On-site processing of single chromosomal DNA molecules using optically driven microtools on a microfluidic workbench

Abstract

We developed optically driven microtools for processing single biomolecules using a microfluidic workbench composed of a microfluidic platform that functions under an optical microscope. The optically driven microtools have enzymes immobilized on their surfaces, which catalyze chemical reactions for molecular processing in a confined space. Optical manipulation of the microtools enables them to be integrated with a microfluidic device for controlling the position, orientation, shape of the target sample. Here, we describe the immobilization of enzymes on the surface of microtools, the microfluidics workbench, including its microtool storage and sample positioning functions, and the use of this system for on-site cutting of single chromosomal DNA molecules. We fabricated microtools by UV lithography with SU-8 and selected ozone treatments for immobilizing enzymes. The microfluidic workbench has tool-stock chambers for tool storage and micropillars to trap and extend single chromosomal DNA molecules. The DNA cutting enzymes DNaseI and DNaseII were immobilized on microtools that were manipulated using optical tweezers. The DNaseI tool shows reliable cutting for on-site processing. This pinpoint processing provides an approach for analyzing chromosomal DNA at the single-molecule level. The flexibility of the microtool design allows for processing of various samples, including biomolecules and single cells.

Optically driven microtools with an antibody‐immobilised surface for on‐site cell assembly

“…Subsequently, antibody modification to the microtools was performed with the method previously reported for enzyme immobilisation. [ 12 ] To introduce carboxyl groups onto the microtool surface, the microtools were treated with UV ozone cleaner [ 17 ] (UV253V8, Filgen) at 20 Pa O 2 for 30 min. Next, 50 mM N‐hydroxysuccinimide (NHS, Sigma‐Aldrich) and 100 mM 1‐ethyl‐3‐ (3‐dimethylaminopropyl) carbodiimide hydrochloride (EDC, Sigma‐Aldrich) were dispensed onto the wafer and incubated [ 18 ] at 20–25°C for 30 min.…”

“…The air trapped in the storage chamber was absorbed into the PDMS as previously reported. [ 12 ] This induces flow from the main channel to the storage chambers, which carries the microtools into the chambers. Once the air is removed from the storage chambers, the flow in the main channel does not enter the chambers, where the tools are stored.…”

“…Subsequently, antibody modification to the microtools was performed with the method previously reported for enzyme immobilisation. [12] To introduce carboxyl groups onto the microtool surface, the microtools were treated with UV ozone cleaner [17] the amino groups in the avidins to the carboxyl groups on the SU-8 surface. After washing with PBS, 1 M ethanolamine (Tokyo Chemical Industry Co., Ltd.) was dispensed and incubated for 30 min to block unreacted groups.…”

“…We previously reported the development of an enzyme‐immobilised microtool for the pinpoint processing of biomolecules, such as a chromosomal DNA molecule. [ 12 ] The microtool was fabricated with a negative photoresist SU‐8, which was driven by optical tweezers [ 13 , 14 , 15 ] and had an enzyme‐immobilised surface. SU‐8 allows for the fabrication of 3D microstructures with a high aspect ratio and is known to have high biocompatibility.…”

Coordination Polymer-Mediated Molecular Surgery for Precise Interconversion of Dicyclobutane Compounds

“…Doctors perform cutting and suturing on specific body parts and scientists can do the same with DNA, − nanomaterials, , C 60 , − and metal organic frameworks (MOFs) , through “molecular surgery”. This strategy allows precise chemical synthesis at the molecular level. − …”