Min-Cheol Lim

A nanoporous membrane-based impedimetric immunosensor for label-free detection of pathogenic bacteria in whole milk

We introduce a nanoporous membrane based impedimetric immunosensor for the label-free detection of bacterial pathogens in whole milk. A simple and rapid method to modify a commercially available alumina nanoporous membrane with hyaluronic acid (HA) effectively reduced the non-specific binding of biomolecules and other cells, and permitted successful immobilization of antibodies. Escherichia coli O157:H7, one of the most harmful food-borne pathogenic bacteria, was tested as a model pathogen in this study. The ionic impedance of electrolytes through nanopores, due to antibody-pathogen interactions, was monitored by impedance spectra and analyzed by normalized impedance change (NIC). The regression equation for the NIC at 1 kHz versus concentration of E. coli O157:H7 (10-10(5)cfu/ml) was obtained, and the detection limit found to be as low as 10 cfu/ml. In addition, the proposed immunosensor was successfully used for the detection of E. coli O157:H7 in whole milk samples with the detection limit as low as 83.7 cfu/ml with 95% probability. The specificity of the immunosensor was also demonstrated using non-target bacteria, including Staphylococcus aureus, Bacillus cereus, and non pathogenic E. coli DH5α. This study shows that a HA-functionalized nanoporous membrane-based impedimetric sensor is capable of detecting pathogenic bacteria in whole milk without any pretreatment. This is a significant step for evaluating the safety of food and environmental samples and other medical diagnostics.

A Low-Noise Solid-State Nanopore Platform Based on a Highly Insulating Substrate

A solid-state nanopore platform with a low noise level and sufficient sensitivity to discriminate single-strand DNA (ssDNA) homopolymers of poly-A40 and poly-T40 using ionic current blockade sensing is proposed and demonstrated. The key features of this platform are (a) highly insulating dielectric substrates that are used to mitigate the effect of parasitic capacitance elements, which decrease the ionic current RMS noise level to sub-10 pA and (b) ultra-thin silicon nitride membranes with a physical thickness of 5 nm (an effective thickness of 2.4 nm estimated from the ionic current) are used to maximize the signal-to-noise ratio and the spatial depth resolution. The utilization of an ultra-thin membrane and a nanopore diameter as small as 1.5 nm allow the successful discrimination of 40 nucleotide ssDNA poly-A40 and poly-T40. Overall, we demonstrate that this platform overcomes several critical limitations of solid-state nanopores and opens the door to a wide range of applications in single-molecule-based detection and analysis.

Elucidation of molecular interactions between lipid membranes and ionic liquids using model cell membranes

Ionic liquids (ILs) are often considered to be green solvents based on their unusual stability, although their toxicity to living organisms has become an emerging issue based on a number of recent studies. We assume that one of the main reasons for this high level of cell toxicity is the molecular interactions between ILs and cell membranes. In this study, we used model cells to demonstrate that ILs can incorporate into lipid membranes, resulting in the perturbation of membrane structure. We employed various methods to elucidate the molecular interactions between cell membranes and ILs. Our results demonstrate that the stability of cell membranes is inversely related to the alkyl chain length and concentration of ILs, providing important information for the design of greener and safer ILs.

Chromatic biosensor for detection of phosphinothricin acetyltransferase by use of polydiacetylene vesicles encapsulated within automatically generated immunohydrogel beads.

We developed a simple and sensitive colorimetric biosensor in the form of microparticles by using polydiacetylene (PDA) vesicles encapsulated within a hydrogel matrix for the detection of phosphinothricin acetyltransferase (PAT) protein, which is one of the most important marker proteins in genetically modified (GM) crops. Although PDA is commonly used as a sensing material due to its unique colorimetric properties, existing PDA biosensors are ineffective due to their low sensitivity as well as their lack of robustness. To overcome these disadvantages, we devised immunohydrogel beads made of anti-PAT-conjugated PDA vesicles embedded at high density within a poly(ethylene glycol) diacrylate (PEG-DA) hydrogel matrix. In addition, the construction of immunohydrogel beads was automated by use of a microfluidic device. In the immunoreaction, the sensitivity of antibody-conjugated PDA vesicles was significantly amplified, as monitored by the unaided eye. The limit of detection for target molecules reached as low as 20 nM, which is sufficiently low enough to detect target materials in GM organisms. Collectively, the results show that immunohydrogel beads constitute a promising colorimetric sensing platform for onsite testing in a number of fields, such as the food and medical industries, as well as warfare situations.

Identifying the Location of a Single Protein along the DNA Strand Using Solid-State Nanopores

Solid-state nanopore has been widely studied as an effective tool to detect and analyze small biomolecules, such as DNA, RNA, and proteins, at a single molecule level. In this study, we demonstrate a rapid identification of the location of zinc finger protein (ZFP), which is bound to a specific locus along the length of a double-stranded DNA (dsDNA) to a single protein resolution using a low noise solid-state nanopore. When ZFP labeled DNAs were driven through a nanopore by an externally applied electric field, characteristic ionic current signals arising from the passage of the DNA/ZFP complex and bare DNA were detected, which enabled us to identify the locations of ZFP binding site. We examined two DNAs with ZFP binding sites at different positions and found that the location of the additional current drop derived from the DNA/ZFP complex is well-matched with a theoretical one along the length of the DNA molecule. These results suggest that the protein binding site on DNA can be mapped or that genetic information can be read at a single molecule level using solid-state nanopores.

Amylosucrase-mediated synthesis and self-assembly of amylose magnetic microparticles

This paper reports a one-step bottom-up approach for the preparation of amylose magnetic beads (AMBs) via enzymatic synthesis and a self-assembly process of amylose and iron oxide nanoparticles. The resulting AMBs were highly effective in the column free purification of target protein with high binding capacity and recyclability.

Biological preparation of highly effective immunomagnetic beads for the separation, concentration, and detection of pathogenic bacteria in milk.

We introduce a system for the efficient separation and concentration of pathogenic bacteria using biologically prepared immunomagnetic beads. Amylose magnetic beads (AMBs) were synthesized by an enzymatic reaction of amylosucrase from Deinococcus geothermalis (DGAS). The simple and rapid conjugation of AMBs and antibodies was achieved by the MBP-SPG fusion protein. MBP (maltose binding protein) binds to the surface of an AMB owing to its intrinsic affinity to the di-glucose in the AMB. SPG (streptococcal protein G) fused to the MBP has specific affinity to the Fc region of the antibody. Anti-Escherichia coli O157 antibodies were conjugated to the AMBs through a MBP-SPG linker without any physical and chemical treatments. The efficiency of separation and concentration of the target E. coli O157:H7 by the functionalized AMBs was revealed by plating counting, conventional polymerase chain reaction (PCR), and real-time RCR analysis. The immuno-AMBs effectively separated and concentrated the target bacteria from a commercial milk sample spiked with known number of bacteria, which was then analyzed by PCR to a detection limit of 10CFU/mL. On the other hand, no PCR product was produced when milk was introduced directly to a PCR reaction. These results show that MBP-SPG is an effective linker and the resulting immuno-AMBs are capable of separating and concentrating the target bacteria from a food matrix.

Enzymatic synthesis of amylose nanocomposite microbeads using amylosucrase from Deinococcus geothermalis

We introduce a biological approach to prepare pure amylose microbeads using the amylosucrase from Deinococcus geothermalis. SWCNTs could be incorporated readily into the amylose structure during enzymatic synthesis to form well-defined amylose–SWCNT composite microbeads through a self-assembly process of the synthesized amylose molecules and SWCNTs.

Effect of short-chain fatty acids on the formation of amylose microparticles by amylosucrase

Amylose microparticles can be produced by self-assembly of amylose molecules through an amylosucrase-mediated synthesis. Here we investigated the role of short-chain fatty acids in the formation of amylose microparticles and the fate of these fatty acids at the end of the reaction. The rate of self-assembly and production yields of amylose microparticles were significantly enhanced in the presence of fatty acids. The effect was dependent on the length of the fatty acid carbon tail; butanoic acid (C4) was the most effective, followed by hexanoic acid (C6) and octanoic acid (C8). The amylose microparticles were investigated by carrying out SEM, XRD, Raman, NMR, FT-IR and DSC analysis. The size, morphology and crystal structure of the resulting amylose microparticles were comparable with those of amylose microparticles produced without fatty acids. The results indicated the carboxyl group of the fatty acid to be responsible for promoting the self-assembly of amylose chains to form microparticles. The fatty acids were eventually removed from the microstructure through the tight association of amylose double helices to form the amylose microparticles.

Inactivation of the virulence factors from 2,3-butanediol-producing Klebsiella pneumoniae

The microbiological production of 2,3-butanediol (2,3-BDO) has attracted considerable attention as an alternative way to produce high-value chemicals from renewable sources. Among the number of 2,3-BDO-producing microorganisms, Klebsiella pneumoniae has been studied most extensively and is known to produce large quantity of 2,3-BDO from a range of substrates. On the other hand, the pathogenic characteristics of the bacteria have limited its industrial applications. In this study, two major virulence traits, outer core LPS and fimbriae, were removed through homologous recombination from 2,3-BDO-producing K. pneumoniae 2242 to expand its uses to the industrial scale. The K. pneumoniae 2242 ∆wabG mutant strain was found to have an impaired capsule, which significantly reduced its ability to bind to the mucous layer and evade the phagocytic activity of macrophage. The association with the human ileocecal epithelial cell, HCT-8, and the bladder epithelial cell, T-24, was also reduced dramatically in the K. pneumoniae 2242 ∆fimA mutant strain that was devoid of fimbriae. However, the growth rate and production yield for 2,3-BDO were unaffected. The K. pneumoniae strains developed in this study, which are devoid of the major virulence factors, have a high potential for the efficient and sustainable production of 2,3-BDO.