a-droplet-based-microfluidic-approach-microsphere-pcr-amplification

A Droplet-Based Microfluidic Approach and Microsphere-PCR Amplification for Single-Stranded DNA Amplicons

Droplet-based microfluidics enable the reliable production of homogeneous microspheres in the microfluidic channel, providing controlled size and ...

Chungbuk National University

As a major human pathogen in the Listeria genus, Listeria monocytogenes causes the bacterial disease listeriosis, which is a serious infection caused by eating food contaminated with the bacteria. We have developed an aptamer-based sandwich assay (ABSA) platform that demonstrates a promising potential for use in pathogen detection using aptamers as analytical bioconjugates. The whole-bacteria SELEX (WB-SELEX) strategy was adopted to generate aptamers with high affinity and specificity against live L. monocytogenes. Of the 35 aptamer candidates tested, LMCA2 and LMCA26 reacted to L. monocytogenes with high binding, and were consequently chosen as sensing probes. The ABSA platform can significantly enhance the sensitivity by employing a very specific aptamer pair for the sandwich complex. The ABSA platform exhibited a linear response over a wide concentration range of L. monocytogenes from 20 to 2×10(6) CFU per mL and was closely correlated with the following relationship: y=9533.3x+1542.3 (R(2)=0.99). Our proposed ABSA platform also provided excellent specificity for the tests to distinguish L. monocytogenes from other Listeria species and other bacterial genera (3 Listeria spp., 4 Salmonella spp., 2 Vibrio spp., 3 Escherichia coli and 3 Shigella spp.). Improvements in the sensitivity and specificity have not only facilitated the reliable detection of L. monocytogenes at extremely low concentrations, but also allowed for the development of a 96-well plate-based routine assay platform for multivalent diagnostics.

Analytical bioconjugates, aptamers, enable specific quantitative detection of Listeria monocytogenes.

We validated a single-stranded, DNA aptamer-based, diagnostic method capable of detecting Lipocalin-2 (LCN2), a biomarker from clinically relevant hepatocellular carcinoma (HCC) patient serum, in the sandwich assay format. Nine aptamers (LCN2_apta1 to LCN2_apta9) for LCN2 were screened with SELEX processes, and a sandwich pair (LCN2_apta2 and LCN2_apta4) was finally chosen using surface plasmon resonance (SPR) and dot blotting analysis. The result of the proposed aptamer sandwich construction shows that LCN2 was sensitively detected in the concentration range of 2.5-500 ng mL(-1) with a limit of detection of 0.6 ng mL(-1). Quantitative measurement tests in HCC patients were run on straight serum and were compared with the performance of the conventional antibody-based ELISA kit. The aptamer sandwich assay demonstrated an excellent dynamic range for LCN2 at clinically relevant serum levels, covering sub-nanogram per mL concentrations. The new approach offers a simple and robust method for detecting serum biomarkers that have low and moderate abundance. It consists of functionalization, hybridization and signal read-out, and no dilution is required. The results of the study demonstrate the capability of the aptamer sandwich assay platform for diagnosing HCC and its potential applicability to the point-of-care testing (POCT) system.

Aptamer-based Sandwich Assay and its Clinical Outlooks for Detecting Lipocalin-2 in Hepatocellular Carcinoma (HCC).

We fabricated droplet-based microfluidic platform for copolymerizable microspheres with acrydite modified DNA probe. The copolymerizable 3-D polyacrylamide microspheres were successfully produced from microcontinuous-flow synthesis with on-channel solidification. DNA copolymerization activity, surface presentation and thermostability were assessed by using fluorescent labeled complementary probe. The binding performance was only visible on the surface area of oligo-microspheres. We show that the resulting oligo-microspheres can be directly integrated into a streamlined microsphere-PCR protocol for amplifying ssDNA. Our microspheres could be utilized as a potential material for ssDNA analysis such as DNA microarray and automatic DNA SELEX process. 

On-Flow Synthesis of Co-Polymerizable Oligo-Microspheres and Application in ssDNA Amplification.

Polycyclic aromatic hydrocarbons (PAHs) are commonly present xenobiotics in natural and contaminated soils. We studied three (phenanthrene, naphthalene, and biphenyl) xenobiotics, catabolism, and associated proteins in  DJ77 by two-dimensional gel electrophoresis (2-DE) analysis. Comparative analysis of the growth-dependent 2-DE results revealed that the intensity of 10 protein spots changed identically upon exposure to the three xenobiotics. Among the upregulated proteins, five protein spots, which were putative dehydrogenase, dioxygenase, and hydrolase and involved in the catabolic pathway of xenobiotic degradation, were induced. Identification of these major multifunctional proteins allowed us to map the multiple catabolic pathway for phenanthrene, naphthalene, and biphenyl degradation. A part of the initial diverse catabolism was converged into the catechol degradation branch. Detection of intermediates from 2,3-dihydroxy-biphenyl degradation to pyruvate and acetyl-CoA production by LC/MS analysis showed that ring-cleavage products of PAHs entered the tricarboxylic acid cycle, and were mineralized in  DJ77. These results suggest that  DJ77 completely degrades a broad range of PAHs via a multiple catabolic pathway.

Proteomic Analysis of Polycyclic Aromatic Hydrocarbons (PAHs) Degradation and Detoxification in  DJ77.

Due to the limitation of rapid development of specific antiviral drug or vaccine for novel emerging viruses, an accurate and rapid diagnosis is a key to manage the virus spread. We developed an efficient and rapid method with high specificity for the Middle East Respiratory Syndrome coronavirus (MERS-CoV), based on one-pot reverse transcription loop-mediated isothermal amplification (one-pot RT-LAMP). A set of six LAMP primers [F3, B3, FIP, BIP, LF (Loop-F), and LB (Loop-B)] were designed using the sequence of nucleocapsid (N) gene with optimized RT-LAMP enzyme conditions: 100 U M-MLV RTase and 4 U  polymerase, implying that the reaction was able to detect four infectious viral genome copies of MERS-CoV within a 60 min reaction time period. Significantly, EvaGreen dye has better signal read-out properties in one-pot RT-LAMP reaction and is more compatible with DNA polymerase than SYBR green I. Isothermally amplified specific N genes were further evaluated using field-deployable microchamber devices, leading to the specific identification of as few as 0.4 infectious viral genome copies, with no cross-reaction to the other acute respiratory disease viruses, including influenza type A (H1N1 and H3N2), type B, human coronavirus 229E, and human metapneumovirus. This sensitive, specific and feasible method provides a large-scale technical support in emergencies, and is also applied as a sample-to-detection module in Point of Care Testing devices.

One-Pot Reverse Transcriptional Loop-Mediated Isothermal Amplification (RT-LAMP) for Detecting MERS-CoV.

The potential copper binding sites in aptamers have been predicted on the basis of secondary structures and the binding affinity of aptamers with copper. Out of the 4 aptamers (Cu-A1 to Cu-A4) selected by SELEX and examined in the present study, the Cu-A2 aptamer shows the highest binding affinity to copper with the lowest K value of 1.83 × 10 M. In order to confirm the binding of copper to the proposed region, the binding affinity was experimentally validated using mutation and deletion analysis. We have confirmed that the high G-C pairing patterns and short stem-interval distance play important roles in copper binding. Aptamer specificity was also verified against diverse heavy metals. We also demonstrate an Aptamer Integrated Recovery Platform (AIRP) to recover copper from acidic mine drainage. AIRP can be easily regenerated at least 20 times without significant deterioration of the retrieval performance. To the best of our knowledge, AIRP is the first demonstration of copper specific recovery using aptamers. This can be scaled up and would have diverse applications in metal contaminated water treatment, recovery and as a potential biosensor for environmental analysis, monitoring, and risk assessment.

Defining the copper binding aptamotif and aptamer integrated recovery platform (AIRP).

In this paper, a Whole-Bacteria SELEX (WB-SELEX) strategy was adopted to isolate specific aptamers against . Real-time PCR amplification and post-SELEX experiment revealed that the selected aptmers possessed a high binding affinity and specificity for . Of the 21 aptamers tested, the C(t) values of the SS-3 and SS-4 aptamers (Ct = 13.89 and Ct = 12.23, respectively) had the lowest value compared to other aptamer candidates. The SS-3 and SS-4 aptamers also displayed a binding affinity () of 39.32 ± 5.02 nM and 15.89 ± 1.77 nM, respectively. An aptamer-based fluorescent biosensor assay was designed to detect and discriminate  cells using a sandwich complex pair of SS-3 and SS-4. The detection of  by the aptamer based fluorescent biosensor platform consisted of three elements: (1) 5'amine-SS-4 modification in a 96-well type microtiter plate surface (-oxysuccinimide, NOS) as capture probes; (2) the incubation with  and test microbes in functionalized 96 assay wells in parallel; (3) the readout of fluorescent activity using a Cy5-labeled SS-3 aptamer as the detector. Our platform showed a significant ability to detect and discriminate  from other enteric species such as ,  and other  species (, ). In this study, we demonstrated the feasibility of an aptamer sensor platform to detect  in a variety of foods and pave the way for its use in diagnosing shigellosis through multiple, portable designs.

Detecting and Discriminating Shigella sonnei Using an Aptamer-Based Fluorescent Biosensor Platform.

We demonstrate an aptablotting assay method that involves direct and indirect aptabody recognition. Nanoscale single-stranded DNA aptamers against GST and DIG-tags are utilized as aptabodies (GST-2 and DIG-1, respectively), and the GST-2 aptabody binding site, or aptatope, as predicted by a MOE-docking simulation of the protein-aptamer complex, shows the interaction of the GST-2 aptabody at the catalytically active region. The aptabody-aptatope interaction was evaluated by an in vitro enzyme inhibitory analysis. The binding capacity of the GST-2 aptabody was assessed by dot-blot, EMSA and SDS-PAGE/electroblot analyses, and the results showed that the aptabodies interact with both the native mono-/dimeric form and the denatured GST form on a membrane. The use of aptabodies can overcome the obstacles of current immunoblot assays, and these molecules are easily assessable via ELISA systems. Moreover, the hybridization of aptabodies and antibodies (hybrid-aptablotting) may have considerable impacts on the design of bioassay platforms.

Aptabody-aptatope interactions in aptablotting assays.

Sphingobium chungbukense DJ77 is a Gram-negative bacterium has metabolic capability of producing exopolysaccharide (EPS) as a potential reducing and stabilizing agent for metallic nanoparticle synthesis. In this study, we investigated the genomic and proteomic analysis to verify metabolic pathway and involved genes and enzymes related to EPS biosynthesis in S. chungbukense DJ77. End-sequencing results of randomly selected fosmid library, which were prepared from high molecular weight DNA of S. chungbukense DJ77, showed identity to sequences from genes related the EPS biosynthesis pathways in several bacteria. We also observed that proteomic responses in S. chungbukense DJ77 by heterogeneously expressing gelA and gelN involved in gellan biosynthesis in Sphingomonas elodea. Comparative two-dimensional gel electrophoresis revealed that both GelA and GelN altered internal expression levels of proteins involved in EPS biosynthesis in S. chungbukense DJ77. The results might provide the genomic and proteomic evidences for presence of EPS biosynthesis pathways in S. chungbukense DJ77.

Genomic and Proteomic Evidences for Exopolysaccharide Biosynthesis in  DJ77.

Shigella sonnei isolate invasion plasmid antigen protein, IpaH, was successfully expressed in recombinant overexpression bacterial system. The soluble expression IpaH was enhanced with molecular chaperon co-expressed environment. Specific aptamer IpaH17 was isolated through the SELEX process and showed fM binding affinity. IpaH17-SPR biosensor platform was involved to verify the binding sensitivity and specificity. The IpaH concentration dependent IpaH17-SPR sensor response was highly linear with a linear regression constant of 99.4% in the range between 0 and 100 ng/mL. In addition, S. sonnei revealed the specific RU value and detected in a real-time manner within 1 hour. Our study indicated that IpaH17-SPR sensor can allow for rapid, sensitive and specific determination of Shigella sonnei virulent factor.

Aptamer-Immobilized Surface Plasmon Resonance Biosensor for Rapid and Sensitive Determination of Virulence Determinant.

In this paper, whole-bacteria SELEX (WB-SELEX) strategy was adopted to isolate specific aptamers against Vibrio parahaemolyticus. Round selection for V. parahaemolyticus was conducted 11 rounds, including two negative selection rounds. It was determined through real-time PCR amplification and post-SELEX experiment. The selected aptmers had high binding property and specificity to V. parahaemolyticus. Of 28 aptamers tested, VPCA-apta#1 had the highest binding affinity compared to other aptamer candidates obtained. To detect V. parahaemolyticus, aptamer based SPR biosensor platform was constructed and pathogenic bacteria sensing was conducted in two steps. The first step was to construct 5'-biotinylated VPCA-apta#1 binding probe. The second step was to incubate V. parahaemolyticus and test microbes in functionalized SA sensor chip in parallel. Our platform showed significant activity for detecting and discriminating V. parahaemolyticus from other enteric species such as Escherichia coli, Listeria monocytogenes, Sigella sonnei, and Vibrio fischeri. This is the first report on the use of whole-SELEX to isolate DNA aptamers specific for V. parahaemolyticus. We demonstrated the feasibility of using aptamer platform for the detection of V. parahaemolyticus in various food supplies. It might be used in multiple points of care for diagnosing Vibriosis.

Surface Plasmon Resonance Aptamer Biosensor for Discriminating Pathogenic Bacteria .

Aptamer-based paper strip sensor for detecting Vibrio fischeri was developed. Our method was based on the aptamer sandwich assay between whole live cells, V. fischeri and DNA aptamer probes. Following 9 rounds of Cell-SELEX and one of the negative-SELEX, V. fischeri Cell Aptamer (VFCA)-02 and -03 were isolated, with the former showing approximately 10-fold greater avidity (in the subnanomolar range) for the target cells when arrayed on a surface. The colorimetric response of a paper sensor based on VFCA-02 was linear in the range of 4 × 10 to 4 × 10 CFU/mL of target cell by using scanning reader. The linear regression correlation coefficient ( R) was 0.9809. This system shows promise for use in aptamer-conjugated gold nanoparticle probes in paper strip format for in-field detection of marine bioindicating bacteria.

Aptamer-Based Paper Strip Sensor for Detecting Vibrio fischeri.

Bacterial exopolymer Levan (β-(2,6) polyfructan) synthesized by levansucrase has attracted interest for various applications due to its low intrinsic viscosity compared with other polysaccharides. We report a novel levansucrase (Lsc) isolated from Sphingobium chunbukense DJ77 and verify its biochemical characteristics by comparative analysis of molecular docking analysis (MOE) and catalytic residue analysis. The complete sequence of the Lsc encoding gene ( lsc) was cloned under the direction of the T7 promoter and purified in an Escherichia coli BL21 (DE3) protein expression system. The enzyme activity analysis and ligand docking MOE study of S. chungbukense DJ77 Lsc revealed that Arg 77, Ser112, Arg 195, Asp196, Glu257, and Gln275 were involved in the sucrose binding and splitting as well as transfructosylation activity. A catalytic comparison of Lsc of S. chungbukense DJ77 with the results of site-directed mutational analysis indicated that Gln275 may coordinate a favorable substrate binding environment, offering broad pH resistance in the range of 5-10. The results suggest that the recombinant E. coli carrying S. chungbukense DJ77 Lsc might produce levan under the regular growth conditions with less need for pH manipulation.

Molecular Docking Analysis and Biochemical Evaluation of Levansucrase from Sphingobium chungbukense DJ77.

Apple Scar Skin Viroid (ASSVd), a nonprotein coding, circular RNA pathogen is relatively difficult to detect by immunoassay. We report here a one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay to improve selectivity for diagnostic use in detecting ASSVd in plants. ASSVd RT-LAMP was accelerated using loop primers and was found to be highly sensitive with a detection limit of 10 copies of cDNA-ASSVd within 30 min. Real-time LAMP and melting curve analysis could differentiate between the true-positive LAMP amplicons and false-positive nonspecific primer amplification products. The optimized RT-LAMP was then followed by the addition of nonthiolated AuNP:poly-adenine (A10)-ASSVd LAMP barcodes, showing a high authentication capacity with colorimetric changes. This type of barcoding assay is a potential alternative for rapid and multiple viroid diagnosis, providing for visible sensing in the field that can be applied to viroid-free planting.

Ji-Young Ahn

College of Natural Sciences

Chungbuk National University

A Droplet-Based Microfluidic Approach and Microsphere-PCR Amplification for Single-Stranded DNA Amplicons

Ji-Young Ahn

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}College of Natural Sciences

Chungbuk National University

A Droplet-Based Microfluidic Approach and Microsphere-PCR Amplification for Single-Stranded DNA Amplicons

College of Natural Sciences