Rapid Quantification of Beta Cell Secretion using Electrochemical Zn - PowerPoint Presentation

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Rapid Quantification of Beta Cell Secretion using Electrochemical Zn

2+ Sensors and Integration with Microphysiological Systems

Emma L. McBride*1,2, James Nolan*3, Hyowon Hugh Lee3, Sherry L. Harbin1,4

1

1Weldon School of Biomedical Engineering, College of Engineering, Purdue University2Medical Scientist/Engineer Training Program, Indiana University School of Medicine3Weldon School of Biomedical Engineering, Center for Implantable Devices, Birck Nanotechnology Center, Purdue University4Department of Basic Medical Sciences, College of Veterinary Medicine, Purdue University*These authors contributed equally to this work.

Abstract:

Beta (β) cell replacement therapy is an experimental treatment for certain individuals with Type 1 Diabetes aiming to restore functional glucose-stimulated insulin secretion (GSIS). Islets are transplanted within 1 to 3 days of isolation, leaving little time for evaluation of quality. Product release criteria often do not include measures of β-cell function, due to the time-consuming nature of GSIS assays and subsequent immunoassays for detection of insulin. Moreover, post-transplantation stimulation indices have been reported to correlate poorly with transplant outcomes, and thus, rapid potency assays for prospective and predictive testing are critically needed. Microphysiological systems are an emerging approach to study cells and organoids in more physiologically relevant microenvironments

in vitro

. Biosensors offer short detection times with sensitivities comparable to traditional immunoassays, and furthermore, may be readily incorporated into microfluidic devices for on-line measurement of dynamic cellular secretion. Here, we propose an electrochemical sensor for Zn

2+

, which is co-released with insulin. The sensor will work by anodic stripping voltammetry (ASV), an established technique for measuring trace Zn

2+

. We will rapidly prototype the sensor using direct writing of carbon ink, which will be functionalized with Nafion and bismuth layers. Future studies will evaluate the sensor’s potential to measure β-cell secretory products at high temporal resolution (<1 minute) when integrated with a microphysiological system featuring perfusion and oligomeric collagen encapsulation of islets. If successful, this system may be applied to other areas of active diabetes research, including drug screening or maturation of stem cell-derived β-cells.

β-cell replacement therapy aims to restore functional Glucose-Stimulated Insulin Secretion to individuals with Type 1 Diabetes

1Potency measures ability to produce a desired clinical effect, and is required by the FDA for quality control

to ensure recipients receive the highest quality islets2Phase 3 Clinical Trial used Static GSIS assay for potency evaluation, but results were not included in product release criteria and have been shown to correlate poorly with transplant outcomes3Dynamic GSIS assay consistently provides higher quality data about insulin release4 but perifusion machines are expensive, require complex set-up, and generate too many samples to process prior to transplantation

Diabetic Nude Mouse Bioassay is gold standard measure of potency but requires up to a month of observation before diabetes reversalThere is a critical need for rapid islet potency assays for prospective and predictive function testing.2

MOTIVATIONhttp://www.biorepdiabetes.com/product/perifusion-system/Quintana, et al. OBM Transplantation. 2018[1] Hering, et al.

Diabetes Care

. 2016; [2] US FDA, 2009; [3]

Ricordi

, et al.

Diabetes

. 2016; [4] Alcazar and Buchwald.

Front endo

. 2019

β

-Cell Replacement Therapy

Dynamic Perifusion System

We

hypothesize

that integration of these 3 components will provide islet potency information more rapidly and more representative of in vivo function than standard GSIS assays. 3HYPOTHESIS

Microphysiological systems aim to reduce gap between in vitro and in vivo models

Nearly all microfluidic systems currently developed for islets use off-line ELISA or on-line competitive immunoassay to measure insulin release, which are expensive and time-consuming1,2

Adewola

, et al.

Biomed microdevices

. 2010

Glieberman

, et al.

Lab Chip

. 2019

[1] Becker, et al.

Biomaterials

. 2019; [2]

Castiello

, et al.

Lab Chip

. 2016

-1.4 V

-0.4 V

4

Modified from Kim, et al.

Electrochem

commun

. 2015

[1] Fu, et al.

Curr

Diabetes Rev

. 2013

Cleaning

Deposition

Stripping

Zn

2+

2e

-

Zn

0

Zn

2+

2e

-

Zn

0

Zn

2

+

Zn

0

2e

-

Potential [V]

Time

Anodic Stripping Voltammetry of Zn

2+

Electrode Layers:

Bismuth

Nafion

Carbon ink

METHODS

Sensors can be tailored to target analyte

Electrochemical sensors are low-cost, readily miniaturized for integration into microfluidic devices

Zn

2+

is co-released with insulin from secretory granules at steady ratio of 2 ions per 6 molecules insulin

1

Anodic stripping voltammetry is commonly used for trace metal detection

Chemical information is converted into measurable electrical signal through movement of electrons

Change in current output correlates with [Zn

2+

]

5

RESULTS

The electrical response to increasing concentrations of Zn2+ is shown for the following conditions: (

A) Commercial Bismuth electrode, bottom left, in acetate buffer (pH 4.6), 10 min preconcentration, (B) Custom Carbon/Nafion/Bismuth electrode, bottom right

, in acetate buffer (pH 4.6), 2 min preconcentration, (C) Custom Bismuth/Nafion/Carbon electrode in Krebs Ringer Buffer (pH 7.3), which is used for GSIS assays, 2 min preconcentration. (D) The calibration curve for condition shown in (C) shows the electrical signal increasing linearly with Zn2+ in the range of 0.1 to 2 mg/L. A: Commercial Sensorhttp://www.dropsens.com/en/screen_printed_electrodes_pag.html 10 mm

C:

Custom Sensor in Krebs Buffer

D:

Calibration Curve from (C)

B:

Custom Sensor in Acetate Buffer

Acknowledgements

Harbin Laboratory (PI: Sherry Harbin)Laboratory of Implantable Microsystems Research (PI: Hyowon Hugh Lee)

IU CDMD Islet and Physiology CoreFundingLeslie Bottorff Fellowship ProgramBioengineering Interdisciplinary Training in Diabetes Research Program (NIDDK T32)IU/Purdue Medical Scientist Training Program (NIGMS T32)

FUTURE DIRECTIONS6

PrototypingComputational modeling of fluid dynamics

Optimize sensitivity and temporal resolution

Verification with Insulin ELISA

Evaluate correlation with diabetic nude mouse bioassay

Future Applications

Drug screening

Simultaneous imaging

Maturation and evaluation of stem-cell derived

β

-cells

Electrochemical Zinc Sensor

Islet Microphysiological System

Integrated Device

Islets encapsulated in Oligomeric type I collagen

Control, working, and reference electrodes

Syringe pumps with increasing glucose solutions for dynamic stimulation