DOT ELISA LOC:

DOT ENZYME LINKED

IMMUNOSORBANT ASSAY LAB ON A
CHIP DEVICE

Pui Choi
Kalie Edelstein
Zac Malberg
PURPOSE
| Minimize reagent volume and waste
| Quicken the response time

| Improve process control

| Reduce costs

| Create a safer platform for biological studies

| Provide portability

| Parallelization and high throughput analysis

ELISA METHODS
| There are many different forms of ELISA to
detect antigen or antibody based on antibody-
antigen interactions.
| An unknown amount of antigen is affixed to a
surface while a specific antibody linked with an
enzyme is washed over the surface allowing it to
bind to the surface antigen. A substance is added
that triggers a detectible signal from the enzyme.
| Current methods utilize a microtiter plate format
and require a relatively high amount of reagent
and sample, heating, repetitive reagent
dispensing, agitation, and spectrophometers for
enzyme detection.
DOT ELISA
| The dot ELISA, a qualitative ELISA test, can be
performed more quickly with the end detection
done visually.
| Dot ELISA is a micro ELISA utilizing antigen
“dotted” onto nitrocellulose filter discs that has
been used for 25 years.
| Because of its relative speed and simplicity, the
dot ELISA is an attractive alternative to
standard ELISA for the initial fabrication of a
LOC ELISA bioMEMS device.
FABRICATION
| Polystyrene Chip
| Straight channels made via CNC machining

| Surface modified via allydextran

| Thin film of PS between two PS sheets to allow

flow
| Nitrocellulose in well
TESTING
o Dot Elisa is qualitative
o Various antigens
o Surface treatments
o Various viscosities
o Positive and Negative controls with varying
incubation times
o Attempt to reduce noise
IMPROVEMENTS
| Automatic temperature control
| Automatic washing

| Standard Buffer properties

| Microcontroller reusability
LIMITATIONS
| Bubble Formation in Channels

| Non-specific binding

| Dot ELISA only qualitative

BUBBLES FORMATION IN
CHANNELS
| Caused by difference in fluid properties of buffers

| Non-uniform distribution of reagents

| Causes faint lines in results

y Reduction in colormetric signal
NON-SPECIFIC BINDING
| Binding of secondary or non-target molecules on
reaction surface
y Low signal-to-noise ratio

| Better immobilization of monoclonal antibody

y Surface modification
DOT ELISA
| Dot ELISA is only qualitative.

| Other ELISA methods, such as sandwich ELISA

would provide quantitative measurements.
BIOCOMPATIBILITY
| This device is an in vitro diagnostic device
y Classified under 21 CFR 886 Subpart F:
Immunological test systems
y Class I device
BIOCOMPATIBILITY
| Polystyrene
y Used in the medical device market
y Biocompatible
| Nitrocellulose
y Widely used for antibody immobilization
y Used by Steinhauer et al. to enhance
biocompatibility of surfaces for antibody
microarrays
|
“Nitrocelluloseis also a proven material well
known for its high protein adsorption capacity
and for providing a biocompatible environment.”
REFERENCES
Albert F., M. Toner. Celluar Micropatterns on Biocompatible Materials. Biotechnol. Prog.
14 (1998), p.338-392.

Steinhauer C., A. Ressine, G. Marko-Varga, T. Laurell, C.A.K. Borrebaeck and C. Wingren.

Biocompatibility of surfaces for antibody microarrays: design of macroporous silicon
substrates. Anal. Biochem. 341 (2005), p. 204.

Surface modification of polystyrene biochip for biotin-labelled protein/streptavidin or

neutravidin coupling used in fluorescence assayXin Gao 1, Hans Jörg Mathieu 1 *, Manfred
Schawaller 21Materials Institute, Swiss Federal Institute of Technology Lausanne (EPFL),
CH-1015 Lausanne, Switzerland

An integrated digital microfluidic lab-on-a-chip for clinical diagnostics on human

physiological fluids.Srinivasan V, Pamula VK, Fair RB. Department of Electrical
Engineering, Duke University, 130 Hudson Hall, Durham, NC 27708, USA.
vijay@ee.duke.edu

Real sample analysis on microfluidic devices. Crevillén AG, Hervás M, López MA, González
MC, Escarpa A. Department of Analytical Chemistry and Chemical Engineering,
University of Alcala, E-28871 Alcala de Henares, Madrid, Spain 
QUESTIONS?