Versatile Low-cost Modular Microfluidic Arrays for Cancer

Diagnostics

James F. Rusling

1-4

, Colleen Krause

,Brunah Otieno

, Karteek Kadimisetty

,Chi Tang

Abhay Vaze

and Gregory Bishop

Department of Chemistry (U-3060), University of Connecticut, 55 N. Eagleville Rd., Storrs, Connecticut 06269, U.S.A.

Department of Cell Biology, University of Connecticut Health Center, Farmington, Connecticut 06032, U.S.A.

Institute of Materials Science, University of Connecticut, 97 N. Eagleville Road, Storrs, Connecticut 06269, U.S.A.

School of Chemistry, National University of Ireland, Galway, Ireland

Keywords: Cancer, Biomarkers, Multiplex, Protein Detection, Microfluidics, Nanoparticles.

Abstract: Accurate, sensitive, multiplexed detection of biomarker proteins in serum and tissue holds significant

promise for personalized cancer diagnostics and therapy monitoring. Here we describe fabrication details of

a modular microfluidic system featuring a small chamber for on-line protein capture from serum by

magnetic beads, positioned upstream of a nanostructured multi-sensor array chamber to achieve high

sensitivity for up to eight proteins, with the ability to expand to many more proteins. Microfluidic chambers

are made by templating PDMS channels on machined aluminum molds to avoid lithography, and mounted

in hard plastic housings equipped with inlet and outlet lines and interfaced with valves. Gold immunoarrays

fabricated by screen or ink-jet printing, or wet chemical etching of gold films utilize amperometry or

electrochemiluminescence (ECL) detection. These arrays are interfaced with microfluidics to achieve well-

controlled mass transport leading to excellent signal/noise and unprecedented sensitivities. With interest in

low cost point of care (POC) systems, we developed a module to also facilitate automated microfluidic

reagent and sample delivery utilizing an open source microcontroller and micropumps, with ECL detection

by camera.

1 INTRODUCTION

Measuring diagnostic panels of multiple proteins in

serum holds great promise for future personalized

cancer screening and therapy monitoring (Manne,

Srivastava and Srivastava, 2005) (Rusling, Kumar

and Gutkind, et al. 2010)

(Ludwig and Weinstein,

2005) (Rusling, 2012) (Rusling, Munge, et al. 2013).

It is necessary to measure multiple proteins in

samples from each patient because a single protein

biomarker is subject to too much individual

variability to give highly accurate predictions. Thus,

the potential to measure concentrations of panels of

biomarker proteins for cancer diagnostics has

created great interest in the biomedical community

for some time (Kulasingam and Diamandis, 2008).

(Hanash, Pitteri, and Faca 2008) (Giljohan and

Mirkin, 2009) (Hanash, Baik and Kallioniemi,

2011). Unfortunately, broad realization of such

diagnostic strategies has yet to be achieved. This is

due in a large part to the lack of suitable

inexpensive, sensitive devices to measure multiple

biomarker proteins in patient samples, as well as the

lack of fully validated panels for cancer diagnosis.

For clinical or point-of-care (POC) use, technical

simplicity of protocols and low cost are essential.

Emerging aspects of nanotechnology and materials

science combined with microfluidics provide

exciting new opportunities to design and fabricate

such devices (Rusling, Munge, et al. 2013).

This

paper will describe development of prototype

modular microfluidic systems capable of

ultrasensitive detection of multiple serum proteins

(Figure 1). Unlike our previous publications

describing multiplexed protein detection with some

of these systems, the present paper will focus on

fabrication, system optimization, and new

automation aspects of the devices.

F. Rusling J., Krause C., Otieno B., Kadimisetty K., Tang C., Vaze A. and Bishop G..

Versatile Low-cost Modular Microﬂuidic Arrays for Cancer Diagnostics.

DOI: 10.5220/0004707400720076

In Proceedings of the International Conference on Biomedical Electronics and Devices (BIODEVICES-2014), pages 72-76

ISBN: 978-989-758-013-0

 2014 SCITEPRESS (Science and Technology Publications, Lda.)

2 SYSTEMS FOR PROTEIN

DETECTION

For protein detection, a 100 L modular cylindrical

chamber for on-line protein capture from

serum on magnetic beads is positioned upstream

of a nanostructured multi-sensor array in a 60 L

chamber to achieve high sensitivity for up to eight

proteins (Figures 1 and 2) (Otieno et al., 2014) with

the ability to expand to many more proteins. The

microfluidic chambers are made by templating

PDMS channels on machined aluminum molds to

avoid lithography. The PDMS slabs are mounted in

hard plastic housings equipped with inlet and outlet

lines to construct the desired chambers, and

interfaced with valves. These modular microfluidic

immunoarrays utilizing amperometry or

electrochemiluminescence (ECL) detection

chambers interfaced with microfluidics achieved

well-controlled mass transport leading to excellent

signal/noise and unprecedented sensitivity.

Amperometric multi-sensor array chips were

fabricated by ink-jet printing of 4 nm alkylthiol gold

nanoparticles (€0.20/chip) (Krause, et al., 2013)

commercial screen printing of carbon and coating

with 5 nm glutathione-gold nanoparticles (€7/chip)

(Chikkaveeraiah, et al., 2011), wet-etching to

fabricate gold CD arrays (Tang, et al., 2012), or, for

ECL, microwell-patterning of pyrolytic graphite

chips (€0.20/chip) (Sardesai, et al., 2013). A simple

print/heat/peel method was developed to transfer

computer printed patterns onto the arrays to create

hydrophobic wells as small as 10 nL around each

sensor. Briefly, a toner pattern is printed by a laser

jet onto high gloss paper, and transferred onto the

array substrate in a heat press. These toner

nanowells can hold 1 L solutions to facilitate

building nanostructures and attaching antibodies on

sensor elements while avoiding cross-contamination.

Figure 1: Prototype modular microfluidic system for on-line protein capture and amperometric detection using magnetic

beads. (A) Capture chamber in which target proteins are captured on-line from the sample by heavily labeled enzyme-

antibody-magnetic beads to form protein-bead bioconjugates. These are washed in the chamber while held magnetically,

then transported into the detection chamber (B) in the modular microfluidic system (C). The magnet (D) traps bioconjugate

beads in the channel during injection of sample and washing, and is removed for transfer of beads to the detection chamber.

Changes in the system for ECL detection include the replacement of multi-electrode array with a solid 2.5x2.5 cm pyrolytic

graphite chip with an array of microwells featuring carbon nanotube forests and replacement of the detection chamber with

a similar chamber that features a optical window on the top to facilitate detecting ECL light with a CCD camera.

VersatileLow-costModularMicrofluidicArraysforCancerDiagnostics

These arrays are fitted into the microfluidic PDMS

detection channel, which is attached to a syringe

pump or micropumps, with associated valves to

deliver reagents and samples to the correct

chambers. Magnetic control is used to hold magnetic

beads bioconjugated with antibodies and enzyme

labels in the capture chamber.

Figure 2: Modules of amperometric microfluidic arrays

with on-line protein capture on magnetic beads. (A)

Capture chamber featuring a PDMS-defined 100 L oval

cylinder sandwiched between two hard PMMA plates. A

tiny magnetic stir bar is included for mixing and

redispersing beads. The target proteins are captured on-

line from the sample in this chamber by heavily labeled

enzyme-antibody-magnetic beads to form protein-bead

bioconjugates. These are washed, and then transported

into (B) detection chamber housing an 8 AuNP-coated

electrode sensor array (on right, sensor elements on top,

contacts on bottom) coated with capture antibodies.

Subsequently, samples are injected and flow times

adjusted so as to fill the sample chamber. Washes

are done to minimize non-specific binding, and then

sent to waste. Then, magnetic control is released,

and the beads are transported to the detection

chamber, where a stop-flow incubation period

allows capture by antibodies on the sensors. After

washing, an enzyme activator/mediator solution is

injected to provide low noise, peak-shaped

responses, as shown for a mixture of interleukin (IL)

proteins IL-6 and IL-8 in serum (Figure 3).

Our multilabel approaches feature massively

enzyme- or ECL-dye-labeled particles that greatly

amplify signals for analyte proteins. Detection limits

as low as 5 fg mL

-1

(~200 aM) have been achieved

for simultaneous measurement of four oral cancer

biomarker proteins in a few L of serum in about 1

hr (Malhotra, et al., 2012). Applications to

biomarker proteins for oral cancer, metastasis, and

inflammation have been pursued. For detection of

metastasized cancer during surgery, high sensitivity

can be traded for speed to achieve immunoassays in

8 min (Krause, et al., 2013). The modular system

can be adapted to other measurements such as

oxidized DNA and metabolic toxicity screening by

changing the active films on the detectors.

3 AUTOMATED REAGENT

AND SAMPLE

INTRODUCTION

POC protein detection will require adding further

automation to the above prototypes. We have thus

most recently designed an automated reagent and

sample delivery module for ECL detection of

proteins. The system features six microfluidic

channels that lead to a detection chamber featuring a

patterned pyrolytic graphite-SWCNT chip (Figure

4). Detection is facilitated by an ECL dye embedded

into 100 nm silica nanoparticles and coated with

antibodies to provide amplification and low fg/mL

detection levels using a CCD camera. The entire

device costs ~€500, excluding the CCD camera.

Microwells were fabricated on the PG chip using the

print/heat/peel technology described above.

Integrated micropumps, one per channel, were

connected to a portable sample/reagent loading

cassette with preloaded, serum samples, wash

buffers and dye-silica nanoparticles equipped with

detection antibodies. These air-separated solutions

are pumped into the six-channel measurement

chamber chip with SWCNT wells containing capture

antibody). A microcontroller open-source electronics

prototyping platform (Arduino) interface provides

fully automated control of flow and incubation

times. A panel of 4 cancer biomarkers can be

measured at clinical levels using this approach.

4 CONCLUSIONS

We described inexpensive, versatile microfluidic

devices for multiple protein detection designed in a

BIODEVICES2014-InternationalConferenceonBiomedicalElectronicsandDevices

Figure 3: Amperometric immunoarray results for protein mixtures in undiluted calf serum using an 8-electrode screen-

printed carbon array with AuNP-coated sensors in the device in Figure 1 at -0.2 V vs. Ag/AgCl. Measurements were done

with an 8-electrode CH Instruments potentiostat. Peaks are developed by injecting a mixture of 1 mM HQ and 0.1 mM

to mediate and activate the horseradish peroxidase (HRP) labels on the magnetic beads for (A) IL-6 and (C) IL-8, and

calibration plots for (B) IL-6 and (D) IL-8. Error bars represent standard deviations (n=4) (Adapted from Otieno et al.,

2014).

Figure 4: Modular microfluidic array for ECL detection of proteins showing hardware including automated sample and

reagent loading cassette. The microfluidic array is placed under a CCD camera in a dark box and connected to a power

supply for ECL measurements.

VersatileLow-costModularMicrofluidicArraysforCancerDiagnostics

modular fashion with no lithography required, using

commercial valves and pumps when possible.

Printing, wet etching and print patterning have been

used to fabricate very inexpensive nanostructured

array chips. These devices can be set up in almost

any laboratory at a relatively low cost. However,

additional simplicity of operation is a goal for full

POC implementation. Our specific application is

detection of multiple proteins from serum and cell

lysates for cancer diagnostics, and this can be

achieved at detection limits down to 5 fg/mL

(attomolar levels), up to 200 times lower than

existing commercial multiplexed protein detection

systems (Rusling J. F., Kumar C. V., Gutkind J. S.,

et al. 2010).

ACKNOWLEDGEMENTS

This work is financially supported by grant

EB014586 from the National Institute of

Biomedical Imaging and Bioengineering (NIBIB),

NIH, USA.

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BIODEVICES2014-InternationalConferenceonBiomedicalElectronicsandDevices