QUANTITATIVE BIOCHEMICAL ASSAY ON A SURFACE

MICROFLUIDIC DEVICE

Ravi Prakash, Dipankar Chugh and Karan V. I. S. Kaler

Department of Electrical and Computer Engineering, Schulich School of Engineering, University of Calgary

2500 University Drive NW, Calgary, (T2N 1N4), Canada

Keywords: Surface Microfluidics (SMF), Dielectrophoresis (DEP), Biochemical Assay, DNA, Lab-on-a-chip, Droplets.

Abstract: Quantitative analysis of chemical and biochemical molecules is an important requirement in many

biochemical assays and can be a challenging task in microfluidic systems due to the smaller sample

volumes. In the present study, we report on the detection and quantification of nucleic acid samples

contained in nanoliter (nL) and picoliter (pL) droplets, formed by employing a DEP based surface

microfluidic system. This surface microfluidic system utilizes non-uniform AC electric fields for dispensing

multiple, nanoliter (and picoliter) sized aliquots of samples and reagents, which can furthermore be

individually addressed, transported and mixed on-chip in a controlled and parallel fashion. Quantification of

dsDNA samples is carried out using a fluorescence based Quant-IT

™

PicoGreen

assay, performed on the

surface microfluidic chip, while the low-level fluorescence emissions are quantified using a photo-multiplier

tube. Our findings show that sample DNA concentrations remain uniform across the dispensed droplets,

although the volume of droplets can be varied as per requirements. Experimental results furthermore prove

that our DEP based and electric field assisted on-chip mixing methodology is at par with conventional

mixing strategies such as vortexing, stirring etc. and more readily achieved compared to conventional closed

channel microfluidic systems.

1 INTRODUCTION

Advances in microfabrication of microfluidic

devices has given rise to the capability of

performing biochemical assays and analysis using

small amount of samples, giving rise to the concept

of lab-on-a-chip (LOC), in which sample pre-

treatment, transportation, reaction, separation and

detection can be achieved on a common platform.

These microfluidic LOC devices provide significant

advantages over conventional approaches in

handling, processing and sample analysis. The

miniaturization specifically provides low

consumption of reagents and samples, portability,

low power consumption, high throughput screening,

disposability, low cost and potential for automated

and remote operation as a result of further system

level integration.

Until recently, such microfluidic devices have

employed microchannels etched in glass, silicon or

other polymeric materials, to handle and process low

amounts of fluidic samples, aided by valves and

pumps to transport the sample or reagents (Hong and

Quake 2003). In contrast, a new class of surface

microfluidic (SMF) devices have emerged, which

have circumvented the need for microchannels to

confine fluids and furthermore require no external or

on-chip pumping or valving. These SMF devices

employ AC electric fields, typically in the low to

mid-frequency regions of the frequency spectrum, to

manipulate aqueous dielectric media, in form of

droplets, on top of suitably tailored hydrophobic

surfaces. The actuation and manipulation of liquids

and droplets in these devices specifically leverage

the motive forces of dielectrophoresis (DEP) or

Electrowetting (EW) to affect sample handling and

offer an attractive alternative to conventional closed

channel microfluidic devices, by providing high

speed dispensing of multiple, equi-volume,

nanoliter sized sample droplets (Pollack et al. 2002,

Jones 2001).

More recently, Chugh and Kaler have shown the

integration of Liquid DEP (L-DEP) based droplet

dispensing with the Droplet DEP (D-DEP)

conveyance scheme to demonstrate automated

binary mixing of two different equi-volume liquid

sample droplets at specific reaction sites (Chugh and

Prakash R., Chugh D. and V.I.S. Kaler K. (2010).

QUANTITATIVE BIOCHEMICAL ASSAY ON A SURFACE MICROFLUIDIC DEVICE.

In Proceedings of the Third International Conference on Biomedical Electronics and Devices, pages 51-58

DOI: 10.5220/0002589500510058

 SciTePress

Kaler 2008, 2009). In this article we report on

further enhancements to the L-DEP sample handling

and manipulation scheme, including the ability of

dispensing different quantities of reagents, by

employing a tapered L-DEP electrode structure. The

tapered L-DEP electrodes, detailed in later sections,

were designed to facilitate automated dispensing of

different sets of daughter droplets from the same

parent but differing in volume. This automated

variable volume sample droplet dispensing scheme

is especially beneficial for chip based biochemical

assays that require different amounts of sample or

reagents to be mixed or titrated.

The utility and capabilities of the variable

volume droplet dispensing is further demonstrated

by performing a DNA-PicoGreen® assay, facilitated

by the judicious integration of three sets of fishbone

shaped electrodes, for transporting the dispensed

sample and reagents droplets to specific reaction

sites. The effectiveness of mixing of the DNA and

PicoGreen (PG) sample droplets was quantified by

detecting the fluorescence signal emitted as a result

of DNA-PG binding using a photomultiplier tube

(PMT). Quantitative gradient of DNA sample was

clearly reflected in fluorescent intensity gradient in

different mixed droplets. Further details of the SMF

devices and measurement systems used to validate

the variable volume dispensing and subsequent

manipulation of the daughter droplets is provided in

the materials and methods sections.

2 BACKGROUND

Dielectrophoresis (DEP) is an electromechanical

phenomenon, manifested as result of the interaction

of the spatially non-uniform electric fields with

polarizable materials (Pohl 1978). Non-uniform

electric fields act on polar molecules, impelling

them to regions of higher field strength. Although

the origins of DEP can be dated back to the late 19

century (Pellat 1895), it is more recently that DEP

has been usefully employed in SMF devices as a

means of manipulating and transporting polarizable

liquids on-chip (Ahmed and Jones 2006). The liquid

and droplet DEP actuation methodology, underlying

physical principles and electrode design parameters

have been reported previously (Jones et al. 2001,

Gunji et al. 2004). In the present study, a tapered L-

DEP electrode structure was utilized for dispensing

variable volume droplets as shown in Figure 2.1

(Chugh and Kaler 2009). These L-DEP electrodes,

serve as a fluidic track for the ‘parent’ sample

droplet pipetted at one end of the structure. On

applying an AC voltage across the coplanar

Figure 2.1: Tapered L-DEP actuation scheme 1.

electrodes, a liquid jet is formed from the sample

droplet and conveyed rapidly towards the opposite

end, covering regions of high field intensity. Once

the jet has reached the opposite end, the applied

voltage is removed and the jet disintegrates into

smaller sized ‘daughter’ droplets at the semi-circular

bump sites, as shown in Figure 2.1. The spacing

between the bumps and jet break-up is governed by

Rayleigh’s instability criteria (

Lord Rayleigh 1879).

Further manipulation and transport of these

dispensed daughter droplets was achieved through

droplet-DEP actuation, developed by (Gunji et al.

2004).

In the present study, we have leveraged liquid

and droplet DEP sample actuation schemes to

dispense, transport, mix nano/picoliter droplets and

execute biochemical assays on our DEP based SMF

device.

3 EXPERIMENTAL

3.1 Device Fabrication

Surface microfluidic devices, used in the present

study, were fabricated using commercially available

4” silicon wafers passivated by 5μm film of SiO

The device consists of L-DEP and D-DEP

microelectrode structures, fabricated in two different

metal layers (Aluminum) separated by a dielectric

film (600nm thick Si

), sandwiched between them

for electrical isolation. For the ‘bottom’ layer

electrodes, a 200nm thick layer of Al was sputtered

on the passivated silicon substrate. Microelectrode

structures were patterned using standard

photolithography procedure (Thirukumaran and

Kaler 2007). Thereafter, a thin layer of silicon

nitride (600 nm) was deposited on top of the Al

microelectrodes using plasma enhanced chemical

vapor deposition (PECVD) technique. A second

layer of Al (200nm) was sputter deposited and

BIODEVICES 2010 - International Conference on Biomedical Electronics and Devices

patterned to obtain the ‘top’ layer microelectrode

structures, followed by a second Si

deposition

(400 nm) using PECVD. Finally, the top surface of

the microfluidic chip was rendered hydrophobic by

spin coating a thin layer (~ 0.1 μm) of Teflon® AF

2400 (DuPont Inc., USA), which is critical for

reliable D-DEP actuation (Gunji et al. 2004).

3.2 Sample Preparation

Detection and quantification of dsDNA samples was

carried out using a fluorescence based Quant-IT™

PicoGreen

assay (Molecular Probes™, Invitrogen,

USA). PicoGreen

(PG) is a well-known fluorescent

nucleic acid stain that selectively binds to dsDNA

(Zipper et al. 2004). PG has a large enhancement in

its fluorescence emission on binding to dsDNA, with

excitation and emission maxima at 488nm and

520nm, while unbound PG has virtually no

measurable fluorescence. A 200ng/μL stock DNA

sample was prepared by suspending lyophilized

plasmid DNA samples of pUC57 (GenScript, USA)

in 1mM Tris solution, prepared fresh in de-ionized

(DI) water on the day of the experiment. The pH of

the Tris solution was adjusted to 7.5 using a 50mM

MES solution. Dilutions of the stock DNA sample

were made in Tris-MES solution. Final plasmid

DNA concentrations were measured using a

NanoDrop UV-Visible spectrophotometer (Thermo

Scientific, USA). PG

reagent was supplied as a 1mL

concentrated solution in dimethylsulfoxide (DMSO).

Based on the supplier’s protocol and concentration

of DNA sample used for on-chip assays, a working

sample of the dye was prepared by making a 4 fold

dilution of the concentrated dye (in DMSO) in Tris-

MES solution. A small amount of Tween

20 was

added to all samples to minimize surface adsorption.

3.3 Experimental Setup

The experimental setup is comprised of a reflected

fluorescence microscope system (BX51, Olympus,

Japan), optically coupled to a CCD color camera

and a Photomultiplier Tube (H7468-01, Hamamatsu,

Japan) to facilitate fluorescence intensity

measurements. A block diagram of the optical setup

is schematically illustrated in Figure 3.3.1. The CCD

camera was replaced by a high-speed camera

(MS70K, Mega Speed, Canadian Photonics Inc.) for

recording Liquid and Droplet-DEP actuations, when

required. Electrical connections to the SMF chip

were enabled using spring loaded contact pins and

high voltage relays (9104 series, COTO Technology,

USA) assembled on a printed circuit board (PCB).

The PCB was mounted on-top of the SMF chip and

CCD

PMT

50/50 beam splitter

ex. filter

470-490 nm

em. filter

λ > 520 nm

dichroic mirror

500 nm

surface

μfluidic chip

parent sample

droplet

20X objective

motorized XYZ stage

Power

Amplifier

Signal

Generator

LabVIEW

Interface

PCB with

contact pins

daughter droplet

Data extraction

field iris

diaphragm

light source

CCD

PMT

50/50 beam splitter

ex. filter

470-490 nm

em. filter

λ > 520 nm

dichroic mirror

500 nm

surface

μfluidic chip

parent sample

droplet

20X objective

motorized XYZ stage

Power

Amplifier

Signal

Generator

LabVIEW

Interface

PCB with

contact pins

daughter droplet

Data extraction

field iris

diaphragm

light source

Figure 3.3.1: Schematic of the experimental setup.

the completed assembly was housed on a motorized

microscope stage (Optiscan

ES103, Prior

Scientific, USA).

A signal generator (TGA 1244, TTi, UK) and

high-voltage amplifier (Precision Power Amplifier

5205A, Fluke) provided the AC voltages required

for liquid and droplet actuations. Furthermore, a

software driver, developed in LabVIEW (National

Instruments, USA) was utilized to program the

signal generator for timed and sequential application

of AC voltages and controlling liquid and droplet

DEP actuations.

3.4 Device Operation

In the present study, two different sets of

experiments were performed to investigate (a) the

variations in DNA (pUC57) concentration, in the

individual daughter droplets, dispensed using the

tapered L-DEP electrode structure (scheme 1) and

(b) on-chip droplet dispensing and mixing of DNA

and PG daughter droplets, together with

fluorescence intensity measurements (scheme 2).

For the first set of experiments, utilizing scheme

1, a DNA+PG sample was prepared off-chip, in

micro-centrifuge tube and vortexed to thoroughly

mix the two components. A 1 μL droplet of this

mixed sample was manually pipetted at one end of

the tapered L-DEP electrode as shown in Figure 2.1.

An AC voltage (500 Vrms @ 100 kHz) is briefly

(40–100 ms) applied across the L-DEP electrodes to

dispense nano and picoliter sized ‘daughter’

droplets. The amount of DNA (moles) in each

daughter droplet can be co-related to the intensity of

QUANTITATIVE BIOCHEMICAL ASSAY ON A SURFACE MICROFLUIDIC DEVICE

fluorescence emissions observed from the daughter

droplet. Thus to study DNA concentration

uniformity in the dispensed daughter droplets of

different volume, each droplet was individually

observed using the optical set-up illustrated in

Figure 3.3.1. Fluorescent emission from each

daughter droplet is detected and quantified using a

PMT. To ensure consistency and measure low-level

fluorescent emissions from the daughter droplets, the

PMT is operated at a constant high gain value

(

0.7 10× ) with a fixed optical path and constant

light intensity. A field iris diaphragm was used to

restrict the diameter of the incident light beam

illuminating the daughter droplets, which excludes

extraneous light from entering the objective, thereby

improving signal to noise ratio (S/N). The iris

aperture was adjusted to circumscribe the largest

daughter droplet (formed on the w = g = 40 μm L-

DEP electrode) and kept same for all other smaller

daughter droplets.

Figure 3.4.1: 1x1 matrix of tapered L-DEP and D-DEP

structures for on-chip assay (Scheme 2).

In the second set of experiments, utilizing

scheme 2, unmixed sample droplets (1 μL) of DNA

and PG are individually pipetted over L-DEP

structure, as shown schematically in Figure 3.4.1.

For executing on-chip sample and reagent mixing,

fishbone shaped D-DEP electrodes are integrated

with the L-DEP electrodes. Daughter droplets of

both DNA and PG are formed by employing the

tapered L-DEP electrode, as mentioned previously.

For mixing the DNA and PG daughter droplets, a

lower frequency AC voltage (120 Vrms @ 20 Hz) is

applied across the D-DEP electrodes, which

transports the daughter droplets from semi-circular

bumps. Selected video frames showing the

integrated liquid and droplet actuation scheme are

illustrated in Figure 4.4.1. All experiments were

performed under a low viscosity silicone oil bath

(200

FLUID, 5CST, Dow Corning) to prevent

Figure 3.4.2: Volumetric measurement of daughter

droplets.

rapid evaporation of daughter droplets.

Volumetric measurement for the dispensed

daughter droplets was required in order to correlate

the PMT output (I

) with the DNA concentration.

Since the top surface of the SMF chip is coated with

Teflon®, resulting in a highly hydrophobic surface,

droplets dispensed on the surface assume a nearly

spherical shape (contact angle ~110

). However,

droplet’s contact angles have strong dependence on

biomolecular species (such as enzymes, proteins,

cells etc.), which can get adsorbed on the surface

and reduce the contact angle (Prakash and Kaler

2008). Thus in order to estimate daughter droplet

volumes, a visual inspection of droplets under high

magnification objective (20X) was performed. A

visual inspection helped distinguish and measure the

two different radii of curvature of the daughter

droplets (r: radius of curvature at the plane where the

droplet contacts the surface and R: actual radius of

the spherical droplet) as shown in Figure 3.4.2. The

measured radii values were used to formulate and

quantify the volume (V) of the daughter droplets

using Eqn. 1.

000

sin (1 )

VrdddrR

πϕ

θφ

φφθ π

−

===

−

==+

∫∫∫

(1)

4 RESULTS AND DISCUSSION

4.1 Sub-nanoliter Droplet Dispensing

and Bio-sample Quantification

A tapered L-DEP structure (refer scheme1) was used

to dispense droplets of mixed DNA-PG sample with

volumes 2.4 nL, 0.9 nL and 0.25 nL, as shown in

Figure 4.1.1. The droplet volumes were estimated

using equation 1, described above. In order to

confirm that sample concentration remains invariant

for all the dispensed droplets irrespective of the

droplet volume, photocurrent values corresponding

to different sized daughter droplets and 4 different

DNA concentrations were measured and are

summarized in Table 1.

BIODEVICES 2010 - International Conference on Biomedical Electronics and Devices

Figure 4.1.1: Bright field and fluorescent images of

different sized daughter droplets containing DNA-PG

complex; (a, b) Bright field and fluorescent image of all

the three sections of tapered L-DEP electrode; (c, d)

Bright field and fluorescent image of 40-30 tapered

electrode section; (e, f) Bright field and fluorescent image

of 30-20 tapered electrode section.

In general, fluorescence emission from the DNA-

PG complex is reported to be linearly proportional to

the quantity of DNA (Singer et al. 1997). Therefore,

since I

is directly related to the amount of DNA (or

number of moles) in daughter droplets, the ratio

/vol. provides a direct measure of sample DNA

molar concentration for each daughter droplet.

Figure 4.1.2: Plots showing PMT output for different sized

daughter droplets of different conc. DNA-PG solutions.

These values, along with the ratio of PMT

photocurrent (I

) and droplet volume are tabulated in

Table 1. We found that the ratio (I

/vol.) remains

constant for all the different sized daughter droplets

(corresponding to a specific DNA concentration)

confirming that the DNA concentration in each of

these daughter droplets remains invariant. This was

further evident from the plot of output current (I

)

vs. measured droplet volume, shown in Figure 4.1.2,

Table 1: Experimental results extracted using scheme 1.

Sample

w = g

(micron)

(μA)

(nL)

/vol.

Correlation

factor

25 ng/μL

DNA with

50X PG

40-40 10.97 2.4 4.57 5.47

30-30 3.99 0.9 4.43 5.65

20-20 1.19 0.25 4.77 5.24

50 ng/μL

DNA with

50X PG

40-40 22.77 2.5 9.11 5.49

30-30 8.76 0.9 9.73 5.14

20-20 2.21 0.25 8.83 5.66

75 ng/μL

DNA with

50X PG

40-40 33.71 2.4 14.05 5.34

30-30 12.57 0.9 13.96 5.37

20-20 3.24 0.25 12.98 5.78

100 ng/μL

DNA with

50X PG

40-40 43.68 2.4 18.20 5.49

30-30 16.53 0.9 18.36 5.45

20-20 4.63 0.25 18.52 5.40

Variance 0.04

where a constant slope (I

/vol.) was observed for

each of the four different DNA sample

concentrations (25, 50, 75 and 100 ng/μL).

From this plot and Table 1, we then estimate a

correlation factor to normalize I

/vol. ratio against

different DNA concentrations, the inverse of which

provides a measure of photo-current per picogram of

DNA sample. The above findings suggest that using

tapered L-DEP scheme, we can dispense multiple

sample droplets with different amounts (moles) of

DNA sample, while keeping the concentration same

as the parent sample. DNA concentrations were

quantified in the dispensed daughter droplets ranged

from 2.4 nL to 0.25 nL, a capability that is not

readily achieved by today’s microfluidic devices.

4.2 Reliability of the L-DEP Scheme

for Multiplexed Applications

Having shown that DNA concentrations are

invariant for daughter droplets dispensed using L-

DEP actuation, we further assess the reliability and

repeatability of DEP based droplet dispensing

scheme. For this, we conducted several experiments

on simple L-DEP dispensing scheme (scheme 1)

with the same parent sample conc. (50 ng/μL DNA-

PG) to see the reliability and repeatability of our

scheme in dispensing uniform conc. daughter

droplets.

The results of 6 different set of experiments with

the same parent DNA sample droplet over different

but, identical L-DEP electrode structures are shown

plotted in Figure 4.2.1. The results clearly show a

QUANTITATIVE BIOCHEMICAL ASSAY ON A SURFACE MICROFLUIDIC DEVICE

reliable dispensing of 6-8 daughter droplets with

equal concentration (confidence of fit ~92.5%).

The high speed of L-DEP actuation (40 msec for

dispensing an array of sub-nanoliter droplets) and

the hydrophobic Teflon® coated surface minimizes

surface adsorption (Prakash and Kaler 2008) for

biological sample and the accuracy in the size and

concentration of dispensed daughter droplets makes

the scheme suitable for use in an on-chip

multiplexed biochemical assay device.

Figure 4.2.1: Plot showing repeatability of L-DEP based

sub-nanoliter droplet dispensing scheme.

4.3 On-chip Mixing of Constant

Volume Sample and Reagent

Droplets using D-DEP

One of the objectives of our DEP based SMF device

is to extend the idea of mixing to implement chip-

based multiplexed assays where several different

samples and reagents are mixed in all possible

combinations in a parallel, automated fashion.

Effective mixing of samples and reagents is an

essential step involved in all chemical and

biochemical applications. Mixing on-chip is not as

readily achieved compared to macroscopic mixing

of samples, where vortexing or stirring actions could

be easily exploited. This is due to the small size, low

sample/reagent volumes and laminar flow

characteristics of the conventional closed-channel

microfluidic devices resulting in a slow and

diffusion limited mixing. In contrast, SMF devices

are capable of mixing sub-nanoliter volumes of

sample and reagent droplets more efficiently (Fair et

al. 2003). We use D-DEP actuation, where electric

field mediated stirring action (electroconvective

effects) takes place during droplet transportation and

facilitates mixing once the droplets come in contact

and merge. To demonstrate this, we used an

integrated Liquid and Droplet-DEP structure (shown

boxed in Figure 3.4.1) which was elaborated in the

experimental section. Three different concentrations

of the DNA sample along with PG dye were used.

Roughly 0.6 nL volume of DNA sample and the PG

dye was first dispensed using the L-DEP dispensing

scheme. The DNA sample and PG daughter droplets

thus formed were then moved towards the

reaction/mixing site using D-DEP actuation.

Fluorescence emissions were recorded in real-time

as the two daughter droplets mixed and are shown in

Figure 4.3.1. We observed a steady increase in the

PMT output which indicates that all the three

different conc. of DNA sample demonstrate a nearly

similar mixing or, ligand binding kinetics (evident

from the identical slope of PMT current vs. time

curves).

Figure 4.3.1: Plot showing time domain data extracted

from PMT illustrating binary mixing of sample and

reagent daughter droplets.

The PMT output finally saturates and remains

constant, indicating complete and thorough mixing

of DNA sample with the PG reagent. The entire

mixing assay was complete within 4-5 sec from

manual pipetting of the parent sample droplets.

These results clearly suggest that on-chip mixing

can be more readily achieved using our DEP based

SMF device as compared to conventional closed-

channel microfluidic devices which require large

sample volumes (in mL), longer (in mm) and wider

mixing channels, larger mixing time and furthermore

sophisticated pump and valve arrangements (Park et

al. 2006).

4.4 On-chip Variable Volume,

Multiplexed DNA-PicoGreen®

Assay

Having demonstrated the capability of our SMF

device in achieving some of the key sample handling

requirements for an on-chip assay system including,

(1) dispensing arrays of sub-nanoliter sample and

BIODEVICES 2010 - International Conference on Biomedical Electronics and Devices

Figure 4.4.1: Micrographs demonstrating results of on-

chip DNA quantification assay; (a, b)L-DEP actuation and

droplet dispensing; (c) mixed daughter droplets; (d, e, f)

Unmixed DNA sample droplets (along tapered L-DEP

electrodes) and mixed DNA-PG droplets; (g-l) bright field

and fluorescent images of the mixed DNA-PG droplets

showing the fluorescent intensity gradient.

reagent droplets with controllable sample mass and

uniform concentration, and (2) efficient mixing of a

pair of dispensed sample and reagent droplets, we

now demonstrate an on-chip nucleic acid assay by

implementing a 1x1 matrix of tapered L-DEP and D-

DEP electrode structures illustrated in scheme 2

(Figure 3.4.1).

Four different concentrations (25, 50, 75 and 100

ng/L) of DNA sample were actuated over one of

the two tapered L-DEP electrodes to dispense

multiple daughter droplets with uniform

concentrations but varying volumes and different

moles of DNA. On the other tapered L-DEP

electrode, PG sample was actuated in a similar

fashion.

Three different sets of paired fishbone electrodes

were used to simultaneously transport sample and

reagent droplets from each of the three different

steps of the tapered L-DEP electrode structure to the

corresponding reaction sites under the influence of

an externally applied electric field, to facilitate

DNA-PG sample mixing (Figure 3.4.1). The entire

process was conducted within 2-4 seconds of

actuation (Figure 3.4.1 and Figure 4.4.1).

Figure 4.4.2: Plots showing PMT output for on-chip DNA-

PG assay using experimental scheme 2, for different DNA

sample concentrations.

Table 2: Results extracted from on-chip DNA-PG assay

experiments employing scheme 2.

DNA

conc.

w = g

(micron)

(μA)

vol.

(nL)

/vol.

Correlation

factor

ng/μL

50X

40-40 5.96 1.4 4.26 5.87

30-30 2.78 0.6 4.63 5.40

20-20 1.13

0.1

4.51 5.54

ng/μL

40-40 12.44 1.4 8.88 5.63

30-30 5.49 0.6 9.15 5.46

20-20 2.15

0.1

8.60 5.81

ng/μL

40-40 17.85 1.4

12.7

5.88

30-30 7.71 0.6

12.8

5.83

20-20 3.25

0.1 13.0

5.77

100

ng/μL

40-40 24.50 1.4

17.5

5.71

30-30 11.26 0.6

18.7

5.33

20-20 4.47

0.1 17.8

5.59

Variance 0.04

The PMT output current (I

) corresponding to the

mixed droplets was plotted against the volume of the

individual DNA daughter droplets, measured prior to

mixing and shown in Figure 4.4.2. The ratio I

/vol.

correlates to the DNA concentration in the dispensed

daughter droplets. Constant slopes for each of the

different DNA concentrations used, indicate that the

ratio of output current (I

) and DNA droplet volume

and hence DNA concentration, remains constant for

each of the mixed droplets. Ip/vol. values and the

corresponding correlation factor for on-chip DNA-

PG assay are reported in Table 2. These values of

correlation factor reported in Table 2 are

furthermore in good agreement with the values of

correlation factor reported in Table 1 for the

corresponding off-chip mixed DNA-PG droplets,

suggesting that on-chip quantification assay is

successfully achieved.

QUANTITATIVE BIOCHEMICAL ASSAY ON A SURFACE MICROFLUIDIC DEVICE

5 CONCLUSIONS

In this study, we have successfully demonstrated the

utility of a tapered electrode structure to

dielectrophoretically dispense variable volume

nanoliter to sub-nanoliter sample droplets (2.4 nL to

0.25 nL) on top of hydrophobic surfaces, with

precision. This tapered droplet scheme was

furthermore interfaced with a fishbone droplet

conveyance scheme to demonstrate its utility in

performing a quantitative, multiplexed assay.

The fluidic sample handling capabilities of the SMF

devices reported in this article may be potentially

leveraged for several purposes including drug

discovery, genomics and pathogen detection. This

SMF scheme can also be multiplexed to an m

matrix to achieve HTS capabilities as an alternative

to the existing close channel technology (Thorsen et

al. 2002). The oil bath submerged experimental

setup can be replaced by using the sub-nanoliter

emulsion dispensing scheme, reported by Prakash

and Kaler (2009).

ACKNOWLEDGEMENTS

The authors gratefully acknowledge the financial

support provided by National Science and

Engineering Research Council of Canada (NSERC),

CMC Microsystems and Micralyne Inc. (Canada) in

support of the research work detailed in this article.

Authors furthermore acknowledge the assistance

provided by the Nanofab staff at U of Alberta in

fabricating the SMF devices.

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