Analytical and Bioanalytical Chemistry
Electronic Supplementary Material
Hemoglobin assay for validation and quality control of medical device
reprocessing
Justin Frey, Allan Guan, Zhenyu Li, Steven Turtil, K. Scott Phillips
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Table of Contents
1. SUPPLEMENTARY METHODS
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Fluorescence quantitation of hemoglobin directly on samples in microplate
Page 3
MicroBCA quantitation of hemoglobin directly on samples in microplate
2. SUPPLEMENTARY FIGURES
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S1a
Direct detection of labeled hemoglobin on silicone by
fluorescence
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S1b
Response range used for LOD/LOQ calculation
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S2a
Direct detection of hemoglobin by microBCA assay
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S2b
Comparison of microBCA sensitivity for hemoglobin and BSA
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S3
Kinetics of reflectivity response (570nm)
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S4
Extraction on different medical device materials
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S5
Soiled silicone sample images
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1. SUPPLEMENTARY METHODS
Fluorescence quantitation of hemoglobin directly on samples in microplate. Fluorescently
labeled hemoglobin was prepared following the protein labeling procedure of [22]. Known
amounts (25-1000ng) of labeled hemoglobin were spotted on 6mm silicone coupons in the wells
of the microplate. The samples were dried, 100µL of DI water was added to each well, and
fluorescence was quantified using a fluorescence microplate reader (Tecan Infinite M1000, ex:
490nm, em: 520nm). Testing of extracted surfaces (unknowns) for residual soil was performed
with this same procedure.
MicroBCA quantitation of hemoglobin directly on samples in microplate. Known amounts
of hemoglobin (0-10µg) were spotted onto 6mm silicone coupons in microplate wells. The
samples were dried and then 100µL BCA working solution was pipetted into each well, along
with 50µL PBS. The plate was sealed with an adhesive cover and incubated for 15min at 60°C in
90% relative humidity. After incubation, the liquid phase of each well was transferred into a
clear bottom 96-well plate and absorbance was measured at 562 nm.
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2. SUPPLEMENTARY FIGURES
Fig. S1 Direct detection of labeled hemoglobin on 6mm silicone coupons by fluorescence. a)
Calibration showing linear response from 0-0.5µg
0 0.5µg hemoglobin. b) Data used for LOD/LOQ
calculation from 0-2.5ng
2.5ng hemoglobin. Dotted lines show linear fit and 90% confidence limits
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Fig. S2 a) microBCA assay calibration showing linear response from 0-10µg
0 10µg hemoglobin. b)
Comparison of microBCA assay sensitivity for hemoglobin and albumin. Open circles:
hemoglobin; Closed circles: albumin. Dotted lines represent linear fit
f
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Fig. S3 a) Evolution of TMB test strip 570nm reflectivity response over time for 0, 37, 370, and
3700ng hemoglobin. All times are increased by the time between spotting the strip and the first
instrument
trument measurement (about 30s)
Fig. S4 Reflectivity assay quantitation
uantitation of hemoglobin soils extracted from several sample
coupon materials: a) silicone; b) Teflon; c) stainless steel
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Fig. S5 Top:: dried soil released from the silicone samples without dissolving. Bottom: curvature
of silicone samples (left) induced by surface tension upon drying of soil. Relative lack of surface
tension upon drying of pure hemoglobin (right)
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