Visualizing Insulin 

Structure Under Shear 

Stress

Andre Chen

Mentor: Grethe Jensen

NIST Center for Neutron Research

5 nm
Insulin is a hormone that regulates glucose 

intake by cells. 

Patients with diabetes are insulin-deficient. 

Diabetes is treated by injection of engineered 

insulin analogues.
Insulin injection

Monohexamers

Dihexamers

Rods

Capillary 

Membrane

Monohexamers

Dissociation Dimers

Monomers

Insulin in Bloodstream

INSULIN THERAPEUTICS

OBJECTIVE

Long-acting insulin drug for 

once-daily dosing Hexamers

Rod-like Structures

Subcutaneous

Tissue

FRONT SIDE
Subcutaneous Injection Blood Vessels

Shear rates on order of 

105-106 s-1

Shear stresses as high as 

9.8 N/m2

Next Step: Does 

Shear Change 

Insulin Structure?

Viscosity η

Velocity v

z

z

r

Shear Rate (s-1)

dv

z

/dr
Possible Outcomes

Rod Lengthening/Breaking Alignment with Applied Shear

Interactions

Shear

Repulsion Attraction

Shear

Dihexamers Monohexamers
Project Overview

Insulin samples prepared:

 Insulin concentrations (mM): 0.6 | 1.2 | 1.8 

 NaCl concentrations (mM): 30 | 150 | 450

Samples sheared at different shear rates to probe 

their viscosity

Small-angle neutron scattering applied during 

shear for structural data

Objective: Determine if shear affects insulin self-assembly

Formulation

Physiological

Screens 

electrostatic 

repulsions
Viewing Nanostructure With Small 

Angle Neutron Scattering

2ᶊ

Source Scattered beam

Transmitted 

beam

Sample

Incident beam

BeamstopDetector

Scattering vector: q = 4πsin(ᶊ)/λ

ki

ks

q

Scattering Intensity I(q)
Why SANS?

Probes appropriate length 

scale to view:

○ Structure

○ Orientation of structures

Isotropic orientation Alignment in flow

Image from http://cns.che.udel.edu/

5 Hexamers per 

rod

20 Hexamers per 

rod

10 Hexamers per 

rod
Rheology Measurements

Instrument: Anton Paar 

MCR 502 Rheometer

Shear Rates: 0-3500 s-1

Sample 

Solution

Titanium

Bob

Titanium 

Couette Cell

Titanium

Bob

Sample 

Solution

Tangential 

Velocity 

Profile

Front View Top View 

Images:

Rheometer:

http://www.anton-paar.com/ca-en/products/details/rheome

Cup and Bob:

ter-mcr-102-302-502/

https://ciks.cbt.nist.gov/~garbocz/SP946/node14.htm
SANS Measurement

2ᶊ

Source ki

ks Scattered beam

Transmitted 

Incident beam beam

BeamstopDetector

Radial 

Measurements

Tangential 

Measurements

Sample 

under 

shear
Insulin

NaCl

Zinc

D2O

Phenol

Phosphate 

Buffer

Sample Preparation

30 mM 150 mM 450 mM 0.6 mM 1.2 mM 0.6 mM 1.2 mM 1.8 mM 0.6 mM 1.2 mM 

1.8 mM 

NaCl Concentrations

Insulin Concentrations

+

+

+

+

+ +

-

-

-

Na+

Cl- ClNa+

NaCl ions 

screening charges

ClCl-
Rheology: Viscosity vs. Shear Stress

Insulin 

Concentration

Volume 

Percentage

0.6mM

1.2mM

1.8mM

0.44%

0.88%

1.32%

30mM NaCl

σ*= 1.3 Pa

150mM NaCl

σ*= 0.22 Pa

450mM NaCl

σ*= 0.19 Pa
0.6mM Insulin

450

150

30

mM NaCl

Radial 

Configuration

Tangential 

Configuration

SANS Data: Increasing Salt 

Concentration

x5

-1 slope

30mM NaCl

150mM NaCl

450mM NaCl

+

Isotropic Orientation

35%
Higher Order Structures: SAXS Comparison

450mM NaCl

SANS Data

SAXS Data

Quadratic 

ordering

Layering
Shear Thickening 

Dependence on Shape

30mM NaCl

150mM NaCl

450mM NaCl

0.6mM Insulin

450

150

30

mM NaCl

x5

-1 slope

30mM NaCl

150mM NaCl

450mM NaCl

+

35%
SANS Data: Increasing Salt Concentration

0.6mM Insulin

450

150

30

mM NaCl

1.8mM Insulin

450

150

mM NaCl

450

150**

1.2mM Insulin

30

mM NaCl

**Shear-induced 

reversible 

structural 

change at 

150mM NaCl, 

3500s-1

x5

30mM NaCl

450mM NaCl 150mM NaCl

+

x5

30mM NaCl

450mM NaCl 150mM NaCl

+

x5

30mM NaCl

450mM NaCl 150mM NaCl

+

Fitted models

With structure factor

Without structure factor
450mM NaCl

30mM NaCl 150mM NaCl

σ*= 1.3 Pa

Electrostatic Interactions?

σ*= 0.22 Pa

σ*= 0.19 Pa

+

+

+

+

-

-

Shear stress 

overcomes 

repulsion at σ*

SHEAR THICKENING

+

+

+

+

-

-

+

+

+

+

-

-

+

+

+

+

-

-

Electrostatic 

Repulsion
450mM NaCl

30mM NaCl 150mM NaCl

σ*= 1.3 Pa

Electrostatic Interactions?

σ*= 0.22 Pa

σ*= 0.19 Pa

+

+

+

+

-

-

Easier to 

overcome 

repulsion 

Higher NaCl = Lower σ* 

+

+

+

+

-

-

+

+

+

+

-

-

+

+

+

+

-

-

Weaker 

Repulsion

With NaCl Screening
Summary of Findings

Structure

Rods form, lengthen, and order with increasing NaCl and 

insulin concentrations

Very little shear dependence

Rheology

Shear-thinning Shear-thickening 

Critical shear stress (electrostatic barrier?)

Future Outlook

Tests with higher shear rates, lower q

More rheological studies
Acknowledgments 

Center for High Resolution Neutron Scattering 

Novo Nordisk

Grethe Vestergaard Jensen, PhD 

Mentor, NIST Center for Neutron Research

Joe Dura, PhD and Julie Borchers, PhD

NCNR SURF Coordinators, NIST Center for Neutron Research

Brandi Toliver, PhD

SURF Program Director, National Institute of Standards and Technology