breath biomarkers targeted in each test are discussed in relation to their physiological production in the human body and the development and implementation of the associated tests. The paper concludes with a brief insight into prospective tests and an outlook of the future direction of breath research.     

利用Breath Figures法制备具有蜂窝状结构的 PMMA/TBT复合膜

以聚甲基丙烯酸甲酯(PMMA)和钛酸四丁酯(TBT)的氯仿溶液为铸膜溶液,采用Breath Figures法制备了蜂窝状结构的PMMA/TBT多孔阵列薄膜,采用SEM、JR、XRD对制备的多孔膜进行了表征,探讨了聚合物浓度、PMMA/TBT配比以及水解抑制剂对膜蜂窝状结构的影响.实验结果表明,起始溶液浓度为20～30m...     

Ultrasensitive Organic Humidity Sensor with High Specificity for Healthcare Applications

Humidity sensors have gained immense importance as non‐invasive, wearable healthcare devices for personal care as well as disease diagnostics. However, non‐specificity, poor stability at extreme conditions, and low sensitivity of the humidity sensor inhibit its usage as a health monitoring device. In the present study, N?F containing organic molecule, Selectfluor^TM (F‐TEDA) based humidity sensors with ～1–2 mm long needle‐shaped crystals is fabricated on interdigitated electrodes resulting in excellent performance. The unidirectional growth of crystals led to the formation of a conduction pathway for water molecules across the crystal, which otherwise are non‐conducting. The as‐fabricated humidity sensor at an operational voltage of 0.8 V displays a sensitivity of six orders in magnitude, best reported so far. The sensor does not exhibit any response upon exposure to various volatile organic compounds and reactive gases, indicating remarkable specificity. The sensor is tolerant to high moisture of 95 % for prolonged hours followed by monitoring over several days and degrades to 50 % of its original sensitivity only after continuous exposure for several days. Electrochemical impedance spectroscopy (EIS) shows reversal from resistive to capacitive behavior with increasing humidity levels. The fabricated humidity sensor acts as a healthcare device for breath rate monitoring and touch‐free examination of skin moisture.     

Modular Breath Analyzer (MBA): Introduction of a Breath Analyzer Platform Based on an Innovative and Unique,Modular eNose Concept for Breath Diagnostics and Utilization of Calibration Transfer Methods in Breath Analysis Studies

Exhaled breath analysis for early disease detection may provide a convenient method for painless and non-invasive diagnosis. In this work, a novel, compact and easy-to-use breath analyzer platform with a modular sensing chamber and direct breath sampling unit is presented. The developed analyzer system comprises a compact, low volume, temperature-controlled sensing chamber in three modules that can host any type of resistive gas sensor arrays. Furthermore, in this study three modular breath analyzers are explicitly tested for reproducibility in a real-life breath analysis experiment with several calibration transfer (CT) techniques using transfer samples from the experiment. The experiment consists of classifying breath samples from 15 subjects before and after eating a specific meal using three instruments. We investigate the possibility to transfer calibration models across instruments using transfer samples from the experiment under study, since representative samples of human breath at some conditions are difficult to simulate in a laboratory. For example, exhaled breath from subjects suffering from a disease for which the biomarkers are mostly unknown. Results show that many transfer samples of all the classes under study (in our case meal/no meal) are needed, although some CT methods present reasonably good results with only one class.     

Trypsin immobilization in ordered porous polymer membranes for effective protein digestion

Fast and effective protein digestion is a vital process for mass spectrometry (MS) based protein analysis. This study introduces a porous polymer membrane enzyme reactor (PPMER) coupled to nanoflow liquid chromatography-tandem MS (nLC-ESI-MS/MS) for on-line digestion and analysis of proteins. Poly (styrene-co-maleic anhydride) (PS-co-MAn) was fabricated by the breath figure method to make a porous polymer membrane in which the MAn group was covalently bound to enzyme. Based on this strategy, microscale PPMER (μPPMER) was constructed for on-line connection with the nLC-ESI-MS/MS system. Its capability for enzymatic digestion with bovine serum albumin (BSA) was evaluated with varied digestion periods. The on-line proteolysis of BSA and subsequent analysis with μPPMER-nLC-ESI-MS/MS revealed that peptide sequence coverage increased from 10.3% (digestion time 10 min) to 89.1% (digestion time 30 min). μPPMER can efficiently digest proteins due to the microscopic confinement effect, showing its potential application in fast protein identification and protease immobilization. Applications of on-line digestion using μPPMER with human plasma and urinary proteome samples showed that the developed on-line method yielded equivalent or better performance in protein coverage and identified more membrane proteins than the in-solution method. This may be due to easy accommodation of hydrophobic membrane proteins within membrane pores.     

Human exhaled air analytics: biomarkers of diseases

Over the last few years, breath analysis for the routine monitoring of metabolic disorders has attracted a considerable amount of scientific interest, especially since breath sampling is a non-invasive technique, totally painless and agreeable to patients. The investigation of human breath samples with various analytical methods has shown a correlation between the concentration patterns of volatile organic compounds (VOCs) and the occurrence of certain diseases. It has been demonstrated that modern analytical instruments allow the determination of many compounds found in human breath both in normal and anomalous concentrations. The composition of exhaled breath in patients with, for example, lung cancer, inflammatory lung disease, hepatic or renal dysfunction and diabetes contains valuable information. Furthermore, the detection and quantification of oxidative stress, and its monitoring during surgery based on composition of exhaled breath, have made considerable progress. This paper gives an overview of the analytical techniques used for sample collection, preconcentration and analysis of human breath composition. The diagnostic potential of different disease-marking substances in human breath for a selection of diseases and the clinical applications of breath analysis are discussed.     

呼吸图案法制备蜂窝状有序多孔薄膜及其功能化应用

呼吸图案(BF)法是一种制备微纳米级规整多孔结构的简单、廉价和高效的方法, 它以水滴为模板, 通过水滴有序排列得到蜂窝状有序多孔结构, 其孔的形貌可以通过选择不同的成膜材料和成膜环境等条件得到控制, 所以在分离膜、模板、响应性表面、催化剂、光电材料等研究领域有广阔的发展前景. 但是, 到目前为止, 人们发现由于成膜条件的不同, 多孔膜的孔形貌受多种因素影响, 规整多孔膜形貌的控制机理还不是很明确, 没有一个统一的结论. 本文结合本课题组所做工作及近五年来国内外呼吸图案法制备蜂窝状有序多孔薄膜的研究成果, 对多孔薄膜的形成过程及其影响因素、多孔膜的功能化及应用等方面进行了归纳总结, 试图揭示孔的形成及孔形貌的调控等相关规律, 希望对后续的进一步研究与应用奠定基础.     

Assessment of terbium (III) as a luminescent probe for the detection of tuberculosis biomarkers

A detection method for nicotinic acid, a specific metabolite marker of Mycobacterium tuberculosis present in cultures and patients' breath, is studied in complex solutions containing other metabolites and in biological media such as urine, saliva and breath condensate. The method is based on the analysis of the luminescence increase of Tb³⁺ complexes in the presence of nicotinic acid due to the energy transfer from the excited ligand to the lanthanide ion. It is shown that other potential markers found in M. tuberculosis culture supernatant, such as methyl phenylacetate, p-methyl anisate, methyl nicotinate and 2-methoxy biphenyl, can interfere with nicotinic acid via a competitive absorption of the excitation photons. A new strategy to circumvent these interferences is proposed with an upstream trapping of volatile markers preceding the detection of nicotinic acid in the liquid phase via the luminescence of Tb³⁺ complexes. The cost of the method is evaluated and compared with the Xpert MTB/RIF test endorsed by the World Health Organization.