Concentration Control in Microfluidics for Neuroscience Applications

Presenter: Ali Hashmi

Advisor: Jie Xu Date: 04.03.2014 School of Engineering and Computer Science, Washington State University

Outline

Background
Neurons Traditional research methods in neuroscience The need for chemical concentration control

Concentration control with microfluidics

Piezoelectric actuation Synchronized pumps

Comparison and suggested improvements

Outline

Background
Neurons Traditional research methods in neuroscience The need for chemical concentration control

Concentration control with microfluidics

Piezoelectric actuation Synchronized pumps

Comparison and suggested improvements

Introduction: Neurons
Neurons transmit information use both chemical and electrical impulses Electrical signals travel along axons Neurotransmitters are released at axon terminals Neurotransmitters either chemically excite neighbouring neurons or repress their activity upon absorption at the dendrites Frequency of chemical/electrical impulse are critical

Source: wikipedia

Traditional research methods in neuroscience

Almost all traditional techniques for neural stimulation involve electrical stimulation
e.g. The first patch clamp technique
Source: wikipedia

Recent techniques in neuroscience such as optogenetics involve light as stimuli to induce neuronal activity
Special protein called channel-rhodopsin

Chemical stimulation remains largely ignored !

Source: NY times

Challenging to achieve precise and rapid chemical concentration control in petri-dishes

The need for chemical concentration control

The effects of neurotransmitters remain a mystery

More than 100 neurotransmitters exist
Acetylcholine associated with learning Endorphins with emotions Functions of many are not known

The imbalance in neurotransmitter concentration can be studied with precise concentration control
Might help in deciphering the cause of various diseases

Platforms to test the effects of drugs and other externally administered substances over long durations

Can microfluidics help?

Microfluidics can enable precise control of small volumes of liquids in microchannels

The flows are extremely slow (low Reynolds number) and laminar
makes manipulation of fluids easy

Concentration gradients can be conveniently generated

local chemical concentration can be varied

Xu and Attinger (2008)

Quake lab (Stanford)

Microfluidics & Neuroscience

Co-culture chambers provide chemical cues to study axon/cell-bodies independently

Taylor et al (2006)

Concentration gradients generated for stimulating neurons have mostly been steady-state

Folch Lab

Outline

Background
Neurons Traditional research methods in neuroscience The need for chemical concentration control

Concentration control with microfluidics

Piezoelectric actuation Synchronized pumps

Comparison and suggested improvements

Objectives

To dispense individual packets of chemical (neurotransmitters or drugs) in a microchannel

To achieve absolute control over spatiotemporal concentration profile

Develop dynamic chemical clamp to study the effects of chemicals on cellular activity

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Dynamic clamp : an artificial neuronal environment

A step toward developing a novel dynamic clamp

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Concentration gradients: piezoelectric actuation

Piezoelectric transducers can enable faster actuation
The shape, amplitude and frequency of the input can be conveniently altered Ease of device fabrication (micromilling and softlithography)

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Fabrication - micromilling

Micro milling
roughness ~500 nm resolution ~5 mm suitable for channel > 50 mm

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Soft lithography

roughness < 100 nm resolution < 5 mm

our device

(Whitesides, Harvard, 1998)

Suitable for channel < 100 mm

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Preliminary design

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Chemical waveform

The graph shows a measure of the ink concentration profile. The duty cycle (50%) is apparent by a slower increase in pixel intensity

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Analysis

The figures show a count of pixels representing the plume for dilatation (left image) and compression (right image) of chamber.

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Chemical waveform contd.

The chemical plume expands rapidly at higher actuation frequencies.

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inferences

chemical waveform may be generated for low actuation frequencies and smaller time periods

diffusion causes the plume to disperse

concentration of chemical in the chamber can vary in time

constant perfusion is necessary to refresh the chemical plume

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1st design iteration

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Chemical waveform

The figures show the intensity profile for the periodic shift in boundary at an input frequency of 1 and 5 Hz and the respective spectrogram

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inferences

concentration gradients were generated via shifting boundaries

No individual chemical packets were observed

Negligible difference between surface tension

Possible ways to break the flow

Modify nozzle geometry Tune the input signal

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Photomask design
4 cm

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Pulsed chemical switch

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Chemical waveform

The plot represents the chemical waveform obtained from the pulsed input.

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Inferences

Concentration waveform does not span the entire width of the channel

Might be helpful for single cell chemical stimulation

Not reliable for stimulating a larger neuronal culture

some further improvements

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Concentration gradients: synchronized pumps

Two programmable syringe pumps can be used in conjunction generate a chemical waveform

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Analytical analysis
= . .

Concentration profile for 1st half cycle

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setup validation

concentration profile

least square fit

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Ca2+ ion nanosensor

(A) schematic showing the calcium ion nanosensor, with carbon nanotubes as the sensing elements (B) image showing the actual device

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Experimental test-rig

Image showing the apparatus: syringe-pumps, probe station and semiconductor device analyzer

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Experimental results

Graph showing the resistance change of CNTs for a 1 Hz ramp Input to the synchronized pumps for a total flow-rate of 0.3 mL/min

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Fourier Analysis

Fourier transform of the signal showing the frequency distribution

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Filtered signal

Filtered signal to compensate for the moving average

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Comparison of systems
Piezoelectric based actuation

high frequency waveforms not robust for long durations of operation

mainly due to mechanical cracks developing in piezoelectric transducer

Chemical concentration not distributed across channel

Synchronized pumps

cannot generate chemical waveforms at higher frequencies

stepping period for the servo motors is limited

difficult to change input signal in real-time robust operation for long time durations uniform concentration across channel

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Suggested improvements

Piezoactuator based chemical switch

transducer with higher blocking force and free displacement can be used relaxation time for PDMS can be varied

Synchronized pump based chemical switch

use pumps with smaller stepping time period a mixer can be installed before CNT sensors

Further design alterations

piezoelectric pumps can be used to generate chemical waveforms at higher actuation frequencies

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Journal Publications
H-index = 5, citations = 46
1.

A. Hashmi*, G. Heiman*, G. Yu, M. Lewis, H. J. Kwon, and J. Xu. Oscillating Bubbles in Teardrop Cavities for Microflow Control. Microfluidics and Nanofluidics , 14, Issue 3-4, p. 591-596 (2013). Y. Xu*, A. Hashmi*, G. Yu, X. Lu, H.J. Kwon, X. Chen, and J. Xu. Microbubble Array for OnChip Worm Processing. Applied Physics Letters 102, p. 023702 (2013). A. Hashmi, G. Yu, M. Reilly-Collette, G. Heiman, and J. Xu. Oscillating Bubbles: a Versatile Tool for Lab on a Chip Applications. Lab on a Chip 12(21), p. 4216-4227 (2012). J. Zhao, A. Hashmi, J. Xu, and W. Xue. A Compact Lab-on-a-Chip Nanosensor for Glycerol Detection. Applied Physics Letters 100(24), p. 243109 (2012). A. Hashmi, A. Strauss, and J. Xu. Freezing of a Liquid Marble. Langmuir 28(28), p.1032410328 (2012). A. Hashmi*, Y. Xu*, B. Coder*, P. A. Osborne, J. Spafford, G. E. Michael, G. Yu, and J. Xu. Leidenfrost Levitation: Beyond Droplets. Scientific Reports 2, article number: 797 (2012). A. Bajwa*, Y. Xu*, A. Hashmi, M. Leong, L. Ho, and J. Xu. Liquid Marbles with In-flows and Out-flows: Characteristics and Performance Limits. Soft Matter 8, p. 11604-11608 (2012). C. M. R. Mesias, G. Yu, H.-J. Kwon, J. Zhao, A. Hashmi, J. Gao, W. Xue, J. Xu and A. Dimitrov. Towards a Dynamic Clamp for Neuro-chemical Modalities (under review). J.W. Jeon, L. Zhang, D. D. Laskar, M. I. Nandasiri, A. Hashmi, J. Xu, R. K. Motkuri, C. A Fernandez, J. Liu, J. L. Lutkenhaus, M. P. Tucker, B. Yang and S. K. Nune, Lignin Derived Nanoporous Carbon for Supercapacitor Applications (under review). A. Hashmi, and J. Xu. On the Quantification of Mixing in Microfluidics (under review).

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* represents equal contributors

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Awards and Achievements

10 publications (3 under review) with 5 as the first author and 2 as the second author

H-index of 5 with 46 citations in the past 2 years of study

Fellowship and assistanceship from Stanford Universitys Bioengineering Program for PhD

2 prestigious NSF travel grants worth 1750 $

Best presentation award for ASME IMECE Microfluidics Symposium

Institutional travel grants and presentation award

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