(163b) Electrochemical Point-of-Care Sensing Methods for the Detection of Tuberculosis Volatile Organic Biomarkers in Patient Breath and Condensate

In this presentation two
electrochemical sensing platforms (gas and liquid phase) for the rapid,
point-of-care (POC) detection of tuberculosis (TB) will be discussed. According
to the World Health Organization, tuberculosis is one of the top ten causes of
death worldwide, killing 1.6 million people in 2017 [1]. Up to one third of adults
and the majority of young children with TB are unable to produce sufficient sputum
for traditional culture testing, making the development of non-sputum-based tests
a priority in TB screening and diagnostics.  Rapid screening of tuberculosis by
evaluation of associated volatile organic biomarkers (VOBs) in breath has been
shown to be a promising technology that is significantly faster and more
convenient than traditional sputum culture tests. Methyl nicotinate and methyl
p-anisate have been isolated as specific biomarkers for the mycobacterium tuberculosis,
as they are not found in high concentrations in ambient air or the breath of
healthy patients, but should be readily present in all patients with active TB.
Previous modelling studies [2] suggest that these biomarkers complex with
certain transition metals (such as cobalt and copper) given a particular bias
voltage and oxidation state of the metal. This known interaction was used as
the basis for the sensing platforms described.

The ability to detect these
biomarkers in both liquid (breath condensate) and gas (direct breath) phases is
useful in POC settings for several reasons. First, tuberculosis is especially
prevalent in low-resource communities, with over 95% of cases presenting in
countries classified as low and middle income. Traditional detection methods
such as sputum culture have incubation times of days or weeks, and require
expensive instrumentation, laboratory level spaces and highly trained
technicians. The option of immediately testing collected breath or storing it
for testing at a later time in condensate form provides flexibility for both
patients and technicians. The sample collection can be performed remotely and
non-invasively, making it ideal in high TB burden countries where patients
often live far from medical facilities. The need for repeated visits to medical
facilities for sample delivery and test results is prohibitive to many, and
results in significant patient dropout. It is the hope that these POC sample
collection and test methods will increase access to testing worldwide and remove
the need for centralized testing facilities. Second, although the levels of
TB-specific VOBs are known to be high in extremely sick patients, those at
earlier stages of the disease and pediatric patients do not show such high levels.
The VOBs described are semi-volatile and readily dissolve in water at
relatively high concentrations, making breath condensate a good candidate for
simple concentration of the biomarkers from breath. Multiple detection methods
for a single patient can also help to increase specificity if the sensors are
used in an array. Finally, the nature of the functional metals themselves has
been found to be a determining factor in their use in either a liquid- or
gas-phase sensor. These features will be discussed further in the overview of
each sensing platform.

A solid-state titanium dioxide
nanotube array-based sensor is used to directly detect TB biomarkers in the gas
phase, making it ideal for POC settings when the breath collected can be
immediately tested. Our lab group previously demonstrated the ability of a
cobalt-functionalized version of this sensor to specifically detect both of the
biomarkers specified using a simple two-electrode setup and a
chronoamperometric method [3]. In this configuration, the pure titanium back of
the sensor functioned as both the counter and reference electrode, meaning that
the applied current is highly dependent on the resistance of the nanotubes and
difficult to reproduce from sensor to sensor. To improve reproducibility and
specificity of the sensor, the platform has been modified to use a solid-state,
conductive electrolyte in order to create a gas phase three-electrode system. A
conductive polymer of known resistance is used to separate the functionalized
TNA from the silver-painted counter and reference electrodes. Doing this
ensures more consistent interactions with the analyte of interest between
sensors. In addition to the electrode changes, new scanning potential methods
such as cyclic voltammetry (CV) and square wave voltammetry (SWV) have shown
promise in detecting the analytes more specifically than chronoamperometry. For
example, running cyclic voltammetry using cobalt functionalized sensors shows a
reduction peak at about -0.5 V vs a silver quasi-reference electrode when
exposed to methyl nicotinate vapor. According to the modelling studies
previously referenced, divalent cobalt shows a more specific binding affinity
to the biomarkers than copper, and can be easily attached to the sensor surface
in hydroxide form using methods such as deposition in an ultrasonic bath or
in-situ anodization of the nanotubes. This makes it the primary metal used in
this sensing platform.

In the liquid phase, these
functional metal interactions can be exploited by mixing the sample solution
containing the biomarkers into an electroactive solution (EAS) containing the
functional metal ion, and observing the change electrochemically. Although both
cobalt and copper can be used in the EAS, copper is preferred when performing
liquid electrochemistry because of its highly reversible redox reactions. In
contrast, cobalt shows low reversibility in liquid systems, making it more
difficult to assess the interaction of cobalt with biomarkers of interest using
methods such as cyclic voltammetry (CV). Testing of this platform with a
divalent copper EAS has shown that the cyclic voltammogram of the EAS changes
significantly when as little as 1 mM methyl nicotinate (MN) is added to the
solution. The variation of peak oxidation voltage with MN concentration can be
seen in Figure 1 below. In order to further understand the reactions taking
place and specifically identify the biomarkers, square wave voltammetry (SWV)
is employed to show which oxidation states of the functionalized metal are
complexing with the biomarker of interest. The SWV for the copper EAS alone
shows three distinct peaks for each of the redox reaction couples present in
the voltage window (less than ±1 V). When a small amount of MN is added, the
size, shape and in some cases peak-voltage of each distinct peak is affected
differently, implying a difference in reaction to specific valence states of
copper (Figure 2). When methyl p-anisate (PA) was added at the same
concentration, different interactions were observed. In this way, a “fingerprint”
method can be used to identify biomarkers once their known interaction is
established. 

Figure 1: Change in peak oxidation of copper
(II)with MN concentration

Figure 2: Effect of MN on square wave
voltammogram of copper

References:

[1] World Health Organization, “Tuberculosis Fact Sheet,”
2018. [Online]. Available:
https://www.who.int/en/news-room/fact-sheets/detail/tuberculosis. [Accessed:
12-Apr-2019].

[2] Ray R, Sarma B, Mohanty S, Prisbrey K, Misra M.
Assessment of metals in detection of TB biomarkers: Novel computational
approach. Journal of Materials Chemistry and Physics. 161 (2015) 1-8

[3]  D. Bhattacharyya, Y. R. Smith, S. K. Mohanty, and M.
Misra, “Titania Nanotube Array Sensor for Electrochemical Detection of Four
Predominate Tuberculosis Volatile Biomarkers,” J. Electrochem. Soc., vol. 163,
no. 6, pp. B206–B214, 2016.