antibody colocalization microarray

Low cross-reactivity, high sensitivity assays for disease diagnosis

We have developed a microarrayer system for making a multiplex assay free of liability pairs which lead to false positives in current sandwich immunoassays. We are utilizing this platform for profiling the proteins in complex biological samples with high sensitivity, high selectivity and high reliability. We are applying this platform to the study of patient samples in many areas, notably breast cancer and traumatic brain injury in collaboration with clinicians and researchers in oncology (for more information, please see the separate page on our Breast Cancer Research). Recently, we have developed a handheld version of the platform called the Snap Chip that can collectively transfer antibody droplets from microarray to microarray upon storage of pre-spotted slides, thus avoiding the use of a microarray spotter to perform colocalization immunoassays. 

Emerging Projects:

  • Silver amplification as an alternative to fluorescence: By replacing fluorescence with sliver amplification it becomes possible to use a common $100 desktop scanner for microarray readout.
  • Dispersed assays: We are devising novel strategies to reduce cross-reactivity in bead-based dispersed sandwich assays such as used in the Luminex platform.
  • Ultrasensitive assays: We design and develop novel digital assay formats to further improve the limit of detection of our assays.
Silicon micropins for spotting of antibody solutions on the microarray. Different colours are reflected depending on the angle the pin is held at under the microscope.


Silicon micropins for spotting of antibody solutions on the microarray. Different colours are reflected depending on the angle the pin is held at under the microscope.

Image of the nanoplotter used to prepare microarrays.


Image of the nanoplotter used to prepare microarrays.

Schematic process flow for three sandwich assay formats, namely the ELISA (a singleplex assay), multiplexed sandwich assays (MSA) in microarray format, and the antibody colocalization microarray (ACM).


Schematic process flow for three sandwich assay formats, namely the ELISA (a singleplex assay), multiplexed sandwich assays (MSA) in microarray format, and the antibody colocalization microarray (ACM).


Microfluidic Probes

Tailoring the cellular microenvironment

We have developed a microfluidic probe (MFP) that combines the concepts of microfluidics and of scanning probes and allows the user to interface macroscopic samples with microscopic conduits. Here, liquid boundaries formed by hydrodynamic forces underneath the MFP confine a flow of solution that can be used to process large surfaces and objects by scanning across them. This concept is versatile and has been used for protein microarraying, complex gradient-formation, multiphase laminar-flow patterning, erasing, localized staining of cells, contact-free detachment of a single cell, the study of how immune cells function in the presence of infection and how cancer cells migrate, and localized perfusion of tissue slices. Many constraints imposed by the monolithic construction of microfluidic channels can now be circumvented using an MFP, opening up new avenues for microfluidic processing.

For more information on the microfluidic probe and its advances and use, please see related videos in Nature MaterialsNature Communications and a featured video in AIP Biomicrofluidics.

MFP sketch showing two apertures through which a localized flow can be obtained.


MFP sketch showing two apertures through which a localized flow can be obtained.

Microfluidic quadrupole, with flow lines illustrated by fluorescent beads. This flow pattern is obtained using an MFP with four apertures. 


Microfluidic quadrupole, with flow lines illustrated by fluorescent beads. This flow pattern is obtained using an MFP with four apertures. 

Simulation of a confined flow from the microfluidic probe.


Simulation of a confined flow from the microfluidic probe.


Micro & Nano-Scale Protein Patterning

Studying Cell Navigation & Connectivity

To better investigate the role of concentration gradients of proteins in the guidance of neurons in development, much work has been conducted to replicate these gradients in vitro but has thus far produced tools that are insufficient to properly address in vivo gradients. Since most in vivo gradients are substrate-bound, there is a significant drive to devise a method to replicate such gradients in vitro to better study and understand their functioning. We successfully developed a rapid, low-cost, high throughput nanocontact lift-off process which facilitates the patterning of protein features down to 100 nm. We then introduced novel algorithms to form ordered/randomn and monotonic/non-monotonic digital nanodot gradients (DNGs) with a dynamic range of 3.85 orders of magnitude, exceeding previous reports by almost 2 orders of magnitude. The generation of such patterns will allow us to investigate the role of major guidance proteins with applications in nervous system regeneration. Currently, an extension of this research involves incorporating these guidance cues into microelectrode arrays and neural probes, with aims to improve the cell-electrode interface on these devices and increase the signal quality.

Neuron-astrocyte co-culture prepared on "islands" of protein micro-contact printed on a surface.


Neuron-astrocyte co-culture prepared on "islands" of protein micro-contact printed on a surface.

An array of gradients patterned with e-beam lithography on a silicon master mold. 


An array of gradients patterned with e-beam lithography on a silicon master mold. 

Neuron navigation on a digital nanodot gradient of protein.


Neuron navigation on a digital nanodot gradient of protein.


Capillarics

Pre-Programmed Microfluidic Capillary Circuits for Diagnostics

We developed a library of microfluidic capillary elements consisting of different capillary pumps, valves and fluidic resistors. We propose to use this toolbox to make various autonomous capillary systems and present some of their capabilities, which have never been investigated. One of the highly interesting platforms is a point-of-care device for methicillin-resistant Staphylococcus aureus (MRSA) detection in high-risk patients in hospitals. Current methods for diagnosis of bacterial infections, such as real time PCR and culturing methods are slow and limited in the number of tests which can be run and the length of time required. Using capillaric elements, we are developing a self-powered microfluidic platform that is simple to operate, compatible with common clinic laboratories and can be used at the point-of-care to deliver results within hours and allow hospital staff to isolate patients with resistant infections to contain their spread. 

A capillaric circuit photolithographically patterned on a silicon master mold.


A capillaric circuit photolithographically patterned on a silicon master mold.

Our work on capillarics featured on the cover of Lab on a Chip.


Our work on capillarics featured on the cover of Lab on a Chip.

An example of a possible circuit composed of capillaric elements. This circuit is capable of delivering reagents in a sequential manner without the need for manual input or external equipment and could be used, for example, as a diagnostic test.


An example of a possible circuit composed of capillaric elements. This circuit is capable of delivering reagents in a sequential manner without the need for manual input or external equipment and could be used, for example, as a diagnostic test.


Circulating Tumour Cells

Filter-Based Isolation of Circulating Tumour Cells

Circulating tumour cells (CTCs) are recognized as a powerful indicator for cancer prognosis. As the disease progresses, cancer cells are shed from tumours into the bloodstream, where they may undergo physiological, chemical, and genetic changes before colonizing distant tissues for metastasis. We have designed a silicon filter for the microfiltration of CTCs. These filters are fabricated from silicon-on-insulator wafers using photolithography and deep reactive ion etching. The 12 mm-diameter filters contain up to 600,000 uniform pores in a membrane 15 μm thick. Thus far, we have worked with 7-20 μm circular pores. We have also designed reusable microfluidic cartridges into which these silicon filters can be placed. Single stage cartridges designed for live imaging experiments are laser-cut from acrylic and sealed with o-rings and a silicone gasket, but recently we have made the switch to multi-stage cartridges, which are 3D printed in photoactive resin and can hold up to five filters stacked in series for the capture of a heterogeneous population of CTCs. 

The single stage microfiltration cartridge and image of silicon filters.  (a) A schematic of the custom cartridge for live imaging. (b) The silicon filter, patterned with (c) uniform pores. 


The single stage microfiltration cartridge and image of silicon filters.  (a) A schematic of the custom cartridge for live imaging. (b) The silicon filter, patterned with (c) uniform pores. 

An SEM image of a cancer cell trapped on one of the 15 μm pores of the silicon filters.


An SEM image of a cancer cell trapped on one of the 15 μm pores of the silicon filters.


Global Health

Rapid, Low-Cost Diagnostics for Global Health Applications

Infectious diseases are among the most critical health problems for people living in both the developed and developing world. Emerging pathogens, from viral infections such as HIV/AIDS, SARS, and avian influenza, to bacterial infections such as Cholera and Tuberculosis (TB) are a constant and rapidly evolving threat. We seek to address the need for rapid, low-cost diagnostics by building tools for disease testing outside of the hospital or research lab setting. This requires the use of novel materials and systems which may autonomously generate flow or heat, for example.

We aim for the development of a rapid, sample-to-answer qPCR system for detection of multiple infectious diseases. We take the advantage of self-powered miniaturized disposable reaction chambers to increase the speed of amplification, reduce the sample consumption, and minimize sample preparation steps. 

Another emerging project involves the use of common cotton threads with their capillary-driven wicking properties to carry out immunoassays. The assay results become visible to the eye within a few minutes after the sample application. We have established binding curves for C-reactive protein (CRP) in buffer and diluted serum and a limit of detection of 377 pM was obtained. We have also demonstrated the possibility of multiplexing using three knotted threads coated with antibodies against CRP, Osteopontin, and Leptin proteins. The results suggest that thread is a suitable support for making low-cost, sensitive, simple-to-use, and multiplexed diagnostic tests. This research has lead to several publications, i.e. an article in Lab on a Chip, featured in research highlights; an article in Analytical Chemistry.

Photograph of ICAT cartridge with six cotton threads in parallel, one nylon fiber bundle knotted across, and an absorbent pad placed at the end.


Photograph of ICAT cartridge with six cotton threads in parallel, one nylon fiber bundle knotted across, and an absorbent pad placed at the end.

A proof-of-concept of multiplexing an immunoassay on threads for building diagnostic tests from low cost materials.


A proof-of-concept of multiplexing an immunoassay on threads for building diagnostic tests from low cost materials.

An image of the microfluidic PCR tape designed for use with our portable, lowcost PCR thermocycler for low resource settings. 


An image of the microfluidic PCR tape designed for use with our portable, lowcost PCR thermocycler for low resource settings.