Detection of Airborne Asbestos by Fluorescent-labeled protein probe - PowerPoint PPT Presentation

NEMC2011(Seattle) Aug. 18, 2011 Detection of Airborne Asbestos by Fluorescent-labeled protein probe and its Application to quick Monitoring *Akio Kuroda, Takenori Ishida, Asbestos Maxym Alexandrov, Tomoki Nishimura Hiroshima University, Japan Fluorescent microscopy

Asbestos: silicate mineral fiber serpentine amphibole chrysotile crocidolite amosite （ Mg 6 Si 4 O 10 (OH) 8 ） (Na 2 (Fe 3+ ) 2 (Fe 2+ ) 3 Si 8 O 22 (OH) 2 ) ((Fe, Mg) 7 Si 8 O 22 (OH) 2 )

Asbestos: widely used in construction materials Slate roof Fire retardant Dry wall (Gypsum) Heat insulator 東日本大震災 NEVER 特設ページより

Asbestos: lung cancer and mesothelioma Johnes, IARC Sci Publ. 1980;(30):637-53.1980 30 - 40 years latent period mesothelioma Asbestos exposure 1950 1940 1960 1970 1980 Japan shock 2005 This has come largely as a shock to the Japanese public in 2005. Not all those deaths were workers; many of the deaths were people who lived near factories including family members of workers.

Amount of asbesots in USA and Japan Asbestos (ton) 37 years 750,000 USA 5 million tons of asbestos are 500,000 remained in Japan JAPAN 250,000 32 years 0

Worldwide trends in the mesothelioma Robinson and Lake, N Engl J Med 2005;353:1591-1603.

Why rapid detection method of asbestos is required?

Conventional method for airborne asbestos Membrane filter asbestos Treat with acetone vapor (transparency) Phase contrast microscopy (PCM) Pump

Phase contrast microscopy (PCM)

Asbestos monitoring (Ministry of Environment) Total fiber concentration 【 Phase contrast microscopy 】 under PCM Less than More than 1 fiber /L 1 fiber /L OK Asbestos 【 Electron microscopy wit EDX 】 or not ? under EDX electron microscopy Time consuming and expensive

Asbestos risk at demolition site Past Asbestos factory At present Demolition site Asbestos risk at demolition site  More than 100 million tons of materials containing asbestos will be dumped until 2035.  Demolition will be completed within a couple of days  Rapid detection method of asbestos is required

Airborne asbestos from earthquake debris ？ 震災アスベスト Tagajyo city, Miyagi Pref.

Quick detection of asbestos Fluorescent Asbestos-binding protein asbestos

Asbestos-binding proteins

Asbestos-binding protein Protein sources Mouse lung Escherichia coli (kDa) Proteins 116 Asbestos 66 45 OmpC OmpA actin 31 DksA 22 HlpA YgiW 14 6 Asbestos- binding protein ！

DksA: chrysotile-binding protein Enzyme asbestos detection DksA Alkaline phosphatase (AP) purple DksA Asbester TM Substrate Kd = 3.5 nM Asbestos (Chrysotile) Asbestos (Chrysotile) 0 0.005 0.01 0.05 0.1 0.5 1 (mg)

Chrysotile detection in the materials 1.Construction materials Example + Asbester TM Dry wall (asbestos or non-asbestos) 2.Centrifuge and remove supernatant ペレット 3.Extraction of Asbester TM 5.Addition A 600 of substrate 4.Transfer supernatant to a new tube Chrysotile content (%)

Specificity of Asbester (DksA-AP fusion) Indistinguishable Materials Composition Bind by X-ray diffraction Chrysotile Mg 6 Si 4 O 10 (OH) 8 + Antigorite Mg 6 Si 4 O 10 (OH) 8 - Talc Mg 3 Si 4 O 10 (OH) 2 - Amosite (Mg,Fe) 7 Si 8 O 22 (OH) 2 - NaFe +3 2 Fe +2 Crosidolite 3 Si 8 O 22 (OH) 2 - Glass wool CaO-P 2 O 5 -SiO 2 -Al 2 O 3 - Silica SiO 2 - Titanium oxide TiO 2 - Silicon carbide SiC - Magnesium hydro Mg(OH) 2 -(-/+) Cement SiO 2 , CaO, etc - Rockwool SiO 2 , CaO, etc - Dry wall CaSO 4 -

Tosaka, et al., Langmuir 2010, 26(12), 9950–9955

How does DksA recognize asbestos? DksA Chrysotile 5.5 nm + + + + Mg-OH + + 2-3 nm + + + + - - - - Chrysotile Antigorite SiO 2 Mg 6 Si 4 O 10 (OH) 8 Mg 6 Si 4 O 10 (OH) 8

HNS: amphibole asbestos-binding protein Binding 137 1 Amphibole asbestos HNS Wollastonite Aluminum silicate Titanium oxide Silicon carbide Amphibole asbestos Wollastonite Amphibole Silicon carbide asbestos Amphibole asbestos (Amosite, Silicon carbide Crocidolite, etc) Asbestos (amphibole asbestos)-binding region

Specificity Fiber DksA HNS (modified) Chrysotile Bound Crocidolite Bound Amosite Bound Asbestos Anthophyllite Bound Tremolite Bound Actinolite Bound Glass wool Fine glass fiber Rockwool Fire proof fiber (RF1) Non- Fire proof fiber (RF2) Aluminum silicate fiber asbestos Titanium potassium Slightly bound Silicon carbide whisker Bound Bound Titanium oxide whisker Wollastonite

Detection of asbestos under fluorescence microscopy

Fluorescent microscopy (FM)

Modification of protein with fluorescence Fluorescein 491nm Fluorescent molecule Asbestos- binding 521nm protein Asbestos

Detection of airborne asbestos under FM asbestos Filter membrane One drop of fluorescent- Transparency label protein Conven New -tional FM PCM

FM SEM (electron microscopy) (same field) EDX Chrysotile

SEM FM 30 nm single chrysotile fibril was detected under FM

Double staining of asbestos HNS- ＦＩＴＣ（ green ） DksA- Ｃｙ３（ red ）

Combination of phase-contrast and fluorescence microscopy (PCM-FM fusion)

PCM

Fluorescent microscopy Light for FM Light for PCM

①② ③ ④

①② ③ ④

① non-asbestos ② asbestos ③ asbestos ④ non-asbestos

Detection of Airborne Asbestos by Fluorescent-labeled protein probe and its Application to quick Monitoring 1. We discovered asbestos-binding proteins. 2. We developed a fluorescence microscopy-based method for selective and highly sensitive detection of two different types of asbestos. 3. The diameter of the thinnest asbestos fibers visualized under fluorescence microscopy was 30-35 nm. 4. Then we proposed PCM and FM fusion analysis. 5. This method could be used for on-site quick monitoring of airborne asbestos, for example, during demolition work.