F lusiTex

Fluorescence sensing integrated into medical textiles

 Current Wound Monitoring

Wound healing involves a complex series of biochemical events. Though our

understanding of the healing process has improved, we still use low tech dressings.

Current Methods
Full or partial removal of wound pad
Visual Observation
Skin Irritation
Increased chances of infection
Only qualitative information
Collecting biochemical information
Highly invasive
Expensive
 The Business Case

$12bn worldwide market aimed solely at wound care

Wound monitoring consists less than 1% of this
industry
6 weeks treatment of a chronical wound
35,000 CHF

Benefits in sensing the wound include

Reduce hospitalization time
Provide better treatment
Prevents amputation
 Working Principle

We want to build a wound healing system that can monitor the healing
process and providing quantitative data.

Non-invasive
TOF camera

One fluorescence signal

per sensing parameter
per wound area

Sensing layer
pH,
metabolites,
oxygen,
Wound
Pad Structure

material
proteins and enzymes
E
E
metabolites and enzymes,
H + , O2

Several detection spots on the pad (microfabricated):

pH
Oxygen
Various metabolites / enzymes
etc.
 The Team

Dr. Luciano Boesel

Sensing chemistry and matrix development

Dr. Greta Faccio

Enzyme engineering and coupling

Dr. Stefano Cattaneo

Fluorescent lifetime camera development

Prof. Bradley Nelson

Coating/microfabrication of wound pad

Prof. Brigitte von Rechenberg

In vivo evaluation of wound pad
 Wound parameters

pH Glucose
Depends on time course
Bio-marker to assess the
and wound-stage.
Normal healing -> pH Oxygen wound status.
Infected wounds:
increases during
granulation and concentration of around
decreases afterwards to 0.31.0 mM.
reach a value of 4-6. 1) Controls cell Non-infected wounds:
Impaired healing -> proliferation concentration of around
2) Anti-bacterial 5.07.6 mM.
the pH oscillates between
activities
pH 7-8
3) Creating new blood
vessels
(angiogenesis),
which is essential to
the growth and
survival of repaired
The sensors

Non-invasive
TOF camera

One fluorescence signal

per sensing parameter
per wound area

Sensing layer
Parameter
Wound
 Fluorescent pH-marker

5(6)-Carboxynaphthofluorescein Fluorescence intensity / a.u.

700
pH = 8
600 pH =
7.7
500 pH =
7.3
400 pH = 7
pH =
300 6.7
200 pH =
6.3
100 pH = 6
pH =
0 5.5
620 720 pH = 5

Wavelength / nm

700
Fluorescence Intensity / a.u.
600
500
400
300
200
100
0
5 5.5 6 6.5 7 7.5 8
pH
 Enzimatic glucose sensing

Principle of glucose sensor functioning Storage and stability of funtionalized matrix

Glucose oxidase HRP, 30C

Glucose + O2 Gluconolactone + H2O2

Horseradish peroxidase

H2DCF + H2O2 DCF + H2O

Sensors after immobilization on alginate

Reaction with glucose solutions

GOx, 30C

glucose 0 0.02 0.5 2 5 10 [mM]

Non-enzymatic glucose sensing
 Oxygen Sensors Fabrication

Ethyl cellulose

PtOEP
1 cm

Alginate gel
Long Luminescence decay times
Luminescence brightness of the probe
Can be detected @ low
concentration
Solubility in the polymeric matrices
Chemical stability and photostability
Commercial or synthetic availability
 Oxygen Sensor System

In the presence of oxygen the lifetime of PtOEP is quenched

Advantage of fluorescence lifetime vs intensity:

independent of dye concentration, excitation light fluctuations, etc.
 Controlling sensors positions

Currently we are also working to precisely position the sensors in the gel
matrix.
Antinode

Top view PZT Reflector

Pressure
Node node

/2
UV excitation

Sensors

Side view

PZT 15
m
The Lifetime Camera

Non-invasive
TOF camera

One fluorescence signal

per sensing parameter
per wound area

Sensing layer
pH,
metabolites,
oxygen,
Wound
 Fluorescence lifetime imaging

Fluorescence lifetime: the time at which the

intensity has decayed to 1/e of the original
value.
FlusiTex FLIM prototype:
Compact, cost-effective,
simple
Standalone system with
integrated light source
Suitable for decays from nano-
to microseconds
Conventional FLIM
setup:
Bulky
Complex
Expensive
 MATLAB GUI Software

Phase-resolved
intensity images

Phase shift Fluorescence

Lifetime (ns)
 FLIM Oxygen Measurements

Ocean Optics RedEye

0 50 % Oxygen Sensing Patch 100
% %

3000

2500
Lifetime [ns]

2000

1500

1000

500
0 10 20 30 40 50 60 70 80 90 100
Oxygen O2 Concentration [%]
Testing in Animal Models
Lactate
pH
Glucose
O2
Pig
Aim 1: testing fresh wounds
Feasibility for wound sensor
pad
Feasibility of parameters
Wound model
Aim 1: Standardized wound on
dorsum of animal; size 4 x 4cm2;
comparison wound pad to
standard wound bandage,
histology of wound healing
Pig
Aim 2: testing infected wounds
Feasibility of primary closure
Monitoring over time
Wound model
Aim 2: Standardized wound on
tibia directly above bone; size 4 x
4cm2; infection with S. aureus;
comparison wound pad to
standard wound bandage
https://www.eqalix.com/technology-and-pip
 WP5 : In Vivo Evaluation

Standardized deep wounds

Dorsum of mini-pigs
4 deep wounds, ~16cm2 each (<1%TBSA)
Standardized depth with dermatome

Treatment groups:
Group 1: Standard of Care
Group 2: Flusitex without molecular sensors (scaffold only)
4 cm
Group 3: Flusitex dressing

4 cm
Wound area
Imaging area
6mm diameter
 WP5 : In Vivo Evaluation

Treatment and tracking timeline

Endpoint: Wound closure or Day 21
Few dressing changes. Daily imaging.
Imaging border zones and center of each wound dressing
Wound imaging parameters (pO2, pH, metabolites)
Wound healing (Epithelialisation, wound surface area, granulation tissue)

Proof of concept: Infected wounds

Inoculation with Pseudomonas Aeruginosa at Day 2
Tracking of infection parameters (local swabs, systemic markers)
Antibiotic therapy after infection establishment
Same treatment distribution as non-infected animals
Achievements and Future Efforts

Achievements

Development of:
o pH sensor
o biomarker sensors
o oxygen sensor

Coupling or integration of the sensor systems to/into the coating matrix

Development of hand-held prototype for fluorescence lifetime imaging in

the nanosecond range

Future Efforts

Microfabrication of the functional matrix on a wound pad

In vitro and in vivo analysis of the monitor pads

Outcome - Gateways

FlusiGate
Flawa, Kenzen, Schoeller,
TheranOptics:
Project to develop a ratiometric
sensor for wound monitoring
based on pH responsive dyes
and a point fluorescent detector

FlusiSafe
U-NICA Solutions AG : project to
investigate the potential of the
FLIM imager for anti-
counterfeiting & product
protection
 Prof. Bradley Nelson
Dr. Luciano Boesel
Dr. Daniel Ahmed
Dr. Claudio Toncelli
Dr. Chen Xiangzhong
Dr. Alina Osypova
Dr. Guido Panzarasa

Thanks!
Dr. Stefano Cattaneo
Dr. Greta Faccio Mr. Christoph Hofer
Dr. Dagmara Jankowska Dr. Hans-Rudolf Graf
Dr. Bernhard Schaffer

Prof. Brigitte von Rechenberg

Dr. Salim Darwiche