INTRODUCTION: This presentation focuses on the development of a non-invasive surveillance test based on an innovative skin patch for collection of sweat metabolites, which provides a convenient and easy-to-use platform for monitoring patients with cystic fibrosis (CF). CF is an autosomal recessive respiratory disease caused by mutation in the cystic fibrosis transmembrane regulator (CFTR) gene. The CFTR gene codes for the CFTR protein, which is an ion channel that controls the movement of chloride ions in and out of epithelial cells. Individuals affected with CF tend to experience periods of worsened lung function, which is referred to as pulmonary exacerbations (PE). In the event of PE, there is an acute worsening of lung function, which can lead to reduced responses to treatment. It is critical to effectively manage and prevent the onset of PE. Metabolites from various biofluids (e.g., blood and sputum) have been recognized as potential biomarkers for the management of PE. While useful, these biological sources can limited due to lack of ease of access at all ages. The proposed skin patch will collect sweat, which is a non-invasive biofluid that is easily accessed regardless of age. The absorbent in the patch is created from cellulose threads, which themselves provide a means by which the collected metabolites can be analyzed directly without prior sample preparation. This presentation will discuss the (i) creation of the skin patch, (ii) development of a novel solvent system that allows detection of both acidic and basic metabolites in a single experiment, and (iii) direct thread spray ambient ionization mass spectrometry method for metabolite characterization.
OBJECTIVE: The objective of this study is to develop a thread-based skin patch for prediction of PE in patients with chronic cystic fibrosis. Patients will wear the patch for a minimum of 24 h, after which the used patch will be mailed to us for subsequent analysis. We plan to detect the 5 selected metabolites in sweat samples directly from the thread substrate. To determine the efficacy of the selected metabolites as predictive biomarkers for the onset of PE, we will take samples before and after treatment. Data will be statistically analyzed by comparing the predictive power of the individual molecules versus the combination. We will determine the least number of metabolites that can provide the highest combined predictive effect.
METHOD: The patch will be created similarly to bandage where cellulose fabric of individual cellulose threads (inner layer) will be placed on an adhesive release liner. The assembly of the cellulose material and the adhesive tape will in turn be placed on an outer base layer (e.g., polyurethane adhesive). The inner cellulose layer will absorb the sweat from the skin once the patch is placed on the body (e.g., on the shoulder or forehead). The specific cellulose absorption material will be optimized by changing the thread density. When possible, the cellulose material will be pre-treated to remove endogenous chemicals that might be present in the commercial fabric. Individual cellulose threads in which the sweat has been absorbed will be removed from the patch and inserted into a glass capillary. The application of appropriate solvent enables selective extraction of interested analytes from the sweat sample. In this project, we employed direct thread spray ionization by adding 4 M NH4OH to the spray solvent MeOH:H2O (80:20, v:v) and applied a 5kV voltage. Method optimization was achieved artificial sweat where we spiked 200 μM of each metabolite individually. Structural characterization was performed using collision-induced dissociation in tandem MS (MS/MS). About 10 μL of artificial sweat was used in all preliminary studies.
RESULTS: The current work is a collaboration between our laboratory and Nationwide Children’s Hospital (NCH) in Columbus, OH. A previous study using liquid chromatographic mass spectrometry analysis of sweat collected with a commercial Wecor Macroduct® Sweat Collection System revealed the following 5 metabolites to hold the most potential as predictive biomarkers for the onset of PE before and after antibiotic treatment: 2-piperidone, thymidine, adipic acid, succinic acid and 2-hydroxy-3-methylbutyric acid. The goal of the current work was to translate this result into a clinical device for non-invasive surveillance of CF patients. The first step in this process was to evaluate the possibility of directly analyzing the sweat from the thread absorbing substrate. This was achieved in two separate experiments: (1) direct ionization of all metabolites from a single thread using a single spray solvent. We identified 4 M NH4OH in MeOH:H2O (80:20, v:v) to be effective at ionization both the acidic and basic compounds through a wrong-way ionization mechanism. The details of this new spray system and its implementation in thread spray MS will be discussed. (2) We performed MS/MS to determine diagnostic fragment ions that can be used to identify each metabolite in the presence of others. Based on these diagnostic ions, we will discuss sensitivity and accuracy of the method in this presentation. We have used the skin patch to collect from healthy volunteers and recorded comparable ion profiles for sweat obtained using Macroduct® from the same two people. This result and those obtained from CF patients will discussed in detail.
CONCLUSIONS: These results suggest that thread substrate is a suitable medium for collection sweat. Thread spray mass spectrometry provided an effective method for analysis of raw sweat directly from the thread substrate. Sample is found to be stable for at least 90 days after collection.