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Future Genomics 2023
•March 22 - 23, 2023 Kimpton Marlowe Hotel, Cambridge, Mass
March 22, 2023
PROTEASE DETECTION ON BLOOD SPOT CARDS FOR FUTURE
COMPANION DIAGNOSTICS
Michael J Heller
Geert Schmid-Schoenbein, Rebekah White, Jayanth Shankara Narayanan, Jorge de la Torre, Sahand Salari-Namin, Heather A.
Farris, Nicole Nolan, Augusta Modestino, Ben Sarno, Jean Lewis, Sean Hamilton, Kyle Gustafson, Paul Mills and Thomas Kipps -
UCSD
Dr. Andrew Senyei - Enterprise Partners
University of California San Diego
Depts Bioengineering and Nanoengineering & Moores Cancer Center
La Jolla, CA 92093 mheller@ucsd.edu
Proteases in Cancer and Other Diseases
In the case of Pancreatic (PDAC) and Colon Cancer the digestive proteases Trypsin,
Chymotrypsin Elastase and other degradative enzymes are often found in the blood at
elevated levels.
Digestive proteases are also
agents of further pathology
• Activation of other proteases
• Cancer progression & metastasis
• Cell receptor cleavage
• Widespread tissue damage
• Contributes to disease morbidity
PDAC – Activation of pancreatic
proteases and leakage into the blood
stream
Colon Cancer – Leakage of the active
digestive enzymes from the gut into
the blood stream – Leaky Gut
Syndrome/Autodigestion
12 Blood Tests to Date
Handheld Prototype
Devices
Protease Biomarker Detection
Liquid Biopsy Assays, Devices, Early Publications & US Patents (MJ Heller & G Schmid-Schoenbein – UCSD)
Inflammatory
Disorders
Cancer
Infectious
Diseases
Cardiovascular
Disorders Sepsis
Coagulation
Disorders
• Proteases as new biomarker for cancer
• Human studies type 2 diabetes, shock, hypertension and coagulation
• Pre-clinical studies in mice/rats for shock and heart failure
• Scientific Publications
• Verification of Autodigestion Hypothesis
• 3 Issued US Patents and two new patent application (UCSD)
Rapid Protease Activity Assay in Whole Blood, Plasma or Serum
“Greatly enhanced sensitivity – one protease produces many fluorescent signals “
+
Blood Charge-Changing Fluorescent
Peptide Substrate
+
Negative cleaved
fragments
Positive cleaved
fluorescent fragments
30-60 Minute Reaction of Protease and Net Negative
Charged- Changing Fluorescent Peptide Substrates
Protease
Substrate
Anal. Chem. 2010, 82, 8251–8258
Fluorescent Charge-Changing Peptide Substrates
Trypsin Specific Charge-Changing Peptide Substrate
Acetyl-N-Asp-Gly-Asp-Ala-Gly-Arg/Ala-Gly-Ala-Gly-Lys-BFL-C-Amide
Cleavage Site Fluorophore
(-) (-) (+)
+H3N
+H3N
COOH-
-
COOH-
-
-
Fast and Simple Electrophoretic Separation
Load blood & peptide sample mixture
into electrophoretic PAGE gel device*
Gel
Wells Samples
Gel
Well Sample
* A variety of microgel electrophoretic
devices have also been used
+H3N
+H3N
+H3N
-
-
COOH-
- COOH-
-
Fluorescent Detection after Electrophoretic Separation
Electrophoretic
Separation
Blood, plasma proteins
and uncleaved
negatively charged
fluorescent peptide
substrates are
effectively removed
“Sample Preparation”
Cleaved positively
charged fluorescent
peptide product
fragments migrate into
gel for “fluorescent
detection”
(+) charges fluorescent peptides
Imaging and Results
Advantages
• Detects protease activity
• Enhanced sensitivity due to amplified fluorescent signal
• Uses only a single drop (5ul-10ul) of whole blood, plasma or
serum
• Simple mini/micro gel electrophoretic formats
• Provides 30-60 minute “Sample to Answer “
Fluorescent band intensity indicates level of protease activity
Type II Diabetes - Published results for metabolic disease and co-morbidities that accompany type II
Diabetes, evidence for elevated trypsin, chymotrypsin, elastase and MMP2/9 activity and cleavage of
insulin receptors.
Modestino AE, Skowronski EA, Pruitt C, Taub PR, Herbst K, Schmid-Schönbein GW, Heller MJ & Mills PJ; “Elevated Resting and
Postprandial Digestive Proteolytic Activity in Peripheral Blood of Individuals With Type-2 Diabetes Mellitus, With Uncontrolled
Cleavage of Insulin Receptors”, Journal of the American College of Nutrition; 2019; https://doi.org/10.1080/07315724.2018.1545611
MMP Inhibitor - Published results on efficacy of a metalloproteinase protease inhibitor in
spinal cord injured dogs
Levine JM, Cohen ND, Heller MJ, Fajt VR, Levine GJ, Kerwin SC, Trivedi AA, Fandel TM, Werb W, Modestino A and
Noble-Haeusslein LJ, “Efficacy of a Metalloproteinase Inhibitor in Spinal Cord Injured Dogs”, PLOS ONE, V9, Issue 5,
e96408, May 2014
Proteases in Cancer
In addition to digestive proteases in PDAC and Colon Cancer many other proteases are
elevated and appear in other Cancers
PROTEASE DETECTION ON BLOOD SPOT CARDS FOR FUTURE COMPANION DIAGNOSTICS
Multi-Omic BioMarkers: cf/ct-DNA (Mutations & Methylation); cf-RNA (mRNA, miRNA);
Exosomes and EV Protein Biomarkers; Nucleosomes and Others (Proteases???)
Are detectable levels present for early cancer stages and for different cancers ???
Apoptotic
& high molecular
weight cf/ct-DNA
Mutations and Methylation
Mitochondria,
Nucleosomes
Necrotic Cell
Debris,
Protease
Biomarker Improved
Detection
Why Proteases may be the Best Biomarkers for Early Cancer Detection?
Genomic (DNA, RNA, Epigenetics) → Exosomal Proteins Multi-Omics → Proteases ?
The Pancreas and Digestive Enzymes (Proteases, Amylases, Lipases &
Nucleases)
* The Acini cells produce proteases in an in-active form
(Trypsinogen, Chymotrypsinogen, etc., they become
activated upon entering the duodenum.
*
Pancreatic Cancer
Pancreatic ductal adenocarcinoma (PDAC) a highly aggressive lethal malignancy due to the lack of early
diagnosis and limited response to treatments (Stages I – IV)
Protease Work at Knight Cancer/CEDAR/OHSU
Many PDAC patient samples which had elevated levels of cf-DNA and Exosome Biomarkers also had
elevated levels of trypsin and chymotrypsin.
Knight Cancer/CEDAR/OHSU/UCSD - MJ Heller and A Modestino
Healthy Digestion
Low Escape of Digestive Enzymes
Normal Vascular Function
Protein & Receptor
Signaling
Autodigestion
Hypothesis
Cancer (PDAC, Colon), IBS, Crohn’s
Disease, Inflammatory Response, Diabetes,
Shock, Organ Dysfunction, Aging???
Leak of Digestive Enzymes
Cell, Tissue Vascular Failure
Protein Degradation
Receptor Cleavage
The Autodigestion Hypothesis
Leakage of Digestive Enzymes in Cancer and Other Diseases
Professor Geert Schmid-Schoenbein - UCSD Dept Bioengineering
Journal of Pancreatic Cancer Volume 6.1, 2020 DOI: 10.1089/pancan.2019.0014
Trypsin Trojan Horse Hypothesis – Does Trypsin Promote More Aggressive Pancreatic Cancer ?
A Review of the Trypsin-PAR2 Axis to Proliferation, Early Invasion, and Metastasis
Kjetil Søreide, Marcus Roalsø,and Jan Rune Aunan
Journal of Pancreatic Cancer Volume 6.1, 2020 DOI: 10.1089/pancan.2019.0014
Trypsin Trojan Horse Hypothesis – Does Trypsin Promote More Aggressive Pancreatic Cancer ?
A Review of the Trypsin-PAR2 Axis to Proliferation, Early Invasion, and Metastasis
Kjetil Søreide, Marcus Roalsø,and Jan Rune Aunan
Pancreatic Cancer Related Protease Biomarker Detection (UCSD)
Support for Trypsin Trojan Horse Hypothesis
MJ Heller, R White, G Schmid-Schoenbein, J S Narayanan
0
20
40
60
80
100
120
140
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Relative
Fluorescent
Intensity
Trypsin & Chymotrypsin RAPD Results for PDAC Patient
Plasma Samples (4ul sample volume )
021 052 055 - 021 034 062 - 021 052 055 - 021 034 062
Trypsin Chymotrypsin
0
10
20
30
40
50
60
70
80
1 2 3 4 5 6 7 8 9
Relative
Fluorescent
Intensity
MMP-2 and Cathepsin-S RAPD Results for PDAC Patient
Plasma Samples (4ul sample volume )
21 052 055 062 021 052 055 062
MMP-2 Cathepsin-S
PDAC/Protease Inhibitor Research Proposal 2022 (UCSD)
Funded – Jan 1, 2023 Start Date
Rebekah White, Michael J Heller, Geert Schmid-Schoenbein and Jayanth Shankara Narayanan
“Detection of Disease Biomarkers and Therapeutic Effectiveness from Blood Spot Card Samples”
US Provisional Patent US63/386,635
MJ Heller, G Schmid-Schoenbein, R White, J S Narayanan & A Senyei
Invention relates to the detection and analysis of biomarkers for cancer and other diseases from small amounts (~1ul-50ul) of
clinical, (blood, plasma, serum), biological and other samples spotted (deposited) and stored on Blood Spot Cards. Describes novel
procedures, processes, methods, assays, devices, systems, and reagents that eliminate the need to first extract the biomarkers from
the Blood Spot Card for subsequent analysis. Describes a novel process where the reaction of the detection reagents with the
biomarkers occurs within (In-Situ) the blood spot card matrix material itself. After which the reaction products are
electrophoretically extracted and detected. Further describes unique processes which can accelerate the in-situ reaction of the
detection reagents with the biomarkers. Such processes reduce the overall assay time and complexity for carrying out biomarker
detection and analysis using blood spot card samples.
Dried Blood Spots: Applications and
Techniques (Wiley Series on Pharmaceutical
Science and Biotechnology: Practices,
Applications and Methods) 1st Edition
by Wenkui Li (Editor), Mike S. Lee (Editor)
ISBN-13: 978-1118054697
ISBN-10: 1118054695
Blood Spot Saver Cards:
Whatman
Perkin-Elmer
Thomas Scientific
Others
Background on Blood Spot Card Applications and Techniques
Liquid Biopsy Protease Biomarker & Companion Diagnostics Using Blood Spot Cards
New Devices, Assay Methods and Reagents for Research, Clinical Lab, POC and Home Testing Scenarios
General
Research
Lab
Research Lab
Automated
Clinical Lab
Automated
Point of Care
Devices
Hand-Held Home
Care Devices
Blood Draw Tubes Finger Prick
Blood Spot Saver Card
Blood Draw Tubes
Blood Spot Saver Card
Advantages
• $$$ Cost Saving
• Time Saving
• Ease of Use
• Small Sample Size
• Blood, plasma, serum,
urine, etc.
• Longer Sample Storage
(Blood)
• Home/POC
• Enables cost effective time
course studies & diagnostics
(hours, days, weeks)
Disadvantages
• Extraction Process
Cumbersome
• Loss of Biomarker
• Sample Dilution
• Small Sample Size
Advantages and Disadvantages for Using Blood Spot Cards
Research, Clinical Lab, POC and Home Testing Scenarios
Blood Spot Card Trypsin Assay Results
Samples - 5ul buffer and 5ul blood spiked with Trypsin (~15 units) dried and stored for 72 hours
Blood spots samples extracted and placed into 4% Agarose Gel wells
0
10
20
30
40
50
60
70
80
90
1 2 3 4 5
Relative
Fluorescent
Intensity
Trypsin Assay Results Blood Spot Card Buffer
and Blood Samples
Buffer electrophoretic ally Blood electrop
Trypsin electrophoretic ally
Buffer Buffer/Trypsin Blood Blood/Trypsin
Blood Spots
Buffer Spots
Sample
Wells
Sample
Wells
4% Agarose Gel
Michael J Heller, Geert Schmid-Schoenbein, Rebekah White, Jayanth Shankara
Narayanan and Andrew Senyei
1. Place excised sample spots into
wells
2. Add specific protease
fluorescent Charging-Changing
Peptide (CCP) substrates and
buffer
3. React 30-60 minutes
(+) Control (-) Control
Excised Patient Sample Spots
Polyacrylimide
or Agarose
MiniGel
Sample Wells
Polyacrylamide PAGE
MiniGel Format
Sample Wells
For analysis excise
(cut/punch out)
spots from card
New Method for In-Situ Protease Detection on Blood Spot Card Samples
Carried Out in Vertical PAGE Gel Format
(+)
Polyacrylimide
or Agarose
MiniGel
Sample Wells
(-)
Positively (+) charged
fluorescent cleaved CCP
product fragments
+ -
Control Control
CCP substrate
cleavage by
proteases
Apply DC electric
field to separate
cleaved CCP
fragments
Spot patient blood
samples onto card
and dry
Sample Well
Uncleaved
Fluorescent
Substrate
Cleaved (+)
fluorescent
fragment
Cleaved (-)
fragment
Protease
Excised paper
spot
Negatively (-) charged fluorescent un-
cleaved CCP and blood components
(+) Control (-) Control
Excised Patient Sample Spots
Comparison of Classical and New In-Situ Procedure for Protease Detection on Blood Spot
Card Samples
Spot patient blood
samples onto card
and dry
1. Place excised sample spots into wells
2. Add specific protease fluorescent Charging-
Changing Peptide (CCP) substrates and buffer
3. React 30-60 minutes
4. Apply DC voltage for 20-30 minutes
5. Measure fluorescent signals
1. Place excised sample spots into tubes
2. Add extraction buffer
3. Masticate and extract for 30-60 minutes
4. Centrifuge and elute extracted materials
5. Place eluted samples into another tube
6. Add specific protease fluorescent Charging-Changing
Peptide (CCP) substrates and buffer
7. React 30-60 minutes
8. Place aliquots of samples into PAGE gel wells
9. Apply DC voltage for 20-30 minutes
10. Measure fluorescent signals
Positively (+) charged
fluorescent cleaved CCP
product fragments
Negatively (-) charged fluorescent
un-cleaved CCP and blood
components
Classical
Procedure New In-Situ
Procedure
1. Place excised sample spots
into wells
2. Add specific protease
fluorescent Charging-
Changing Peptide (CCP)
substrates and buffer
3. React 30-60 minutes
(+) Control (-) Control
Excised Patient Sample Spots
For analysis excise
(cut/punch out) spots
from card
Spot patient blood
samples onto card
and dry
Negatively (-)
charged
blood components
New Method for In-Situ Protease Detection on Blood Spot Card Samples
Carried Out in a Horizontal Agarose Gel Format.
Sample Well
Uncleaved
Fluorescent
Substrate
Cleaved (+)
fluorescent
fragment
Protease
Excised paper
spot
Cleaved (-)
fragment Horizontal
Agarose
MiniGel
Format
Sample
Wells
CCP substrate
cleavage by
proteases
(+) Control
(-) Control
Positively (+)
charged
fluorescent
cleaved CCP
product
fragments
Apply DC electric
field to separate
cleaved CCP
fragments
Negatively (-)
charged
fluorescent un-
cleaved CCP
(+)
(-)
In-Situ Trypsin Assay from Blood Spot Card
With Trypsin Spiked Blood and Plasma Samples
1 2 3 4 5 6 7 8 9 10 11 12
X Con (-) Con (+) X Blood (-) X Blood (+) X Plasma (-) X Plasma (+) X
White Light Image
4% Agarose Gel
Fluorescent Image
4% Agarose Gel
Sample
Wells Sample
Wells
Blood Cells and
Components
(+)
(-)
Bands from Un-cleaved Negatively Charged
(-) Fluorescent CCP Substrate
Bands from Cleaved Positively Charged (+) Fluorescent
CCP Product Fragments
IMG_4462-226-1206-Trypsin-BH223-pp32-White Light-061422
1 2 3 4 5 6 7 8 9 10 11 12
X Con (-) Con (+) X Blood (-) X Blood (+) X Plasma (-) X Plasma (+) X
IMG_4456-226-1206-Trypsin-BH223-pp32-Fluorescent-061422
IMG_4457-226-1206-Trypsin-
BH223-pp32-Blocked Fluor-
061422
0
5
10
15
20
25
30
35
40
45
50
Relative
Fluorescent
Intensity
Trypsin Spiked -BH-223-Blood & Plasma-1st-pp32-061
Con (-) Con (+) Blood (-) Blood (+) Plasma (-) Plasma (+)
In-Situ Trypsin Assay from Blood Spot Card
With Trypsin Spiked Blood and Plasma Samples
IMAGE J Quantitative Analysis of Image-4457-226-1206 and Resulting Graph
Sample
Wells
Bands from Cleaved
Positively Charged (+)
Fluorescent CCP Product
Fragments
1 2 3 4 5 6 7 8 9 10 11 12
X Con (-) Con (+) X Blood (-) X Blood (+) X Plasma (-) X Plasma (+) X
IMG_4470-226-1206-Chymotrypsin Full Gel Fluorescent Image-061422
IMG_4490-226-1206-Chymotrypsin Final Gel Image White Light-061422
In-Situ Chymotrypsin Assay from Blood Spot Card
With Chymotrypsin Spiked Blood and Plasma Samples
1 2 3 4 5 6 7 8 9 10 11 12
X Con (-) Con (+) X Blood (-) X Blood (+) X Plasma (-) X Plasma (+) X
White Light Image
4% Agarose Gel
Sample
Wells
Blood Cells and
Components
1 2 3 4 5 6 7 8 9 10 11 12
Fluorescent Image
4% Agarose Gel
Sample
Wells
Bands from Un-cleaved Negatively Charged
(-) Fluorescent CCP Substrate
Bands from Cleaved Positively Charged (+) Fluorescent
CCP Product Fragments
X Con (-) Con (+) X Blood (-) X Blood (+) X Plasma (-) X Plasma (+) X
0
5
10
15
20
25
30
1 2 3 4 5 6 7
Relative
Fluorescent
Intensity
Chart Title
IMG_4472-226-1206-
Blocked Fluorescent
Gel Image 1/2 sec
061422
In-Situ Chymotrypsin Assay on Blood Spot Card Blood and Plasma Samples -
IMAGE J Quantitative Analysis of Image-IMG_4472-226-1206 and Resulting Graph
Con (-) Con (+) Blood (-) Blood (+) Plasma (-) Plasma (+)
Sample
Wells
Bands from
Cleaved Positively
Charged (+)
Fluorescent CCP
Product Fragments
1 2 3 4 5 6 7 8 9 10 11 12
X Con (-) Con (+) X Blood (-) X Blood (+) X Plasma (-) X Plasma (+) X
PROTEASE DETECTION ON BLOOD SPOT CARDS FOR FUTURE COMPANION DIAGNOSTICS
PROTEASE DETECTION ON BLOOD SPOT CARDS FOR FUTURE COMPANION DIAGNOSTICS
Combined Protease Detection and
Protease Inhibitor/Therapeutics
Assays
Advantages
• Fast (~30-60 minutes)
• Small sample size 5ul-10ul
• Improved sensitivity due to amplified fluorescent signal
• Whole blood, plasma or serum
• Simple electrophoretic mini/micro gel formats and devices
• Technology applicable to other disease related degradative enzymes (lipases, nucleases, amylases, etc.)
• Blood Spot Card samples
• Easily translatable to a Handheld POC Device for Early Cancer and Other Disease Detection
Continued Efforts
• PDAC Grant - Dr. Rebekah White PDAC studies with patient whole blood samples, plasma and serum samples
• Examine more pre-malignancy PanIN and IPMN samples
• Test protease inhibitors (FUT-175, etc.)
• Combined assay for “Protease Detection with Protease Inhibitor Therapeutic Effectiveness”
• New scientific publications
• New patent applications (UCSD)
Protease Biomarker Liquid Biopsy & Companion Diagnostics
MJ Heller, G Schmid-Schoenbein & R White
1. Lewis J, Alattar A, Akers J, Carter B, Heller MJ, and Chen C,"A Pilot Proof-Of-Principle Analysis Demonstrating Dielectrophoresis (DEP) as a Glioblastoma Biomarker Platform"; Dec 1 2019; Scientific Reports. 9, 1, 10279.
2. Lewis JM, Vyas A, Qiu Y, Messer KS, White R and Heller MJ, “Integrated Analysis of Exosomal Protein Biomarkers on AC Electrokinetic Chips Enables Rapid Detection of Pancreatic Cancer in Patient Blood”, ACS Nano (2018), DOI: 10.1021/acsnano.7b08199
3. Heineck DP, Lewis JM and Heller, MJ, “Electrokinetic device design and constraints for use in high conductance solutions”, Electrophoresis, 2017, DOI 10.1002/elps.201600563
4. Ibsen SD, Wright J, Lewis JM, Kim S, Ko S-Y, Ong J, Manouchehri S, Vyas A, Akers J, Chen CC, Carter BS, Esener SC and Heller MJ, “Rapid Isolation and Detection of Exosomes and Associated Biomarkers from Plasma”, ACS Nano 2017, DOI: 10.1021/acsnano.7b00549
5. Manouchehri S, Ibsen S, Wright J, Rassenti L, Ghia EM, Widhopf IIGF, Kipps TJ and Heller MJ, Dielectrophoretic Recovery of DNA from Plasma for the Identification of Chronic Lymphocytic Leukemia Point Mutations, Internat. J of Hematologic Oncology, May 2016 ,Vol. 5, No.
1, Pages 27-35 , DOI 10.2217/ijh-2015-0009
6. Ibsen S, Sonnenberg A, SchuttC, Mukthavaram R, Yeh Y, Ortac I, Manouchehri S, Kesari S, Esener S and Heller MJ, “Recovery of Drug Delivery Nanoparticles from Human Plasma using an Electrokinetic Platform Technology”, SMALL, 2015, DOI: 10.1002/smll.201500892
7. Lewis J, Heineck D and Heller MJ, “Detecting Cancer Biomarkers in Blood: Challenges for new molecular diagnostic and point-of-care tests using cell-free nucleic acids”, Expert Review Molecular Diagnostics, (2015) doi: 10.1586/14737159.2015.1069709
8. Sonnenberg A, Marciniak JY, Rassenti L, Ghia EM, Skowronski EA, Manouchehri S, McCanna JP, Widhopf II GF, Kipps TJ and Heller MJ, “Dielectrophoretic Isolation and Detection of Cancer Related Circulating Cell Free DNA Biomarkers from Blood and Plasma”,
Electrophoresis, V35, 12-13, pp. 1828-36, July 2014.
9. Sonnenberg A, Marciniak JY, Rassenti L, Ghia EM, Skowronski EA, Manouchehri S, McCanna JP, Widhopf II GF, Kipps TJ and Heller MJ, “Rapid Electrokinetic Isolation of Cancer-Related Circulating Cell Free DNA Directly from Blood”, Clinical Chemistry, 60:3, pp.500-509,
2014
10. McCanna JP, Sonnenberg A, and Heller MJ, Low level epifluorescent detection of nanoparticles and DNA on dielectrophoretic microarrays, J. BioPhotonics , 1–11 (2013) / DOI 10.1002/jbio.201300046
11. Sonnenberg, A, Marciniak JY, McCanna J, Krishnan R, Rassenti L, Kipps TJ and Heller MJ “Dielectrophoretic Isolation and Detection of cfc-DNA Nanoparticulate Biomarkers and Virus form Blood”, Electrophoresis, V34, pp.1076-1084, 2013
12. Sonnenberg, A, Marciniak, JY, Krishnan, R, Heller MJ, “Dielectrophoretic Isolation of DNA and Nanoparticles from Blood”, Electrophoresis V33, 2482-90, 2012
13. Krishnan R and Heller MJ, Rapid Isolation and Detection of Cell Free Circulating DNA and Other Disease Biomarkers Directly from Whole Blood, in Circulating Nucleic Acids in Plasma and Serum, 2011, Chap. 34, pp 247-257, DOI 10.1007/978-90-481-9382-0_34, Print ISBN
978-90-481-9381-3 Online ISBN, 978-90-481-9382-0, Springer Science+Business Media B.V.
14. Heller MJ, Krishnan R and Sonnenberg A, Detection of Cancer Related DNA Nanoparticulate Biomarkers and Nanoparticles in Whole Blood, TechConnect World 2010 Proceedings, Nanotechnology 2010, Vol 3, Chap. 6, Cancer Nanotechnology pp 372-375
15. Krishnan R, Dehlinger DA, Gemmen GJ, Mifflin RL, Esener S and Heller MJ, “Interaction of nanoparticles at the DEP microelectrode interface under high conductance conditions”, Electrochemical Communications, V11, #8, 1661-1666, 2009
16. Krishnan R and Heller MJ, “An AC electrokinetic method for the enhanced detection of DNA nanoparticles”, J. Biophotonics, V2, #4, pp. 253-261, 2009
17. Krishnan R, SullivanBD, Mifflin RL, Esener SC, and Heller MJ, Alternating current electrokinetic separation and detection of DNA nanoparticles in high-conductance solutions, Electrophoresis, v. 29, #9, pp. 1765-1774, May 2008.
Protease Assay Publications (UCSD Heller Lab)
1. Modestino, A.E., et al., Elevated Resting and Postprandial Digestive Proteolytic Activity in Peripheral Blood of Individuals With Type-2 Diabetes Mellitus, With Uncontrolled Cleavage of Insulin
Receptors. J Am Coll Nutr, 2019. 38 (6): p. 485-492.
2. Modestino A, Tyndall M, Yu J, Lefkowitz RB, Schmid-Schönbein GW and Heller MJ, Thrombin Activity Assay in Untreated Whole Human Blood, Electrophoresis, 37, 2248-2256, 2016.
3. Levine JM, Cohen ND, Heller M, Fajt VR, Levine GJ, Kerwin SC, Trivedi AA, Fandel TM, Werb W, Modestino A and Noble-Haeusslein LJ, “Efficacy of a Metalloproteinase Inhibitor in Spinal Cord
Injured Dogs”, PLOS ONE, V9, Issue 5, e96408, May 2014
4. Altshuler AE, Richter MD, Modestino AE, Penn AH, Heller MJ and Schmid-Schonbein GW, Removal of luminal content protects the small intestine during hemorrhagic shock but is not sufficient to
prevent lung injury, Physiological Reports, v1, Is5, e00109, pp.1-18, Oct 2013 (DOI: 10.1002/phy2.109)
5. Frankwich K, Tibble C, Torres-Gonzalez M, Bonner M, Lefkowitz R, Tyndall M, Schmid-Schonbein GW, Villarreal F, Heller MJ, Herbst K. Proof of Concept: Matrix metalloproteinase inhibitor
decreases inflammation and improves muscle insulin sensitivity in people with type 2 diabetes. J Inflamm (Lond) 9: 35, 2012
6. Lefkowitz RB, Schmid-Schönbein GW, and Heller MJ, "Whole Blood Assay for Elastase, Chymotrypsin, Matrix Metalloprotease-2 and Matrix Metalloprotease-9 Activity”, Analytical Chemistry , 82
(19), pp. 8251-8258, 2010
7. Lefkowitz RB, Schmid-Schönbein GW, and Heller MJ, "Whole Blood Assay for Trypsin Activity Using Polyanionic Focusing Gel Electrophoresis", Electrophoresis, V31, pp. 2442-2451, 2010.
8. Lefkowitz RB, Marciniak JY, Hu CM, Schmid-Schönbein GW, and Heller MJ “An Electrophoretic Method for the Detection of Chymotrypsin and Trypsin Activity Directly in Whole Blood”,
Electrophoresis, V31, 403-410, 2010.
Other Recent Publications (UCSD Heller Lab)
Acknowledgments
mheller@ucsd.edu
760-415-1962 (c)
UCSD – Dr. Geert Schmid-Schoenbein, Dr. Rebekah White, Dr. Jayanth Shankara Narayanan,
Dr. Jorge de la Torre, Sahand Salari-Namin, Heather A. Farris, Nicole Nolan, Dr. Augusta Modestino, Dr.
Jean Lewis, Dr. Ben Sarno, D. Sean Hamilton, Dr. Kyle Gustafson, Dr. Paul Mills and Dr. Thomas Kipps
Enterprise Partners - Dr. Andrew Senyei
OHSU/Knight Cancer/CEDAR – Dr Stuart Ibsen, Ella Stimson, Dr. Jesus Bueno Alvarez,, Dr. Terry Morgan,
Dr. Jared Fisher Dr. Missy Wong and Dr. Jason Link

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PROTEASE DETECTION ON BLOOD SPOT CARDS FOR FUTURE COMPANION DIAGNOSTICS

  • 1. Future Genomics 2023 •March 22 - 23, 2023 Kimpton Marlowe Hotel, Cambridge, Mass March 22, 2023 PROTEASE DETECTION ON BLOOD SPOT CARDS FOR FUTURE COMPANION DIAGNOSTICS Michael J Heller Geert Schmid-Schoenbein, Rebekah White, Jayanth Shankara Narayanan, Jorge de la Torre, Sahand Salari-Namin, Heather A. Farris, Nicole Nolan, Augusta Modestino, Ben Sarno, Jean Lewis, Sean Hamilton, Kyle Gustafson, Paul Mills and Thomas Kipps - UCSD Dr. Andrew Senyei - Enterprise Partners University of California San Diego Depts Bioengineering and Nanoengineering & Moores Cancer Center La Jolla, CA 92093 mheller@ucsd.edu
  • 2. Proteases in Cancer and Other Diseases In the case of Pancreatic (PDAC) and Colon Cancer the digestive proteases Trypsin, Chymotrypsin Elastase and other degradative enzymes are often found in the blood at elevated levels. Digestive proteases are also agents of further pathology • Activation of other proteases • Cancer progression & metastasis • Cell receptor cleavage • Widespread tissue damage • Contributes to disease morbidity PDAC – Activation of pancreatic proteases and leakage into the blood stream Colon Cancer – Leakage of the active digestive enzymes from the gut into the blood stream – Leaky Gut Syndrome/Autodigestion
  • 3. 12 Blood Tests to Date Handheld Prototype Devices Protease Biomarker Detection Liquid Biopsy Assays, Devices, Early Publications & US Patents (MJ Heller & G Schmid-Schoenbein – UCSD) Inflammatory Disorders Cancer Infectious Diseases Cardiovascular Disorders Sepsis Coagulation Disorders • Proteases as new biomarker for cancer • Human studies type 2 diabetes, shock, hypertension and coagulation • Pre-clinical studies in mice/rats for shock and heart failure • Scientific Publications • Verification of Autodigestion Hypothesis • 3 Issued US Patents and two new patent application (UCSD)
  • 4. Rapid Protease Activity Assay in Whole Blood, Plasma or Serum “Greatly enhanced sensitivity – one protease produces many fluorescent signals “ + Blood Charge-Changing Fluorescent Peptide Substrate + Negative cleaved fragments Positive cleaved fluorescent fragments 30-60 Minute Reaction of Protease and Net Negative Charged- Changing Fluorescent Peptide Substrates Protease Substrate
  • 5. Anal. Chem. 2010, 82, 8251–8258 Fluorescent Charge-Changing Peptide Substrates Trypsin Specific Charge-Changing Peptide Substrate Acetyl-N-Asp-Gly-Asp-Ala-Gly-Arg/Ala-Gly-Ala-Gly-Lys-BFL-C-Amide Cleavage Site Fluorophore (-) (-) (+)
  • 6. +H3N +H3N COOH- - COOH- - - Fast and Simple Electrophoretic Separation Load blood & peptide sample mixture into electrophoretic PAGE gel device* Gel Wells Samples Gel Well Sample * A variety of microgel electrophoretic devices have also been used
  • 7. +H3N +H3N +H3N - - COOH- - COOH- - Fluorescent Detection after Electrophoretic Separation Electrophoretic Separation Blood, plasma proteins and uncleaved negatively charged fluorescent peptide substrates are effectively removed “Sample Preparation” Cleaved positively charged fluorescent peptide product fragments migrate into gel for “fluorescent detection” (+) charges fluorescent peptides
  • 8. Imaging and Results Advantages • Detects protease activity • Enhanced sensitivity due to amplified fluorescent signal • Uses only a single drop (5ul-10ul) of whole blood, plasma or serum • Simple mini/micro gel electrophoretic formats • Provides 30-60 minute “Sample to Answer “ Fluorescent band intensity indicates level of protease activity
  • 9. Type II Diabetes - Published results for metabolic disease and co-morbidities that accompany type II Diabetes, evidence for elevated trypsin, chymotrypsin, elastase and MMP2/9 activity and cleavage of insulin receptors. Modestino AE, Skowronski EA, Pruitt C, Taub PR, Herbst K, Schmid-Schönbein GW, Heller MJ & Mills PJ; “Elevated Resting and Postprandial Digestive Proteolytic Activity in Peripheral Blood of Individuals With Type-2 Diabetes Mellitus, With Uncontrolled Cleavage of Insulin Receptors”, Journal of the American College of Nutrition; 2019; https://doi.org/10.1080/07315724.2018.1545611
  • 10. MMP Inhibitor - Published results on efficacy of a metalloproteinase protease inhibitor in spinal cord injured dogs Levine JM, Cohen ND, Heller MJ, Fajt VR, Levine GJ, Kerwin SC, Trivedi AA, Fandel TM, Werb W, Modestino A and Noble-Haeusslein LJ, “Efficacy of a Metalloproteinase Inhibitor in Spinal Cord Injured Dogs”, PLOS ONE, V9, Issue 5, e96408, May 2014
  • 11. Proteases in Cancer In addition to digestive proteases in PDAC and Colon Cancer many other proteases are elevated and appear in other Cancers
  • 13. Multi-Omic BioMarkers: cf/ct-DNA (Mutations & Methylation); cf-RNA (mRNA, miRNA); Exosomes and EV Protein Biomarkers; Nucleosomes and Others (Proteases???) Are detectable levels present for early cancer stages and for different cancers ??? Apoptotic & high molecular weight cf/ct-DNA Mutations and Methylation Mitochondria, Nucleosomes Necrotic Cell Debris, Protease Biomarker Improved Detection Why Proteases may be the Best Biomarkers for Early Cancer Detection? Genomic (DNA, RNA, Epigenetics) → Exosomal Proteins Multi-Omics → Proteases ?
  • 14. The Pancreas and Digestive Enzymes (Proteases, Amylases, Lipases & Nucleases) * The Acini cells produce proteases in an in-active form (Trypsinogen, Chymotrypsinogen, etc., they become activated upon entering the duodenum. *
  • 15. Pancreatic Cancer Pancreatic ductal adenocarcinoma (PDAC) a highly aggressive lethal malignancy due to the lack of early diagnosis and limited response to treatments (Stages I – IV)
  • 16. Protease Work at Knight Cancer/CEDAR/OHSU
  • 17. Many PDAC patient samples which had elevated levels of cf-DNA and Exosome Biomarkers also had elevated levels of trypsin and chymotrypsin. Knight Cancer/CEDAR/OHSU/UCSD - MJ Heller and A Modestino
  • 18. Healthy Digestion Low Escape of Digestive Enzymes Normal Vascular Function Protein & Receptor Signaling Autodigestion Hypothesis Cancer (PDAC, Colon), IBS, Crohn’s Disease, Inflammatory Response, Diabetes, Shock, Organ Dysfunction, Aging??? Leak of Digestive Enzymes Cell, Tissue Vascular Failure Protein Degradation Receptor Cleavage The Autodigestion Hypothesis Leakage of Digestive Enzymes in Cancer and Other Diseases Professor Geert Schmid-Schoenbein - UCSD Dept Bioengineering
  • 19. Journal of Pancreatic Cancer Volume 6.1, 2020 DOI: 10.1089/pancan.2019.0014 Trypsin Trojan Horse Hypothesis – Does Trypsin Promote More Aggressive Pancreatic Cancer ? A Review of the Trypsin-PAR2 Axis to Proliferation, Early Invasion, and Metastasis Kjetil Søreide, Marcus Roalsø,and Jan Rune Aunan
  • 20. Journal of Pancreatic Cancer Volume 6.1, 2020 DOI: 10.1089/pancan.2019.0014 Trypsin Trojan Horse Hypothesis – Does Trypsin Promote More Aggressive Pancreatic Cancer ? A Review of the Trypsin-PAR2 Axis to Proliferation, Early Invasion, and Metastasis Kjetil Søreide, Marcus Roalsø,and Jan Rune Aunan
  • 21. Pancreatic Cancer Related Protease Biomarker Detection (UCSD) Support for Trypsin Trojan Horse Hypothesis MJ Heller, R White, G Schmid-Schoenbein, J S Narayanan 0 20 40 60 80 100 120 140 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Relative Fluorescent Intensity Trypsin & Chymotrypsin RAPD Results for PDAC Patient Plasma Samples (4ul sample volume ) 021 052 055 - 021 034 062 - 021 052 055 - 021 034 062 Trypsin Chymotrypsin 0 10 20 30 40 50 60 70 80 1 2 3 4 5 6 7 8 9 Relative Fluorescent Intensity MMP-2 and Cathepsin-S RAPD Results for PDAC Patient Plasma Samples (4ul sample volume ) 21 052 055 062 021 052 055 062 MMP-2 Cathepsin-S
  • 22. PDAC/Protease Inhibitor Research Proposal 2022 (UCSD) Funded – Jan 1, 2023 Start Date Rebekah White, Michael J Heller, Geert Schmid-Schoenbein and Jayanth Shankara Narayanan
  • 23. “Detection of Disease Biomarkers and Therapeutic Effectiveness from Blood Spot Card Samples” US Provisional Patent US63/386,635 MJ Heller, G Schmid-Schoenbein, R White, J S Narayanan & A Senyei Invention relates to the detection and analysis of biomarkers for cancer and other diseases from small amounts (~1ul-50ul) of clinical, (blood, plasma, serum), biological and other samples spotted (deposited) and stored on Blood Spot Cards. Describes novel procedures, processes, methods, assays, devices, systems, and reagents that eliminate the need to first extract the biomarkers from the Blood Spot Card for subsequent analysis. Describes a novel process where the reaction of the detection reagents with the biomarkers occurs within (In-Situ) the blood spot card matrix material itself. After which the reaction products are electrophoretically extracted and detected. Further describes unique processes which can accelerate the in-situ reaction of the detection reagents with the biomarkers. Such processes reduce the overall assay time and complexity for carrying out biomarker detection and analysis using blood spot card samples.
  • 24. Dried Blood Spots: Applications and Techniques (Wiley Series on Pharmaceutical Science and Biotechnology: Practices, Applications and Methods) 1st Edition by Wenkui Li (Editor), Mike S. Lee (Editor) ISBN-13: 978-1118054697 ISBN-10: 1118054695 Blood Spot Saver Cards: Whatman Perkin-Elmer Thomas Scientific Others Background on Blood Spot Card Applications and Techniques
  • 25. Liquid Biopsy Protease Biomarker & Companion Diagnostics Using Blood Spot Cards New Devices, Assay Methods and Reagents for Research, Clinical Lab, POC and Home Testing Scenarios General Research Lab Research Lab Automated Clinical Lab Automated Point of Care Devices Hand-Held Home Care Devices Blood Draw Tubes Finger Prick Blood Spot Saver Card
  • 26. Blood Draw Tubes Blood Spot Saver Card Advantages • $$$ Cost Saving • Time Saving • Ease of Use • Small Sample Size • Blood, plasma, serum, urine, etc. • Longer Sample Storage (Blood) • Home/POC • Enables cost effective time course studies & diagnostics (hours, days, weeks) Disadvantages • Extraction Process Cumbersome • Loss of Biomarker • Sample Dilution • Small Sample Size Advantages and Disadvantages for Using Blood Spot Cards Research, Clinical Lab, POC and Home Testing Scenarios
  • 27. Blood Spot Card Trypsin Assay Results Samples - 5ul buffer and 5ul blood spiked with Trypsin (~15 units) dried and stored for 72 hours Blood spots samples extracted and placed into 4% Agarose Gel wells 0 10 20 30 40 50 60 70 80 90 1 2 3 4 5 Relative Fluorescent Intensity Trypsin Assay Results Blood Spot Card Buffer and Blood Samples Buffer electrophoretic ally Blood electrop Trypsin electrophoretic ally Buffer Buffer/Trypsin Blood Blood/Trypsin Blood Spots Buffer Spots Sample Wells Sample Wells 4% Agarose Gel Michael J Heller, Geert Schmid-Schoenbein, Rebekah White, Jayanth Shankara Narayanan and Andrew Senyei
  • 28. 1. Place excised sample spots into wells 2. Add specific protease fluorescent Charging-Changing Peptide (CCP) substrates and buffer 3. React 30-60 minutes (+) Control (-) Control Excised Patient Sample Spots Polyacrylimide or Agarose MiniGel Sample Wells Polyacrylamide PAGE MiniGel Format Sample Wells For analysis excise (cut/punch out) spots from card New Method for In-Situ Protease Detection on Blood Spot Card Samples Carried Out in Vertical PAGE Gel Format (+) Polyacrylimide or Agarose MiniGel Sample Wells (-) Positively (+) charged fluorescent cleaved CCP product fragments + - Control Control CCP substrate cleavage by proteases Apply DC electric field to separate cleaved CCP fragments Spot patient blood samples onto card and dry Sample Well Uncleaved Fluorescent Substrate Cleaved (+) fluorescent fragment Cleaved (-) fragment Protease Excised paper spot Negatively (-) charged fluorescent un- cleaved CCP and blood components
  • 29. (+) Control (-) Control Excised Patient Sample Spots Comparison of Classical and New In-Situ Procedure for Protease Detection on Blood Spot Card Samples Spot patient blood samples onto card and dry 1. Place excised sample spots into wells 2. Add specific protease fluorescent Charging- Changing Peptide (CCP) substrates and buffer 3. React 30-60 minutes 4. Apply DC voltage for 20-30 minutes 5. Measure fluorescent signals 1. Place excised sample spots into tubes 2. Add extraction buffer 3. Masticate and extract for 30-60 minutes 4. Centrifuge and elute extracted materials 5. Place eluted samples into another tube 6. Add specific protease fluorescent Charging-Changing Peptide (CCP) substrates and buffer 7. React 30-60 minutes 8. Place aliquots of samples into PAGE gel wells 9. Apply DC voltage for 20-30 minutes 10. Measure fluorescent signals Positively (+) charged fluorescent cleaved CCP product fragments Negatively (-) charged fluorescent un-cleaved CCP and blood components Classical Procedure New In-Situ Procedure
  • 30. 1. Place excised sample spots into wells 2. Add specific protease fluorescent Charging- Changing Peptide (CCP) substrates and buffer 3. React 30-60 minutes (+) Control (-) Control Excised Patient Sample Spots For analysis excise (cut/punch out) spots from card Spot patient blood samples onto card and dry Negatively (-) charged blood components New Method for In-Situ Protease Detection on Blood Spot Card Samples Carried Out in a Horizontal Agarose Gel Format. Sample Well Uncleaved Fluorescent Substrate Cleaved (+) fluorescent fragment Protease Excised paper spot Cleaved (-) fragment Horizontal Agarose MiniGel Format Sample Wells CCP substrate cleavage by proteases (+) Control (-) Control Positively (+) charged fluorescent cleaved CCP product fragments Apply DC electric field to separate cleaved CCP fragments Negatively (-) charged fluorescent un- cleaved CCP (+) (-)
  • 31. In-Situ Trypsin Assay from Blood Spot Card With Trypsin Spiked Blood and Plasma Samples 1 2 3 4 5 6 7 8 9 10 11 12 X Con (-) Con (+) X Blood (-) X Blood (+) X Plasma (-) X Plasma (+) X White Light Image 4% Agarose Gel Fluorescent Image 4% Agarose Gel Sample Wells Sample Wells Blood Cells and Components (+) (-) Bands from Un-cleaved Negatively Charged (-) Fluorescent CCP Substrate Bands from Cleaved Positively Charged (+) Fluorescent CCP Product Fragments IMG_4462-226-1206-Trypsin-BH223-pp32-White Light-061422 1 2 3 4 5 6 7 8 9 10 11 12 X Con (-) Con (+) X Blood (-) X Blood (+) X Plasma (-) X Plasma (+) X IMG_4456-226-1206-Trypsin-BH223-pp32-Fluorescent-061422
  • 32. IMG_4457-226-1206-Trypsin- BH223-pp32-Blocked Fluor- 061422 0 5 10 15 20 25 30 35 40 45 50 Relative Fluorescent Intensity Trypsin Spiked -BH-223-Blood & Plasma-1st-pp32-061 Con (-) Con (+) Blood (-) Blood (+) Plasma (-) Plasma (+) In-Situ Trypsin Assay from Blood Spot Card With Trypsin Spiked Blood and Plasma Samples IMAGE J Quantitative Analysis of Image-4457-226-1206 and Resulting Graph Sample Wells Bands from Cleaved Positively Charged (+) Fluorescent CCP Product Fragments 1 2 3 4 5 6 7 8 9 10 11 12 X Con (-) Con (+) X Blood (-) X Blood (+) X Plasma (-) X Plasma (+) X
  • 33. IMG_4470-226-1206-Chymotrypsin Full Gel Fluorescent Image-061422 IMG_4490-226-1206-Chymotrypsin Final Gel Image White Light-061422 In-Situ Chymotrypsin Assay from Blood Spot Card With Chymotrypsin Spiked Blood and Plasma Samples 1 2 3 4 5 6 7 8 9 10 11 12 X Con (-) Con (+) X Blood (-) X Blood (+) X Plasma (-) X Plasma (+) X White Light Image 4% Agarose Gel Sample Wells Blood Cells and Components 1 2 3 4 5 6 7 8 9 10 11 12 Fluorescent Image 4% Agarose Gel Sample Wells Bands from Un-cleaved Negatively Charged (-) Fluorescent CCP Substrate Bands from Cleaved Positively Charged (+) Fluorescent CCP Product Fragments X Con (-) Con (+) X Blood (-) X Blood (+) X Plasma (-) X Plasma (+) X
  • 34. 0 5 10 15 20 25 30 1 2 3 4 5 6 7 Relative Fluorescent Intensity Chart Title IMG_4472-226-1206- Blocked Fluorescent Gel Image 1/2 sec 061422 In-Situ Chymotrypsin Assay on Blood Spot Card Blood and Plasma Samples - IMAGE J Quantitative Analysis of Image-IMG_4472-226-1206 and Resulting Graph Con (-) Con (+) Blood (-) Blood (+) Plasma (-) Plasma (+) Sample Wells Bands from Cleaved Positively Charged (+) Fluorescent CCP Product Fragments 1 2 3 4 5 6 7 8 9 10 11 12 X Con (-) Con (+) X Blood (-) X Blood (+) X Plasma (-) X Plasma (+) X
  • 37. Combined Protease Detection and Protease Inhibitor/Therapeutics Assays
  • 38. Advantages • Fast (~30-60 minutes) • Small sample size 5ul-10ul • Improved sensitivity due to amplified fluorescent signal • Whole blood, plasma or serum • Simple electrophoretic mini/micro gel formats and devices • Technology applicable to other disease related degradative enzymes (lipases, nucleases, amylases, etc.) • Blood Spot Card samples • Easily translatable to a Handheld POC Device for Early Cancer and Other Disease Detection Continued Efforts • PDAC Grant - Dr. Rebekah White PDAC studies with patient whole blood samples, plasma and serum samples • Examine more pre-malignancy PanIN and IPMN samples • Test protease inhibitors (FUT-175, etc.) • Combined assay for “Protease Detection with Protease Inhibitor Therapeutic Effectiveness” • New scientific publications • New patent applications (UCSD) Protease Biomarker Liquid Biopsy & Companion Diagnostics MJ Heller, G Schmid-Schoenbein & R White
  • 39. 1. Lewis J, Alattar A, Akers J, Carter B, Heller MJ, and Chen C,"A Pilot Proof-Of-Principle Analysis Demonstrating Dielectrophoresis (DEP) as a Glioblastoma Biomarker Platform"; Dec 1 2019; Scientific Reports. 9, 1, 10279. 2. Lewis JM, Vyas A, Qiu Y, Messer KS, White R and Heller MJ, “Integrated Analysis of Exosomal Protein Biomarkers on AC Electrokinetic Chips Enables Rapid Detection of Pancreatic Cancer in Patient Blood”, ACS Nano (2018), DOI: 10.1021/acsnano.7b08199 3. Heineck DP, Lewis JM and Heller, MJ, “Electrokinetic device design and constraints for use in high conductance solutions”, Electrophoresis, 2017, DOI 10.1002/elps.201600563 4. Ibsen SD, Wright J, Lewis JM, Kim S, Ko S-Y, Ong J, Manouchehri S, Vyas A, Akers J, Chen CC, Carter BS, Esener SC and Heller MJ, “Rapid Isolation and Detection of Exosomes and Associated Biomarkers from Plasma”, ACS Nano 2017, DOI: 10.1021/acsnano.7b00549 5. Manouchehri S, Ibsen S, Wright J, Rassenti L, Ghia EM, Widhopf IIGF, Kipps TJ and Heller MJ, Dielectrophoretic Recovery of DNA from Plasma for the Identification of Chronic Lymphocytic Leukemia Point Mutations, Internat. J of Hematologic Oncology, May 2016 ,Vol. 5, No. 1, Pages 27-35 , DOI 10.2217/ijh-2015-0009 6. Ibsen S, Sonnenberg A, SchuttC, Mukthavaram R, Yeh Y, Ortac I, Manouchehri S, Kesari S, Esener S and Heller MJ, “Recovery of Drug Delivery Nanoparticles from Human Plasma using an Electrokinetic Platform Technology”, SMALL, 2015, DOI: 10.1002/smll.201500892 7. Lewis J, Heineck D and Heller MJ, “Detecting Cancer Biomarkers in Blood: Challenges for new molecular diagnostic and point-of-care tests using cell-free nucleic acids”, Expert Review Molecular Diagnostics, (2015) doi: 10.1586/14737159.2015.1069709 8. Sonnenberg A, Marciniak JY, Rassenti L, Ghia EM, Skowronski EA, Manouchehri S, McCanna JP, Widhopf II GF, Kipps TJ and Heller MJ, “Dielectrophoretic Isolation and Detection of Cancer Related Circulating Cell Free DNA Biomarkers from Blood and Plasma”, Electrophoresis, V35, 12-13, pp. 1828-36, July 2014. 9. Sonnenberg A, Marciniak JY, Rassenti L, Ghia EM, Skowronski EA, Manouchehri S, McCanna JP, Widhopf II GF, Kipps TJ and Heller MJ, “Rapid Electrokinetic Isolation of Cancer-Related Circulating Cell Free DNA Directly from Blood”, Clinical Chemistry, 60:3, pp.500-509, 2014 10. McCanna JP, Sonnenberg A, and Heller MJ, Low level epifluorescent detection of nanoparticles and DNA on dielectrophoretic microarrays, J. BioPhotonics , 1–11 (2013) / DOI 10.1002/jbio.201300046 11. Sonnenberg, A, Marciniak JY, McCanna J, Krishnan R, Rassenti L, Kipps TJ and Heller MJ “Dielectrophoretic Isolation and Detection of cfc-DNA Nanoparticulate Biomarkers and Virus form Blood”, Electrophoresis, V34, pp.1076-1084, 2013 12. Sonnenberg, A, Marciniak, JY, Krishnan, R, Heller MJ, “Dielectrophoretic Isolation of DNA and Nanoparticles from Blood”, Electrophoresis V33, 2482-90, 2012 13. Krishnan R and Heller MJ, Rapid Isolation and Detection of Cell Free Circulating DNA and Other Disease Biomarkers Directly from Whole Blood, in Circulating Nucleic Acids in Plasma and Serum, 2011, Chap. 34, pp 247-257, DOI 10.1007/978-90-481-9382-0_34, Print ISBN 978-90-481-9381-3 Online ISBN, 978-90-481-9382-0, Springer Science+Business Media B.V. 14. Heller MJ, Krishnan R and Sonnenberg A, Detection of Cancer Related DNA Nanoparticulate Biomarkers and Nanoparticles in Whole Blood, TechConnect World 2010 Proceedings, Nanotechnology 2010, Vol 3, Chap. 6, Cancer Nanotechnology pp 372-375 15. Krishnan R, Dehlinger DA, Gemmen GJ, Mifflin RL, Esener S and Heller MJ, “Interaction of nanoparticles at the DEP microelectrode interface under high conductance conditions”, Electrochemical Communications, V11, #8, 1661-1666, 2009 16. Krishnan R and Heller MJ, “An AC electrokinetic method for the enhanced detection of DNA nanoparticles”, J. Biophotonics, V2, #4, pp. 253-261, 2009 17. Krishnan R, SullivanBD, Mifflin RL, Esener SC, and Heller MJ, Alternating current electrokinetic separation and detection of DNA nanoparticles in high-conductance solutions, Electrophoresis, v. 29, #9, pp. 1765-1774, May 2008. Protease Assay Publications (UCSD Heller Lab) 1. Modestino, A.E., et al., Elevated Resting and Postprandial Digestive Proteolytic Activity in Peripheral Blood of Individuals With Type-2 Diabetes Mellitus, With Uncontrolled Cleavage of Insulin Receptors. J Am Coll Nutr, 2019. 38 (6): p. 485-492. 2. Modestino A, Tyndall M, Yu J, Lefkowitz RB, Schmid-Schönbein GW and Heller MJ, Thrombin Activity Assay in Untreated Whole Human Blood, Electrophoresis, 37, 2248-2256, 2016. 3. Levine JM, Cohen ND, Heller M, Fajt VR, Levine GJ, Kerwin SC, Trivedi AA, Fandel TM, Werb W, Modestino A and Noble-Haeusslein LJ, “Efficacy of a Metalloproteinase Inhibitor in Spinal Cord Injured Dogs”, PLOS ONE, V9, Issue 5, e96408, May 2014 4. Altshuler AE, Richter MD, Modestino AE, Penn AH, Heller MJ and Schmid-Schonbein GW, Removal of luminal content protects the small intestine during hemorrhagic shock but is not sufficient to prevent lung injury, Physiological Reports, v1, Is5, e00109, pp.1-18, Oct 2013 (DOI: 10.1002/phy2.109) 5. Frankwich K, Tibble C, Torres-Gonzalez M, Bonner M, Lefkowitz R, Tyndall M, Schmid-Schonbein GW, Villarreal F, Heller MJ, Herbst K. Proof of Concept: Matrix metalloproteinase inhibitor decreases inflammation and improves muscle insulin sensitivity in people with type 2 diabetes. J Inflamm (Lond) 9: 35, 2012 6. Lefkowitz RB, Schmid-Schönbein GW, and Heller MJ, "Whole Blood Assay for Elastase, Chymotrypsin, Matrix Metalloprotease-2 and Matrix Metalloprotease-9 Activity”, Analytical Chemistry , 82 (19), pp. 8251-8258, 2010 7. Lefkowitz RB, Schmid-Schönbein GW, and Heller MJ, "Whole Blood Assay for Trypsin Activity Using Polyanionic Focusing Gel Electrophoresis", Electrophoresis, V31, pp. 2442-2451, 2010. 8. Lefkowitz RB, Marciniak JY, Hu CM, Schmid-Schönbein GW, and Heller MJ “An Electrophoretic Method for the Detection of Chymotrypsin and Trypsin Activity Directly in Whole Blood”, Electrophoresis, V31, 403-410, 2010. Other Recent Publications (UCSD Heller Lab)
  • 40. Acknowledgments mheller@ucsd.edu 760-415-1962 (c) UCSD – Dr. Geert Schmid-Schoenbein, Dr. Rebekah White, Dr. Jayanth Shankara Narayanan, Dr. Jorge de la Torre, Sahand Salari-Namin, Heather A. Farris, Nicole Nolan, Dr. Augusta Modestino, Dr. Jean Lewis, Dr. Ben Sarno, D. Sean Hamilton, Dr. Kyle Gustafson, Dr. Paul Mills and Dr. Thomas Kipps Enterprise Partners - Dr. Andrew Senyei OHSU/Knight Cancer/CEDAR – Dr Stuart Ibsen, Ella Stimson, Dr. Jesus Bueno Alvarez,, Dr. Terry Morgan, Dr. Jared Fisher Dr. Missy Wong and Dr. Jason Link