Surface‐Modified Mesh Filter for Direct Nucleic Acid Extraction and its Application to Gene Expression Analysis

Abstract

Rapid and convenient isolation of nucleic acids (NAs) from cell lysate plays a key role for onsite gene expression analysis. Here, this study achieves one-step and efficient capture of NA directly from cell lysate by developing a cationic surface-modified mesh filter (SMF). By depositing cationic polymer via vapor-phase deposition process, strong charge interaction is introduced on the surface of the SMF to capture the negatively charged NAs. The NA capturing capability of SMF is confirmed by X-ray photoelectron spectroscopy, fluorescent microscopy, and zeta potential measurement. In addition, the genomic DNAs of Escherichia Coli O157:H7 can be extracted by the SMF from artificially infected food, and fluorescent signal is observed on the surface of SMF after amplification of target gene. The proposed SMF is able to provide a more simplified, convenient, and fast extraction method and can be applied to the fields of food safety testing, clinical diagnosis, or environmental pollutant monitoring.

Nanoscale control by chemically vapour-deposited polymers

“…The fast and efficient capture of nucleic acids from cell lysate was demonstrated using a conventional stainless steel mesh filter or an extraction microchip conformally modified with iCVD poly(2-dimethyaminomethyl styrene). 153,154 Under the conditions of extraction, this iCVD surface has a positively charged functional group [-N + (CH3)2], producing a strong attractions to negatively charged nucleic acids. This method successfully allowed the identification of the DNA from a specific strain of bacteria in multiple types of spoiled food items.…”

Nanoadhesive Layer to Prevent Protein Absorption in a Poly(dimethylsiloxane) Microfluidic Device

“…The thickness of the nanoadhesive layer is less than 200 nm, so the geometry of the microfluidic channel is fully retained (Figure 1B, left). The iCVD process is known to possess high deposition conformality and preserves geometrical features, as demonstrated by several previous reports [37][38][39]. The excellent conformality of the nanoadhesive layer is therefore highly advantageous considering the vast difference between nanoadhesive thickness (0.05-0.5 μm) and typical microfluidic channel dimensions (10-1000 μm).…”

Multifunctional Printable Micropattern Array for Digital Nucleic Acid Assay for Microbial Pathogen Detection

“…In addition to compartmentalization, the functionalities for in situ lysis of target microbial pathogens and instant recovery of the target nucleic acid critically limit the development of sensitive, specific, and quantitative detection using the digital nucleic acid assay. − The previous work showed that surface modification methods based on polymer thin films with positively charged functionalities can be applied to extract nucleic acids from target pathogens for the genetic analysis. , Although the strong electrostatic characteristics of the positively charged polymer surface may enhance the nucleic acid capturing performance, the surface still lacks the capability for multifunctional modification such as surface energy control, additional functionalization, and micropatterning. Therefore, an alternative functional surface with multifunctionality for an effective DNA capture capability is highly desired.…”

“…To fabricate multifunctional micropattern arrays, a new type of positively charged, multifunctional polymer film of poly­(dimethylaminomethyl styrene- co -hydroxyethyl methacrylate) (pDH) was synthesized in the vapor phase, using the photoinitiated chemical vapor deposition (piCVD), a convenient tool for synthesizing functional copolymers with biocompatibility and mechanical and chemical stability. − The copolymer surface can rupture the bacterial membrane and bind DNA via a dimethylaminomethyl styrene (DMAMS) moiety. In addition, the hydroxyl functionality of hydroxyethyl methacrylate (HEMA) can be utilized for the silane-based surface modification. , The composition of the pDH copolymer was systematically tuned by controlling the monomer flow ratio during the piCVD process. − The hydrophobic octadecyltrichlorosilane (OTS)-treated background and hydrophilic pDH micropattern array surface enabled an aqueous solution to be separated into thousands of individual microdroplets without a complicated fabrication procedure. ,,, The multifunctional micropattern array was applied to a digital nucleic acid assay in which a loop-mediated isothermal amplification (LAMP) was performed for a digitized DNA detection. − The microfluidic chip with the multifunctional micropattern array demonstrated an excellent genetic detection performance with the benefits of a simple fabrication, stable and reliable compartmentalization, and high sensitivity and specificity toward real pathogenic bacteria based on a simple procedure without bulky instruments or additional time-consuming steps.…”