US 20090305319A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2009/0305319 A1 Baudenbacher et al. (43) Pub. Date: Dec. 10, 2009 (54) APPARATUS AND METHODS FOR (21) Appl. No.: 10/213,049 MONITORING THE STATUS OF A METABOLICALLY ACTIVE CELL (22) Filed: Aug. 6, 2002 (76) Inventors: Franz J. Baudenbacher, Franklin, Related U.S. Application Data TN (US); John P. Wikswo, Brentwood, TN (US); R. Robert (60) Provisional application No. 60/310,652, filed on Aug. Balcarcel, Nashville, TN (US); 6, 2001. David Cliffel, Nashville, TN (US); Sven Eklund, Antioch, TN (US); Publication Classification Jonathan Mark Gilligan, (51) Int. Cl. Nashville, TN (US); Owen CI2O 1/02 (2006.01) McGuinness, Brentwood, TN (US); CI2M 3/00 (2006.01) Todd Monroe, Baton Rouge, LA (US); Ales Prokop, Nashville, TN (52) U.S. Cl. ........................................ 435/29: 435/287.1 (US); Mark Andrew Stremler, Franklin, TN (US); Andreas (57) ABSTRACT Augustinus Werdich, Nashville, TN (US) An apparatus and methods for monitoring the status of a cell that consumes oxygen. In one embodiment of the present Correspondence Address: invention, the method includes the steps of confining the cell MORRIS MANNING MARTIN LLP in a sensing Volume, measuring dynamically intracellular or 3343 PEACHTREE ROAD, NE, 1600 ATLANTA extracellular signaling of the cell, and determining the status FINANCIAL CENTER of the cell from the measured intracellular or extracellular ATLANTA, GA 30326 (US) signaling of the cell. Patent Application Publication Dec. 10, 2009 Sheet 1 of 41 US 2009/0305319 A1 108 Biological Ya aga stimulator r c X Generator 109 i 112 1 10 2. System i models 111 114 113 116 Fig. 1 Patent Application Publication Dec. 10, 2009 Sheet 2 of 41 US 2009/0305319 A1 /7 I (X) 20 273 283 L 271 28 1 Fig. 2B Patent Application Publication Dec. 10, 2009 Sheet 3 of 41 US 2009/0305319 A1 Fig. 3B 309 311 -211 X---------X% - H 2 %t YZZ 31 O S&2% O X) % 4%1. 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SNR 4 ev, SNR 4 9,R EEEv. SNRs V Ecy. SNRs liee. SNRG O evsnra NewsNR4Test > evisit New SNR 2 le. SNR > TestNR6 Nev. SNRs Lev. SR 6 New SNR4 ev. SNR 4 -- - - TO OC CYD. O A. Dow. At Co 7. 9 O 5 Fig. 9 Patent Application Publication Dec. 10, 2009 Sheet 14 of 41 US 2009/0305319 A1 296 996 313996V6 Patent Application Publication Dec. 10, 2009 Sheet 15 of 41 US 2009/0305319 A1 1 OO1 1 OO2 1003 1004 5 2, 2,5 3.0 3,5 4.0 Time in hours Fig. 10 Patent Application Publication Dec. 10, 2009 Sheet 16 of 41 US 2009/0305319 A1 Fig. 1 1A 1166 1162 1151 SS 1129 EŠirl 6. Fig. 11B Fig. 11C 1128 1151 Patent Application Publication Dec. 10, 2009 Sheet 17 of 41 US 2009/0305319 A1 1200 B t 1204 12O6 1215 A 1209 /N 1 1202 12O6 12O7 Fig. 12B Patent Application Publication Dec. 10, 2009 Sheet 18 of 41 US 2009/0305319 A1 1320 1311 Fig. 13A 1309 131 O Fig. 13B 1308 Fig. 13C Patent Application Publication Dec. 10, 2009 Sheet 19 of 41 US 2009/0305319 A1 1424 Patent Application Publication Dec. 10, 2009 Sheet 20 of 41 US 2009/0305319 A1 1503 Fig. 15 Patent Application Publication Dec. 10, 2009 Sheet 21 of 41 US 2009/0305319 A1 16O2 1612 1606 1605 1611 N. Oxidative -1 Phosphorylation Fig. 16 Patent Application Publication Dec. 10, 2009 Sheet 22 of 41 US 2009/0305319 A1 1704 1705 O 60 Time (min) Fig. 17A infuse Cyanide 1701 1 7 O 5 Time (min) Fig. 17B Patent Application Publication Dec. 10, 2009 Sheet 23 of 41 US 2009/0305319 A1 Macrophage - Infuse - endotoxin Time (min) Fig. 18A Hepatocyte 1811 - Infuse -: endotoxin Time (min) Fig. 18B Patent Application Publication Dec. 10, 2009 Sheet 24 of 41 US 2009/0305319 A1 No phenobarbital preexposure 0 Time (min) 60 Fig.19A Phenobarbital preexposure :- infuse VX 191 0 Time (min) 60 Fig.19B Patent Application Publication Dec. 10, 2009 Sheet 25 of 41 US 2009/0305319 A1 2OO1 1 mM 1 OnM 2002 Glucose Glucose G.) 9 9 CO O 5 C s of D CS2 C CD O. d) .9) C E. of 9. O A) 2 CD 0 Time (min) 60 Fig. 20 Patent Application Publication Dec. 10, 2009 Sheet 26 of 41 US 2009/0305319 A1 2101 21 O2 ACtion potential Time Fig. 21 Patent Application Publication Dec. 10, 2009 Sheet 27 of 41 US 2009/0305319 A1 2200 2203 2204 22O1 POtOCO Definition Zoom on Specific- - - Protocol Step St 5E Feature Extraction Lib Bif Broad 2251 Aria O is: Data Point Lib ) E) E-2205 225 GOs Ye 2253 Viral S5 EEEEEE FF-Sun is br- 259 SE OOOO b: Dif stop- 2206 2257 Neuro 4 -4, 2ASS gs oscin funct OO) 4 0 OOOOOO gS. \ CCHist PotocoSelect PotocolSelect PotocoSelect Data Path 1 Path 2 Path 3 22O2 V w 2208 2209 22O7 Fig. 22 Patent Application Publication Dec. 10, 2009 Sheet 28 of 41 US 2009/0305319 A1 230 2351 2301 p 23O3 J 2304 / Feature Space of-7 -- - - - - O2C PGA &: six-is: GK) Classify r - - - - 2305 Confidence Factor: 20% r Metabolic Model-F3) Fit to Feature Space 2nd Order PCA Classify 2313 2314 Patent Application Publication Dec. 10, 2009 Sheet 29 of 41 US 2009/0305319 A1 2400 2405 2404 Celle Mac?ophage Liver Broad Sensors: Heat Oxygen. Free Radca? NADPH.GlukconegenSIS Spectrum Activity feat Extraction:Slope and AU 24O6 24O1 Expected. Both NADPHOvergent Response ity REF: ABLE xxx 24O7 Computed Robustness: 19% 24.08 Gram-Negative Celtine: Macophage-cD4) Bacteria ASSa Sensors: Heat. Oxygen. Fee RadcasADPH 2402 feat Extraction:Siope and AJC Expected: DSCIn on Activation Robustness Factor:40% Path/No Path Celine: intestical Celine Bacteria ASSa Sensots: Heat. Oxygen. Free Radical NADPH 2403 Feat Extraction:Slope and AUC Expected: Discriminor Mel Leyes Robustness Factor:80, Patent Application Publication Dec. 10, 2009 Sheet 30 of 41 US 2009/0305319 A1 2500 2573 Patent Application Publication Dec. 10, 2009 Sheet 31 of 41 US 2009/0305319 A1 500 or -- pt adding acid 1 v mV pass 1 -O-ph adding base2 v mV pass2 -o- pH adding acid3 v nV pass3 -o- pH adding base4 v mV passa 450 - i 8s 400 - IRO, pH g : Electrode 350 - 3. 300 - s s Eo 250 200 - 50 - Trr ninth wr - - - - - - - - - - - - - - - - - - 4 5 6 7 8 9 pH Patent Application Publication Dec. 10, 2009 Sheet 32 of 41 US 2009/0305319 A1 2702 se O - see - - - - - - -- N N - Y - N. N. v Fig.27A 2708 Patent Application Publication Dec. 10, 2009 Sheet 33 of 41 US 2009/0305319 A1 28O1 a. 2808 28 O2 N . 28 05 B -3% s B --isS 2S1s16-- 28 O4 > 2809 - Fig. 28B Patent Application Publication Dec. 10, 2009 Sheet 34 of 41 US 2009/0305319 A1 2808 2804 2801 Patent Application Publication Dec. 10, 2009 Sheet 35 of 41 US 2009/0305319 A1 Fig. 29B Patent Application Publication Dec. 10, 2009 Sheet 36 of 41 US 2009/0305319 A1 2912 2905 29O.