Exposure to atmospheric particulate matter (PM) is strongly linked to increases in morbidity and mortality throughout the world [1
]. Analysis of long-term exposure studies has shown a dose-dependent relationship between PM2.5
mass and both cardiovascular and respiratory mortality [2
]. This relationship also holds for neurological disorders, with a wide scale meta-analysis also showing a link between PM2.5
mass exposure and a significantly increased risk of Alzheimer’s disease as well as increased risk for stroke, dementia, Parkinson’s disease and autism spectrum disorder [3
]. An underlying mechanism thought to be responsible for PM to generate such a diverse range of health impacts is known as oxidative stress.
Oxidative stress is drawn from microbiology [4
], and is defined as an excess of oxidants within a cell leading to disruptions in normal redox processes and cellular function. The oxidants in question are a group of oxygen-centred chemical species known as reactive oxygen species (ROS), which include: O2−.
and ROOH. In the context of air pollution, it states that inhalation and deposition of PM in the lungs can introduce ROS to cells. Their high reactivity allows them to interfere cellular function, resulting in one of three tiers of oxidative stress depending on severity of exposure [5
]. Tier 1 response results in the activation of a cells internal antioxidant defense to deplete excess ROS. If this defense is overwhelmed the cell moves into Tier 2, in which cellular signalling pathways are activated, and the affected cells become inflamed. In cases of extreme oxidative stress, Tier 3 results in the death of exposed cells through apoptosis or necrosis. These cellular responses, coupled with the ability for portions of ultrafine PM to penetrate into the bloodstream and cells [6
], and the important role oxidative stress plays in cellular signalling pathways [7
], forms the basis of the science implicating PM exposure to a diverse range of health outcomes.
An important parameter in the investigation of oxidative stress is the oxidative potential (OP) of PM, which is defined as the total degree to which a PM sample can oxidize components in its environment [5
]. This is directly tied to how much ROS a PM sample can introduce to the body, and thus is related to the degree to which it can induce oxidative stress in exposed cells [8
]. Cellular measurements of OP involve the exposure and examination of animals or lab cultivated cells for markers of oxidative stress; or direct measurement of radicals within cells using chemical probes [9
]. By necessity these methods are complex and difficult to adapt to field measurements for atmospheric pollution exposure; limiting time resolution and making large-scale atmospheric studies challenging. To resolve this, several methodologies have been developed which measure the ROS activity of PM outside of cells.
Acellular methodologies vary considerably in both sensitivity and application. Notable amongst them is electron paramagnetic resonance (EPR) spectroscopy, which is uniquely capable of identifying individual ROS species using a variety of spin traps [10
]. While this is a powerful capability, EPR is an offline methodology that cannot quantify all ROS using a single setup, requires extensive training, and is large, expensive and difficult to deploy in field campaigns. Alternative acellular approaches instead aim to provide a single value representing the cumulative total of ROS present independent of the species present. Systems based on these approaches [13
] are able to collect significantly more data with a higher resolution in the field when compared to EPR. A key challenge in this approach is that ROS introduced through inhalation of PM can be divided into two major categories: 1) PM-bound ROS, defined as ROS present on particles whilst in the atmosphere [17
]; and 2) endogenous ROS, which are ROS generated through chemical interactions between PM and exposed cells [13
]. Both of these sources are distinct and require different methodologies for measurement. Consequently, no single acellular methodology is capable of measuring the total OP of a PM sample; instead different acellular methodologies measure contributions to total OP. This work is concerned with the most common acellular measurements of PM-bound ROS, with in-depth investigations on methodologies for both PM-bound and endogenous ROS found elsewhere [5
The most commonly applied probe for PM-bound ROS is 2,7-dichlorofluorescein diacetate (DCFH-DA), a probe which reacts with certain ROS to form a fluorescent product [21
]. In recent years it has been integrated into several online instruments [15
]. Whilst these systems are a significant improvement over offline techniques, further advancement of the systems is made limited by the DCFH-DA probe in a few key ways, namely: a minimum reaction time of 11 min [15
]; the required use of horseradish peroxidase to catalyse the reaction, which can lead to a non-linear response [21
]; a high rate of autoxidation in probe stock solutions; and the requirement for the PM to be water soluble. To address these points a profluorescent nitroxide (PFN) probe 9,10-bis(phenylethynyl)anthracene-nitroxide (BPEAnit) [26
] was developed and integrated into a system known as the Particle Into Nitroxide Quencher (PINQ) [27
The PINQ is PM-bound ROS instrument which collects PM regardless of composition directly into a solution of DMSO and the BPEAnit probe. The reaction with the probe is diffusion limited, lowering the instrument time resolution to as low as one min while achieving a limit of detection of 0.08 nmol.m−3. This manuscript details the continued development of the Particle Into Nitroxide Quencher (PINQ) into a fully online PM-bound-ROS monitor. In particular, the development, testing and integration of a dedicated flow-through fluorimeter for rapid quantification of the BPEAnit probe is covered. Details on the design, calibration and operation methodology of the instrument are provided; and preliminary high time resolution PM-bound-ROS measurements are presented for both a chamber study and ambient monitoring campaign.