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Single cell impedance spectroscopy label free f analysis of cells

The Coulter counter

Each cell that passes through the aperture reduces the current gives a spike. Cell behaves like an insulating sphere. Measuring the size of the current change is used to size and count the cells.

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Microfabricated devices in silicon

Analysis electronics

electrodes

Koch M et al. Microfabricated electrodes for the construction of a four-electrode impedance based Coulter Counter . J Micromech Microeng 9 159161 (1999).

Frequency spectrum of permittivity for cells in suspension (dielectric spectroscopy)

Two dispersions: p -dispersion - interfacial polarisation (MaxwellWagner polarisation). Characterise cell membrane -relaxation - counterion polarisation, dominated by cell surface charges (little studied)

< 100kHz

Frequency / Hz

100kHz to 10MHz

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Flow through devices (micro flow cytometry)

Broad-band dielectric spectroscopy Average Signal from > 106 cells ~

V(j)

Z mix =

V ( j ) I ( j )

Single cell impedance spectroscopy - microchip with dimensions the size of the cell

Single cell analysis in a fluid flow

Typical voltage signal (at one frequency) as cell passes electrodes

Transit time is sub ms, up to 100 cells/sec

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Discrete SINGLE Frequency Analysis

Integrating Optics
Multi-colour fluorescence AND Impedance peda ce

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Analysis

NO PARTICLE (CELL)

Cube of conducting liquid, electrodes top and bottom

Gap g Electrode area A

A C = o r g R= 1g A

G = (g/A) is the inverse geometrical cell constant of the system, (units m-1).

Impedance (or dielectric) spectroscopy of cells

Z mix =
~

V ( j ) 1 = mix G I ( j ) j

In a system where the field is not uniform, replace G with Gf, which takes into account the field divergence, through a modified geometrical cell constant

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Maxwells Mixture Equation

p m  p + 2 m m =   1 p m  p + 2 m 1 + 2

mix = s

1 + 2f CM 1 fCM

mix

 p = j /

The permittivity (capacitance) and conductivity of the cell

the volume fraction, ratio of particle volume to total volume This equation is approximately correct up to 20% volume fraction. 10m diameter cell in a 20m cube is 7% vol fraction.

Shell model and equivalent electrical circuit

Mem, Mem

m , m

i, i
R d

Cell with membrane

Specific Membrane conductance is nearly zero, ignored Specific Membrane capacitance

CMem = o Mem d

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Circuit analysis & Mixture theory

Electrodes
Chip Stray Capacitance

o m 1 3

m i
2 m + i
1

3 m (1 2 )

m, m
CDL

Cm Rm CMem Ri
CDL Current around cell (medium)

: volume
fraction

9 4 0

Intracellular current path

RCMem

4 9

1 2 m

i
1

We measure Zmix

Z mix =

U ( j ) 1 = mix G I ( j ) j

After some maths, using mixture theory, the complex permittivity can be written as electrical circuit components:

 Z mix =

Rm (1 + j Ri CMem ) j Rm CMem + (1 + j Ri CMem )(1 + j Rm Cm )

The individual components are related to the physical properties of the cells through the following table:

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SUMMARY TABLE of Relationships

Size of cell (given a knowledge of Gf )

Cell membrane capacitance

Typical plot of permittivity vs frequency for interfacial relaxation (-relaxation) due to cell membrane charging typical data measured in traditional dielectric spectroscopy.

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Complete system includes electronic circuitry

Two voltage outputs for each applied frequency Real (X) and Imaginary (Y) impedance amplitude,

Frequency spectra for a single CELL taking into account the electronics

Low Frequency (<1MHz): cell volume (affected by EDL) 18 membrane capacitance Medium Frequency (1MHz-10MHz): High Frequency (>10MHz): internal properties (cytoplasm/nucleus)

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Frequency spectrum for latex beads

Solid object, no membrane Impedance dominated by size (and permittivity at h.f.). hf)

Data obtained at two frequencies simultaneously, reference frequency of 500kHz, other frequency swept.

Scatter plots (TWO frequencies)

Opacity Z@2MHz z/Z@500kHz
5.8m 7.18m

Opacity: ratio of HF impedance to LF impedance. Factors out size variations. Sensitive to changes in membrane properties.

10.0m

Size Z@500kHz

The properties of the beads do not vary much with frequency, hence opacity stays constant. There is almost a linear relationship between size and l.f. impedance

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Human Blood

Erythrocytes
red d bl blood d cells ll ~ RBC

5,000,000 RBC per l 4,000 11,000 Leukocytes per l only 150-400 Monocytes per l
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http://www.andrewlost.com/images/atoms_molecules_page/blood_cells_red_back_21361a.jpg

Blood cells capacitance and size

T-lymphocytes

B-lymphocytes

data from Yang J., et al Biophys J, 78 2000. Measured using ROT and DEP

Monocytes

Granulocytes

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Full Blood Count (FBC)

A generic blood test, performed to diagnose disease (virus, cancer) rule out a clinical problem monitor therapy establish a prognosis screen for disease determine effective drug dosage/prevent toxicity. FBC is used to measure properties of blood cells cells. Normally done by light scattering: Bigger cells scatter more light at small angles Granular cells scatter more light than agranular at large angles

Frequency sweeps for white blood cells

Data obtained at two frequencies simultaneously, reference frequency of 500kHz, other frequency swept.

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Scatter plots for WBCs. Each sub-population purified, then re-mixed

Cell m membrane capacita ance

Size

Single cell impedance

Light scatter (FACS)

Whole human blood Erythrocytes removed by lysis

Cell m membrane capacitan nce

Size

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Smaller particles limit is set by noise in the system To measure smaller particles, requires smaller electrode and channel dimensions, increasing
Platelets Beads

RBC
Platelets (1m) and RBCs (5m)

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Can we increase the sensitivity of the system to detect changes in cell phenotype, i.e. using CD antibody expression Currently performed using fluorescently labelled antibodies

Can this be done electrically? Can we replace the fluorescence label with an impedance label

Conjugation of beads to cells beads are impedance labels

+
CD4 conjugated beads

All other cells (CD4-) CD4+ T-Lymphocyte

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Simulation showing change in impedance with increasing binding of beads

Application in medicine - Counting T lymphocyte sub-population to diagnose HIV AIDS

A small number of the lymphocyte population express an antigen called CD4+. A CD4 count of less than 200 cells/l of whole blood establishes diagnosis of AIDS i.e. the CD4+ cells need to be counted to diagnose HIV AIDS Currently performed using expensive flow cytometry (FACS) and fl fluorescent antibodies ib di Can it be done with impedance labels?

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White blood cell prep.

BEFORE ADDING BEADS

AFTER ADDING BEADS

Whole blood

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Comparison with commercial FACS machine

Cell type T-cells with CD4 beads Lymphocytes (non CD4+) Total Lymphocytes Monocytes beads with FACS (% of total) 18.0 (11243/62312) 12.7 (7914/62312)) 30.7 CD4 0.6 (328/62312) 4.3 (2695/62312) 4.9 64.4 (40132/62312) IS (% of total) 15.6 (1215/7789) 12.9 (1005/7789) 28.5 0.8 (66/7789) 5.4 (421/7789) 6.2 65.4 (5095/7789) 4-8 40-70 20-40 N/A Typical Numbers [Wintrobe 1999] 15-20 %

Monocytes (no beads) Total Monocytes Granulocytes

The electrical double layer

R Electrode CDL CDL Electrode

RDL C

RDL

double layer

bulk electrolyte

double layer

The capacitance of the Double layer can be approximated to CDL = r. In PBS, PBS this gives gives.... For a small electrode the D.L. Capacitance leads to a significant series reactance, reducing the signal. Answer: Increase electrode area, either absolute leads to reduced sensitivity or relative, porous electrodes

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Smooth electrodes in PBS

0.5 cm diameter electrodes

Nanostructured Pt electrodes in PBS

0.5 cm diameter electrodes

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Conclusions Impedance spectroscopy measures the dielectric properties of single cells the effective polarisability given by Maxwell Wagner relaxation. This data can be used to characterise cells impedance impedance labelling labelling of cells provides an electrical analogue of fluorescent labelling for enhanced cell identification

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