F l u s i Te x Fluorescence sensing integrated into medical textiles Markus Bannwarth
Current Wound Monitoring
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
Frost and Sullivan, Medtech insights 2009 $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
Working Principle
Non-invasive TOF camera
One fluorescence signal per sensing parameter per wound area
Sensing layer
Wound
pH, metabolites, oxygen,‌
The Team
Dr. Luciano Boesel
Sensing chemistry and matrix development Dr. Michael Richter
Enzyme engineering and coupling
Fluorescent lifetime camera development
Dr. Stefano Cattaneo
Prof. Bradley Nelson
Coating/microfabrication of wound pad Prof. Brigitte von Rechenberg
In vivo evaluation of wound pad
The Matrix Biocompatibility
Facile Processing
Alginate or agarose
Modification On the market
pH Sensing
Non-invasive TOF camera
One fluorescence signal per sensing parameter per wound area
Sensing layer
Wound
pH
pH Sensing Healing Chronic process wound acute wound
pH-values pH-valuesofofwound wound
88
77 Injury
Injury
66
55
Acute phase
Inflammation
ChronicGranulation phase
Time Time
Dargaville, T. R.; Farrugia, B. L.; Broadbent, J. A.; Pace, S.; Upton, Z.; Voelcker, N. H., Biosens. Bioelectron. 2013, 41, 30-42.
Spontaneous reepithelisation
Fluorescence intensity / a.u.
Fluorescent pH-marker pH = 8 pH = 7.7 pH = 7.3 pH = 7 pH = 6.7 pH = 6.3 pH = 6 pH = 5.5 pH = 5
12 10 8 6 4 2 0 620
640
660
680
700
720
740
pka ≈ 7
Fluorescence Intensity / a.u.
Wavelength / nm 120 100 80 60 40 20 0 5
5.5
6
6.5
pH
7
7.5
8
Biomarker Sensing
Non-invasive TOF camera
One fluorescence signal per sensing parameter per wound area
Sensing layer
Wound
Metabolites, Enzymes,..
Biomarker Sensing
material proteins and enzymes E
E metabolites and enzymes
Several detection spots on the pad (microfabricated): • Various metabolites / enzymes • pH • Oxygen • etc.
Biomarker Sensing Metabolite/compound
detection
Calcium
Cameleon
A
Bicarbonate
Bicarbonate-dependent
A
Iron
superoxide dismutase Ferritin
A
Glucose
Fluorescent glucose
A
binding protein
B
Uric acid
Glucose oxidase Urate oxidase
B
Lactate
Xanthine oxidase Lactate oxidase
B B
Lactate monooxygenase
B
Histamine
Diamine oxidase
B
Bilirubin
Bilirubin oxidase
B
Cholesterol
Cholesterol oxidase
B
Amino acids
Amino acid oxidases
B
Lactate dehydrogenase
Resazurin
C
Alkaline phosphatase
Fluorescein diphosphate
C
Metalloproteases
Fluorescent fusion
D
Neutrophil elastase
protein Fluorescent fusion
D
protein
metabolite sensing
A Intrinsic protein fluorescence
B Coupled enzyme assay
enzyme sensing
C Commercial fluorescent substrate E
D Designed fluorescent substrates E
Protease Sensing S2 S1
FRET
S0
donor
528 nm
YFP fluorescence
S1
485 nm
CFP fluorescence
YFP: 位em = 528 nm
absorbance
CFP: 位ex = 428 nm 位em = 485 nm
S0
acceptor
485 nm
528 nm
200 180
before cleavage after cleavage
160 140 120 100 80 60 40 fluorescence in
20 0
460
480
500
520
540
位 (nm)
560
580
600
Metabolite Sensing
Lactate oxidase
Lactate, glucose,‌
Oxygen Sensing
Non-invasive TOF camera
One fluorescence signal per sensing parameter per wound area
Sensing layer
Wound
Oxygen
Oxygen Sensor System
Excitation band
Emission band
PtOEP
Quenching through
O2
Oxygen Sensor System
Correlate lifetime change with amount of oxygen
Oxygen Sensor Fabrication
Nanopillar formation
Electrodeposition
2 Îźm
The Lifetime Camera
Non-invasive TOF camera
One fluorescence signal per sensing parameter per wound area
Sensing layer
Wound
pH, metabolites, oxygen,‌
The Setup
Objective Pinholes
Focusing lens Diffuser
Mirrors
Laser diode 450 nm
First Sample Measurements
In Vivo Evaluation
Non-invasive TOF camera
One fluorescence signal per sensing parameter per wound area
Sensing layer
Wound
pH, metabolites, oxygen,‌
In Vivo Evaluation Standardized deep wounds Wound parameters recorded until closure (pO2, pH, metabolites) Wound healing semi-quantitative evaluation and tracking (Epithelialisation, wound surface area, granulation tissue)
Infected deep wounds
Inoculation with Staphylococcus Aureus or Pseudomonas Aeruginosa Tracking infection progress (sampling, swabs) Sensing wound parameters (pH, pO2, metabolites, glucose) Antibiotic therapy after infection establishment Tracking wound healing (Semi-quantitatively) and recording parameters Comparing infected and non-infected wound environments Correlating wound healing and monitored parameters
WP5 Sensing: in vitro and in vivo
5.1 Testing wound parameter monitoring (in vitro)
5.2
Design of in vivo analysis
Year 2
Year 3
Achievements and Future Efforts Achievements •
Development of: ● pH sensor ● biomarker sensors ● oxygen sensor
•
Coupling or integration of the sensor systems to/into the coating matrix
•
Development of optical setup for fluorescence lifetime imaging in the nanosecond range
Future Efforts •
Microfabrication of the functional matrix on a wound pad
•
Lifetime imaging of pH values, biomarker concentrations and oxygen content with the lifetime camera
•
In vitro and in vivo analysis of the monitor pads
Prof. Bradley Nelson Dr. Selman Sakar Dr. Chen Xiangzhong
Dr. Luciano Boesel Dr. Markus Bannwarth
Thanks! Dr. Stefano Cattaneo Christoph Hofer
Dr. Michael Richter Dr. Dagmara Jankowska Dr. Greta Faccio
Prof. Brigitte von Rechenberg Dr. Salim Darwiche