Isotope-Selective Gas Sensing Using Photoacoustic Non-Dispersive Spectroscopy †

: The flow of carbons into the citric acid cycle can be readily traced by supplementation with 13 C stable isotope labelled nutrients. However, the quantification of the amount of fully oxidised nutrients to carbon dioxide is a challenging task. This contribution presents an isotope-selective, miniaturized gas detection scheme based on indirect photoacoustic spectroscopy. The results show that low-cost, continuous, in situ monitoring of the isotope ratio in gaseous samples is feasible.


Introduction
The oxidation of high-energy carbohydrates is the dominant energy source of the vast majority of heterotrophic organisms, including all animals.The main compound classes donating their reduced carbon bonds are lipids, sugars, amino acids and, to a lesser extend, nucleotides, all of which converge at the so-called citric acid cycle (TCA, also known as the Krebs cycle), where the full oxidation to carbon dioxide (CO 2 ) takes place.Interestingly, the TCA cycle can also serve as a biosynthetic platform, where carbons from one class of metabolites are shunted into the biosynthesis of another.The utilisation in energy production or biosynthesis is a very dynamic process that integrates the metabolic needs of the cell with the availability of nutrients and oxygen [1,2].While the flow of carbons into the TCA cycle can be readily traced by supplementation with 13 C stable isotope-labelled nutrients, and their detection via mass spectrometric analysis, the quantification of the extent to which these nutrients are fully oxidised to CO 2 poses a significant challenge due to its gaseous nature.Currently, this is mainly done by indirect methods such as the detection of radioactive 14 C from labelled compounds or use of a relatively expensive setup to detect 13 CO 2 [3].There are currently no established methods to accurately detect and quantify nutrient respiration via 13 CO 2 detection in systems relevant for routine lab work such as tissue culture.
However, photoacoustic-based, non-dispersive infrared spectroscopy (NDIR) has been demonstrated to enable highly selective CO 2 sensors at a much-reduced system size due to its better sensitivity as compared to standard NDIR system [4].In this contribution, the scheme is expanded to demonstrate isotope-selective detection of 12 CO 2 and 13 CO 2 , thus paving the way for low-cost, in situ systems for the direct determination of the isotopic

Materials and Methods
The detection scheme relies on a single, mid-infrared light emitting diode (LED) from Hamamatsu (L15895LED) with a central wavelength of 4.2 µm illuminating a detection path length of 5 mm and two hermetically sealed, miniaturized photoacoustic detectors that each include a MEMS microphone (Invensense ICS-40720) and a 500 µm thick sapphire window for optical access, which are filled at 1 bar pressure with 100% CO 2 using standard isotope ratio of the carbon atom 13 C 12 C standard and 100% 13 CO 2 , respectively.To characterize the system, varying concentrations of standard CO 2 and pure 13 CO 2 have been mixed with synthetic air and the photoacoustic signal has been recoded.

Discussion
The sensor response of both system channels is shown in Figure 1 for varying CO 2 concentrations using both standard carbon isotope ratio and pure 13 CO 2 as test gases.Both channels show a response to both CO 2 isotope mixtures, but with a pronounced difference in sensitivity.Since only 12 CO 2 and 13 CO 2 cause signals, the setup enables a straightforward determination of δ 13 C, suitable for many applications that require isotope selective gas sensing applications.

Discussion
The sensor response of both system channels is shown in Figure 1 for varying CO2 concentrations using both standard carbon isotope ratio and pure 13 CO2 as test gases.Both channels show a response to both CO2 isotope mixtures, but with a pronounced difference in sensitivity.Since only 12 CO2 and 13 CO2 cause signals, the setup enables a straightforward determination of δ 13 C, suitable for many applications that require isotope selective gas sensing applications.Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

Figure 1 .
Figure 1.The sensor response to varying CO2 concentrations in dry synthetic air: (a) Using the standard isotope ratio of CO2, the sensitivity of the standard CO2 channel is considerably higher than for the 13 CO2 channel.(b) For the gas sensitive characterization, using pure 13 CO2 gas, the situation is reversed.Author Contributions: G.R.G. and A.O.P. built the sensing system, and performed the experiments and the data analysis; L.M. supported the experimental execution; G.R.G. and S.P. wrote the manuscript; P.K. devised the experiment; S.P. and G.R.G. devised the experimental setup and edited the manuscript.All authors have read and agreed to the published version of the manuscript.Funding: G.R.G. acknowledges funding from the Research Council of Norway under Grant Number 301552 (Upscaling Hotpots).

Figure 1 .
Figure1.The sensor response to varying CO 2 concentrations in dry synthetic air: (a) Using the standard isotope ratio of CO 2 , the sensitivity of the standard CO 2 channel is considerably higher than for the 13 CO 2 channel.(b) For the gas sensitive characterization, using pure 13 CO 2 gas, the situation is reversed.