Image: Microfluidic bioassay devices are currently the preferred diagnostic tools. They measure concentration of disease biomarkers within a patient sample, such as blood, which is passed across a surface containing immobilized bioreceptors to capture the biomarker. They can indicate the likelihood of a disease based on presence/absence or based on comparison of the biomarker concentration in the sample relative to the normal body level (Image courtesy of Okinawa Institute of Science and Technology Graduate University).
Researchers have developed an improved microcontact printing technology to create an optimal disease diagnostic device of bioreceptor arrays for multiplexed microfluidic assays.
Since efficiency of microfluidic bioassay devices relies on how intact and functional the bioreceptors are, immobilizing these bioreceptors without causing damage has proved daunting. Over the last two decades, microcontact printing, which uses a rubber stamp to immobilize the bioreceptors, has been established as a robust method to create a variety of assays with multiple applications. Yet this method also has its flaws, particularly when utilized at the nano scale of proteins and DNA. At this scale, the techniques currently used compromise resolution, whether by deforming the stamp or damaging the bioreceptors, thus yielding data somewhat unmanageable for diagnostics or other applications.
Now researchers at Okinawa Institute of Science and Technology Graduate University (OIST; Okinawa, Japan) have developed a sequence of printing steps that have rectified these issues. For microcontact printing “you need a stamp, an ink, and a surface, and then you create your pattern on your surface. It’s as simple as that,” said paper first author Shivani Sathish, OIST PhD student. The stamp is made of polydimethylsiloxane (a flexible solid similar to the rubber used in everyday stamps), the ink is (3-aminopropyl)triethoxysilane (APTES; a solution composed of silicon- and oxide-containing molecules), and the surface is glass.
After coating the stamp with the ink, the stamp is pressed onto the glass, and then removed after a short incubation. The result is a patterned layer of APTES on the glass—a checkerboard of regions with or without APTES. Next, a microfluidic device (which contains microchannels configured to guide fluid through specified pathways) is sealed over the patterned glass. Finally, the bioreceptors are chemically linked to the APTES regions within the microfluidic channels. The system is now ready for use as a diagnostic assay by delivering a body fluid sample through the microfluidic device attached to the glass.
The APTES solution has convenient chemistry. “Depending on your bioreceptor of interest, you just have to choose the appropriate chemistry to link the molecule with the APTES,” said Ms. Sathish. One stamp can be used to prepare an assay with the ability to immobilize a variety of different bioreceptors for multiplexing. Thus one stamp allows for multiple tests and diagnoses on a single surface. This feature would be advantageous for diagnosing complex diseases such as cancer, which relies on tests that can detect multiple markers to improve the diagnosis.
Ms. Sathish and colleagues first patterned nanoscale features of APTES using ink made of APTES in water, as opposed to harsh chemicals, which eliminated the stamp-swelling issue. Then, they immobilized the bioreceptors onto the surface as the very last step of the process, after patterning the APTES and attaching the microfluidic device. By attaching the bioreceptors as the final step, the researchers avoided exposing them to extreme and damaging conditions. They then demonstrated the efficacy of the final device by running an assay to capture the biomarkers interleukin 6 and human c-reactive protein, which are often elevated during inflammation.
“The final goal is to create a point-of-care device,” said OIST Professor Amy Shen, who headed the study. “If you get your bioreceptors pre-immobilized within microfluidic devices, you can then use them as diagnostic tools as and when required,” Ms. Sathish continued, “[Eventually] instead of having a whole clinical team that processes your sample…we’re hoping that the patients can do it themselves at home.”
The study, by Sathish S et al, was published April 5, 2017, in the journal Analyst.
Okinawa Institute of Science and Technology Graduate University