HKSTP APAC Innovation Summit 2015 Series – Robotics June 25, 2015

Department of Mechanical and Biomedical Engineering,

City University of Hong Kong, Hong Kong SAR

Enabling Mechanical Biomarkers

using Atomic Force Microscopy

King W. C. Lai, Assistant Professor

(kinglai@cityu.edu.hk)

P. 2

Background

• Study of cellular mechanics

• AFM-based nanorobot

Applications in biological research

• Monitoring the neurodegeneration process

• Identification of peripheral neuropathy

Automated patch clamp system

Applications in nanomaterial research

Other robotic research

Conclusion

CONTENT

P. 3

CELL MECHANICAL PROPERTIES Cell integrity

Cell Biology / Genetics, Mr. Foster

Disease state

Nature review Cancer, 2012 BIOCOMM, 2013

Cell division

The mechanical properties of cells have been shown to be useful markers of cell

state by the biophysics community, and potential to achieve biomarkers with:

label-free;

robust and high-throughput;

low-cost and inexpensive.

D. Di Carlo, “A mechanical biomarker of cell state in medicine.,” J. Lab. Autom., vol. 17, no. 1, pp. 32–42, Feb. 2012.

P. 4

CURRENT APPROACHES Micropipette aspiration

BMC Cell Biology, 2008 Journal of Applied Physiology, 2000

Magnetic twisting

cytometry

Optical tweezers

Nature Materials, 2007

Fluidic shear flow

Journal of Biomechanics, 2000

Nanoindenter Drive

direction

PNAS, 2012

Micropillar substrate

P. 5

ATOMIC FORCE MICROSCOPY (AFM)

Drive

direction

AFM

scanning

Drive

direction

AFM indentation

Cell image

Cell mechanical property

Bruker Nano, Santa

Barbara, CA

BioScope Catalyst

atomic force

microscope

P. 6

AFM-BASED NANOROBOT

AFM

Scanning

image

Interactive

Force

Augmented reality

interface

Position &

command

Image

update

Movement

command

Cantilever

Cells

Haptic device

Optical

image

Controller &

SAM

Original control

algorithmController

AFM

scanner

Input (t) Output (t)

Scanning control strategy

Original control

algorithmController AFM scanner

Input (t) Output (t)

Input (t)

Nanorobotic

algorithm

Nanorobot control strategy

P. 7

Neurodegenerative disease is caused by loss of structure or function of

neurons, or neurons death

Examples: Parkinson’s and Alzheimer’s disease

NEURODEGENERATIVE DISEASE

S. Boillée et al., Neuron,

2006.

P. 8

Alzheimer’s disease:

• Lifetime risk between age 65

and 100 is 33% for men and 45% for women

• Yearly cost is about 250

billion US dollars[1]

Detailed disease mechanism is

not fully understood

Treatment for neurodegenerative

disease is under development

NEURODEGENERATIVE DISEASE

[1] K. a Jellinger, B. Janetzky, J. Attems, and E. Kienzl, “Biomarkers for early diagnosis of Alzheimer disease: ‘Alzheimer

Associated gene’--a new blood biomarker?,” Journal of cellular and molecular medicine, vol. 12, no. 4, pp. 1094–117, Aug. 2008.

Posted by JDGopez R.N. on August 8, 2012

in Medical Information, Nursing Blog

P. 9

Limitation of conventional methods

• Investigate the toxin induced neuron death based on the measurement of numbers of dead cells

• Difficult to obtain the real-time information and response occur in the individual cell and study the dynamic process during the neurodegeneration

It is crucial to develop a less invasive approach to monitor the process of neuron death under physiological conditions in real-time

Base on the observation of cell and cytoskeleton

LIMITATION

Normal neuron Synapse detachment

Apoptosis Axon and dendrites

degradation

P. 10

NR1 NR2Cell membrane Cell membrane

Ca2+

Neurotoxicity

GlutamateGlycine

Extracellular side

Cytoplasmic side

NMDA Receptor • Ionotropic glutamate receptor

Concentrated at postsynaptic sites

• Ligand-gated ion channel Mediates Ca2+ influx

• Activation requires of binding of glutamate

and glycine

• May contribute to excitotoxic death of

dopaminergic neurons in Parkinson’s disease Glutamate excitotoxicity

Material • SH-SY5Y cell:

in vitro models of neuronal function and differentiation

commonly used in Parkinson’s and Alzheimer’s disease model

• NMDA: N-methyl-D-aspartate, a neural toxin

Mimic the action of glutamate Inducing excessive Ca2+ influx to result in cell deficiency or death

NMDA INDUCED CELLULAR BEHAVIOR

P. 11

AFM IMAGING AND SURFACE ROUGHNESS

Y. Fang, C. Y. Y. Iu, C. N. P. Lui, C. K. M. Fung, H.-W. Li, K. K. L. Yung, N. Xi, and K. W. C. Lai, “Investigating dynamic structural and

mechanical changes of neuroblastoma cells associated with glutamate-mediated neurodegeneration”, Scientific Reports, vol. 4, 7074, 2014.

After 200 μM treatment: • Neurites were contracted;

• Cell body was shrunken;

• Cytoskeleton was aggregated;

• Surface roughness was increased.

Control cell NMDA treated cell

P. 12

FORCE-INDENTATION MEASUREMENTS Indentation depth: 500 nm

Force-indentation curves: 64

Number of cells for stiffness

comparison:> 30 for each group

Con

trol cell

200 μ

M N

MD

A

treate

d c

ell

**

**

Y. Fang, C. Y. Y. Iu, C. N. P. Lui, C. K. M. Fung, H.-W. Li, K. K. L. Yung, N. Xi, and K. W. C. Lai, “Investigating dynamic structural and

mechanical changes of neuroblastoma cells associated with glutamate-mediated neurodegeneration”, Scientific Reports, vol. 4, 7074, 2014.

P. 13

TIME-DEPENDENT RESPONSE

Y. Fang, C. Y. Y. Iu, C. N. P. Lui, C. K. M. Fung, H.-W. Li, K. K. L. Yung, N. Xi, and K. W. C. Lai, “Investigating dynamic structural and

mechanical changes of neuroblastoma cells associated with glutamate-mediated neurodegeneration”, Scientific Reports, vol. 4, 7074, 2014.

After 50 μM treatment:

• Gradual contraction of cell body and

neruites;

• Gradual increase of Young’s modulus.

• Real-time monitor of neurodegeneration.

Control cell NMDA treated cell

P. 14

IDENTIFICATION OF SIGNALING PATHWAY

Y. Fang, C. Y. Y. Iu, C. N. P. Lui, C. K. M. Fung, H.-W. Li, K. K. L. Yung, N. Xi, and K. W. C. Lai, “Investigating dynamic structural and

mechanical changes of neuroblastoma cells associated with glutamate-mediated neurodegeneration”, Scientific Reports, vol. 4, 7074, 2014.

Induced cell stiffening in NMDA treatment:

P. 15

Trends in Cancer Incidence and Death Rates by Sex,

United States, 1975 to 2010.

Cancer Statistics 2014, CA CANCER J CLIN, 64, 9–29,

2014

Cancer

Surgery

Radiation

Chemotherapy

5-Year Relative

Survival Rates: increased from

49% (1975-

1977) to 68% (2003-2009).

Severe side-

effects: decreased blood

cells,

inflammation of the digestive tract

and hair loss.

Suffering

CANCER THERAPY

P. 16

Chemotherapy: chemicals to kill cells, both cancer and normal cells.

Commercial anti-cancer drugs: vincristine (VCR) and paclitaxel (Taxol).

CHEMOTHERAPY PERIPHERAL NEUROPATHY

50 ng/ml, VCR 500 ng/ml,

Taxol

Immunostaining with neuronal specific marker anti-β-tubulin III, dorsal root ganglion (DRG) from adult C57BL/6 mice (8-12

weeks old).

Anti-cancer drugs Neuropathy: numbness,

tingling, sensation disturbance, and muscle weakness

Withdraw lifesaving therapy

N. P. B. Au, Y. Fang, N. Xi, K. W. C. Lai, and C. H. E. Ma, “Probing for chemotherapy-induced peripheral neuropathy in live dorsal root ganglion

neurons with atomic force microscopy.,” Nanomedicine, vol. 10, no. 6, pp. 1323–1333, Mar. 2014.

P. 17

DOSAGE RESPONSE OF DRG NEURONS

Under vincristine treatment: • Correlation of Young’s modulus to concentration

of vincristine;

• Young’s modulus was acquired from live neuron in physiological-like condition without time-consuming pre-treatment;

• Correlation of microtubules depolymerization to concentration of vincristine;

• Correlation fluorescence intensity to concentration of vincristine.

N. P. B. Au, Y. Fang, N. Xi, K. W. C. Lai, and C. H. E. Ma, “Probing for

chemotherapy-induced peripheral neuropathy in live dorsal root ganglion neurons with atomic force microscopy.,” Nanomedicine, vol. 10, no. 6, pp.

1323–1333, Mar. 2014.

P. 18

Cell-attached recording

Electrode

Frances M. Ashcroft, Ion Channels and Disease, 2000

Single ion channel activity observation

Whole cell recording

Electrode

Katsutoshi Furukawa, et al., Journal of Neurochemistry, 2006

Whole cell recording using patch clamp

Single channel current :1-50 pA Transmembrane current: 50 pA - 10 nA

Patch clamp recording: Nobel prize!

P. 19

(I) Setup (II) Engaging

(III) Gigaseal formation

Procedures of patch clamp

(IV) Break-in

Movement of pipette; voltage pulse and current; pressure of pipette

P. 20

AUTOMATED PATCH CLAMP SYSTEM

P. 21

DEMOSTRATION

P. 24

SERVICE ROBOTS

Autonomous Navigation

Simultaneous Localization and

Mapping (SLAM)

SLAM

Encoder Data

Laser Scan Data

Odometry

Measurement

Scan Matching

Occupancy Grid

Mapping

Robot pose

Demand

P. 25

AFM-based nanorobot can be used to obtain mechanical

biomarker of biological cells (cell morphology, surface

roughness, Young’s modulus)

Monitor real-time response of living cell

They are promising indicators to guide research of

neurodegeneration and chemotherapy, and reveal the

mechanism of drug treatment

CONCLUSION

CTL MK801 MK801+NMDA NMDA0

5

10

15

20

25

30

* *

* ** *

Young's

modulu

s /

kPa

Treatment

* *

P. 26

HKRGC: GRF Grant (CityU139313 and CityU124213)

ITC: ITF Grant (9442009)

CityU: Startup Grant (7200311 and 9610254)

ACKNOWLEDGEMENT

Collaborators:

Prof. Ken K. L. Yung, Department of Biology, Hong Kong Baptist University

Prof. H. W. Li, Department of Chemistry, Hong Kong Baptist University

Dr. Eddie Ma, Department of Biomedical Sciences, CityU of Hong Kong

Dr. Meyya Meyyappan, NASA, USA

P. 27

Thank you!