Applications of Nanotechnology and Chemical Sensors for the Detection and Identification of Multiple Sclerosis, In Comparison to Other Autoimmune and Neurological Diseases by Exhalation Samples
MS is the most common chronic neurological disease affecting young adults, with onset
usually at the age 20-40 years. Women are affected 3-4 times more than men. It is a complex
multi-factorial disease, with underlying both genetic and environmental factors. Different
populations have different susceptibility (Compston and Coles 2008).
The disease is characterized by 2 main phenotypes: relapsing-remitting or progressive
course. Clinical disability is due to destruction of the CNS myelin (mainly
oligodendrocytes) due to 3 processes (Franklin 2002; Franklin and Ffrench-Constant 2008;
Frischer, Bramow et al. 2009):
1. Inflammation- immune cells with aberrant activity invade the brain and spinal cord and
cause destruction of CNS myelin (a process called demyelination and secondary
neurodegeneration - axonal and neuronal loss)
2. Primary neurodegeneration (axonal and neuronal loss) - without prominent inflammation
3. Repair - the inflammatory and neurodegenerative processes are followed by an attempt
of the CNS to repair - however, this partial and incomplete repair is often the basis
of residual deficits and disability (Chandran, Hunt et al. 2008) .
- The acute MS relapse (presented as paralysis, visual loss, etc.) - is considered
to be due to an aberrant acute immune activation and inflammatory process in the
- The chronic accumulating disability - is considered to be due to the
Repair processes are mainly noted after the acute relapse - and recovery of function can be
spontaneous. However, in severe relapses sometimes there is need for STEROID TREATMENT
(Tischner and Reichardt 2007).
Following the increased understanding of the disease, new immunotherapies were developed
(COPAXON /; Interferon -beta )in the last 10-15 years for long term treatment. However
these can attenuate the disease (reduce the number of relapses per year) but do not cure it.
In addition, they are beneficial in only ~40 % of the Relapsing -Remitting patients.
Currently there are no treatments for patients with the Progressive Disease - who have
gradual increased disability (Murray 2006).
Presently there are no biomarkers available for diagnosis and routine follow-up of MS.
Oligoclonal IgG in the CSF - which help confirm the diagnosis, require an invasive
procedure and are not correlated with disease activity nor response to therapy; and MRI,
which allows monitoring of MS activity and response to treatment is too expensive to
routine use (Link and Huang 2006; Murray 2006).
Dr Hossam Haick from the Technion, developed an electronic nose for diagnosis of diseases
via breath samples. Dr Haick's previous studies have shown that an electronic nose based on
gold nanoparticles could form the basis of an inexpensive and non- invasive diagnostic tool
for lung cancer (Peng, Tisch et al. 2009) and kidney diseases (Haick, Hakim et al. 2009).
Research hypothesis Biomarkers of CNS inflammation and/or neurodegeneration and/or CNS
repair can be detected by the "electronic nose" in breath samples of persons with MS.
Identification of biomarkers of:
1. CNS inflammation and CNS-autoimmunity
3. CNS repair
- that may serve as markers for: disease (vs controls), disease activity
(predicting aggressive disease course, predicting Relapse; predicting Malignant vs
Benign MS); response to therapy (Steroid , immunotherapies or neuroprotective
Work plan outline:
Evaluate few groups clinically:
- MS patients at acute relapse pre - vs- after 7 ,30 and 90 days of steroids treatment -
to assess indicators of the acute inflammatory process and of the effects of Steroid
- Relapsing MS patients vs Progressive MS patients vs controls which include healthy
individuals as well as patients suffering from neurological and autoimmune diseases
other than MS - to assess inflammatory vs neurodegenerative indicators.
- MS patients who are Good- vs Poor- Responders to immunotherapy or Steroids.
Alveolar breath of the volunteers is collected using an "offline" method that effectively
separates the endogenous from the exogenous breath volatile biomarkers and excludes the
nasal entrainment. Two bags of 750 ml of breath samples per volunteer are collected in inert
Mylar bags (Eco Medics, Duerten, Switzerland). Vapor sampling was performed by extended
breath sampling into the collection apparatus for 15-20 minutes, with several stops during
this process. The first three minutes of breath sampling are discarded due to the possible
contamination of the upper respiratory air. The subsequent deep air is retained for testing
purposes. The samples are collected with a tube that was introduced in the volunteer's mouth
and connected to the collection bag. All participants provide a signed informed consent to
this study, which is performed following the approval and according to the guidelines of the
Helsinki Committee of Carmel Medical Center and Technion's committee for supervision of
experiments in humans.
CHEMICAL ANALYSIS OF THE BREATH SAMPLES:
Gas-Chromatography/Mass-Spectrometry (GCMS-QP2010; Shimadzu Corporation, Japan), combined
with a thermal desorption system (TD20; Shimadzu Corporation, Japan), is used for the
chemical analysis of the breath samples. A Tenax® TA adsorbent tube (Sigma Aldrich Ltd.) is
employed for pre-concentrating the VOCs in the breath samples. Using a custom-made pump
system, the breath samples from the Mylar bags are sucked up through the TA tube at 100
ml/min flow rate, being then transferred to a thermal desorption (TD) tube (Sigma Aldrich
Ltd.) before being analyzed by GC-MS. The following oven temperature profile was set: (a) 10
min at 35°C; (b) 4°C/min ramp until 150°C; (c) 10°C/min ramp until 300°C; and (d) 15 min at
300°C. An SLB-5ms capillary column (Sigma Aldrich Ltd.) with 5% phenyl methyl siloxane (30 m
length, 0.25 mm internal diameter and 0.5 μm thicknesses) is employed. The splitless
injection mode is used for 2 min, at 30 cm/sec constant linear speed and 0.70 ml/min column
flow. The molecular structures of the VOCs are determined via the standard modular set,
using 10 ppm isobutylene (Calgaz, Cambridge, Maryland, USA) as standard calibration gas
during each run. GC-MS chromatogram analysis is realized using the GCMS solutions version
2.53SU1 post-run analysis program (Shimadzu Corporation), employing the National Institute
of Standards and Technology (NIST) compounds library (Gaithersburg, MD 20899-1070, USA).
Upon interaction between the breath samples and the detector, the volatile organic compounds
adsorb into the organic part of the sensing material The result of this adsorption is
translated to an electrical signal (resistance) that is transmitted to the macro-world
(e.g., the screen of the device) through the (semi-)conductive material found in the same
film. The results are then presented on the computers screen.
An automated system controlled by a custom LabView (National Instruments) program is used to
perform the sensing measurements. The sensors are tested simultaneously, in the same
exposure chamber, using an Agilent 34980A multifunction switch. A Stanford Research System
SR830 DSP lock-in amplifier controlled by an IEEE 488 bus is used to supply the AC voltage
signal (0.2 V at 1 kHz) and to measure the corresponding current (<10μA in the studied
devices). This setup allows for measuring normalized changes in conductance as small as
0.01%. Sensor resistance was continuously acquired during the experiments. Sensing
experiments were continuously performed using subsequent exposure cycles (see SOI, section
Features extraction. For VOC analysis, three parameters are extracted from each sensor
response: (i) the normalized change of sensor resistance at the middle of the exposure (S1);
(ii) the normalized change of sensor resistance at the end of the exposure (S2); and (iii)
the area under the response curve (S3). S1 and S2 are calculated with regard to the value of
sensor resistance prior to the exposure. For breath analysis, two parameters are extracted
from each sensor response (either to the control VOC or to each release of breath sample):
(i) the normalized change of sensors resistance soon after the exposure (S4); and (ii) the
normalized change of sensors resistance at the middle of the exposure (S5). S4 and S5 are
calculated with regard to the value of sensor resistance prior to the exposure. A
compensation and calibration process is posteriorly applied to these parameters to retain
from sensor responses only that information related to their response to the VOCs from the
breath samples. The mean values of the parameters obtained over the two successive exposures
to the same sample are then calculated.
Allocation: Non-Randomized, Intervention Model: Single Group Assignment, Masking: Single Blind (Investigator), Primary Purpose: Diagnostic
Volatile organic compounds in the exhaled breath
Identification of volatile compounds in exhaled breath that differentiate individuals with MS from healthy individuals and from individuals with other autoimmune and neurological diseases
Ariel Miller, MD PhD
Multiple Sclerosis Center Carmel Medical Center
Israel: Ministry of Health