A Membrane Filter-Assisted Mammalian Cell-Based Biosensor Enabling 3D Culture and Pathogen Detection.

We have developed a membrane filter-assisted cell-based biosensing platform by using a polyester membrane as a three-dimensional (3D) cell culture scaffold in which cells can be grown by physical attachment. The membrane was simply treated with ethanol to increase surficial hydrophobicity, inducing the stable settlement of cells via gravity. The 3D membrane scaffold was able to provide a relatively longer cell incubation time (up to 16 days) as compared to a common two-dimensional (2D) cell culture environment. For a practical application, we fabricated a cylindrical cartridge to support the scaffold membranes stacked inside the cartridge, enabling not only the maintenance of a certain volume of culture media but also the simple exchange of media in a flow-through manner. The cartridge-type cell-based analytical system was exemplified for pathogen detection by measuring the quantities of toll-like receptor 1 (TLR1) induced by applying a lysate of P. aeruginosa and live E. coli, respectively, providing a fast, convenient colorimetric TLR1 immunoassay. The color images of membranes were digitized to obtain the response signals. We expect the method to further be applied as an alternative tool to animal testing in various research areas such as cosmetic toxicity and drug efficiency.

Long-term nationwide assessment of polychlorinated dibenzo-p-dioxins/dibenzofurans and dioxin-like polychlorinated biphenyls ambient air concentrations for ten years in South Korea.

In this study, seasonal/regional variations of Polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/DFs) and dioxin like-polychlorinated biphenyls in the ambient air were monitored for ten years (2008-2017) using a high volume air sampler. As a result of strict regulation enforced by Korea Ministry of Environment in 2008, PCDD/DFs concentrations in the ambient air decreased from 0.051 pg I-TEQ Sm-3 in 2009 to 0.014 pg I-TEQ Sm-3 in 2017 which was comparably associated with cut-down of their emission sources from 880.2 g I-TEQ Sm-3 in 2001 to 24.2 g I-TEQ Sm-3 in 2015; revealing that it was only 2.7% against that of 2001. In 2017, mean TEQ concentration level of PCDD/DFs in the air of South Korea was quite low in comparison to its ambient environmental standards of 0.6 pg I-TEQ Sm-3 for PCDD/DFs. Particularly, the sum of PCDD/DFs in the background revealed the lowest level, however, the fraction of octachlorodibenzodioxin among other isomers exposed at the highest level in this study, suggesting that the ambient air quality in the background being studied was severely and persistently impaired by inflowing unknown sources of any possible anthropogenic transboundary migratory air pollutants. Moreover, this study conducted the scientific analysis of the long-term variations in the ambient air and emission sources using principal component analysis. From this of 10 years long-term nationwide assessments for the PCDD/DFs and dl-PCBs in the ambient air, it is possible to prove that South Korean environmental policy to manage POPs has been successfully conducted for the last ten years.

Spatial distribution, source identification, and anthropogenic effects of brominated flame retardants in nationwide soil collected from South Korea.

Soil samples were collected at 61 sites of the national monitoring network for persistent organic pollutants (POPs) in South Korea. The target compounds were brominated flame retardants (BFRs), including polybrominated diphenyl ethers (PBDEs), polybrominated biphenyls (PBBs), hexabromocyclododecanes (HBCDDs), and tetrabromobisphenol A (TBBPA). The mean concentrations of Σ27 PBDEs, Σ3 HBCDDs, and TBBPA in soil were 222, 17.2, and 4.4 ng/g, respectively, but PBBs were not detected. Industrial sites had statistically higher BFR concentrations than suburban sites but no significant difference compared with urban sites. The commercial deca-BDE mixtures were the most likely source of PBDE contamination in the soil samples, with the minor influence of commercial penta-BDE and octa-BDE mixtures. The profiles of HBCDDs in most soil samples differed from those in the powder types of technical HBCDD mixtures, indicating that they are affected by the HBCDDs contained in commercial products and the conversion of HBCDD diastereoisomers (γ-HBCDD to α-HBCDD) in the environment. The concentrations of Σ27 PBDEs, Σ3 HBCDDs, and TBBPA were significantly correlated with population density, gross domestic product, and the number of companies (p < 0.01), indicating a direct impact of anthropogenic activities. Significant correlations among BFRs were determined (0.63 < r < 0.74, p < 0.01), suggesting that these pollutants had similar sources. Relatively good correlations (0.44 < r < 0.98, p < 0.01) between BDE-209 and other light BDEs (except for BDE-71, -77, -126, -156, and -205) might result from the degradation of heavy BDEs under anaerobic and natural sunlight conditions. To the best of our knowledge, this study provides the most comprehensive soil monitoring data for various BFRs in South Korea. Furthermore, it is the first report on soil contamination by deca-BDE, HBCDDs, and TBBPA in South Korea.

Nationwide levels and distribution of endosulfan in air, soil, water, and sediment in South Korea.

We investigated the levels and distribution patterns of α- and ß-endosulfan and endosulfan sulfate in air, soil, water, and sediment samples collected from the South Korean persistent organic pollutants (POPs) monitoring networks. In the air samples, the highest concentrations of the total (Σ3) endosulfan (50.3-611 pg/m3, mean: 274 pg/m3) were observed during summer. Spearman analysis revealed a good correlation between agricultural land area and atmospheric concentrations of Σ3 endosulfan except during winter. Regardless of the season, the ratio of the two isomers (α/ß) was 3.6-4.9 in the air samples, higher than that observed in technical mixtures (2.0-2.3), possibly due to the higher volatility of α-endosulfan, compared to ß-endosulfan. Concentrations of Σ3 endosulfan in the soil samples (n.d.-13.4 ng/g, mean: 0.8 ng/g) were not significantly different except at some stations adjacent to large areas of farmland. The average levels of Σ3 endosulfan in the water and sediment samples were 2.1 ng/L and 0.1 ng/g dw, respectively. In analyzing the four largest rivers, it was observed that a few water stations during spring and fall and sediment stations in fall had high concentrations of the two isomers and endosulfan sulfate, particularly around the Yeoungsan and Nakdong Rivers near large areas of agricultural land. Endosulfan sulfate was dominant at most water and sediment sampling stations. This study demonstrates that the endosulfan found in most environmental compartments most probably derives from agricultural areas despite its ban as a pesticide. On the other hand, given that it was also detected in industrial and urban areas, in which pesticide application does not occur, it can be conjectured that endosulfan is aerially transported at higher temperatures and continuously circulates within the environment.

Distribution and diastereoisomeric profiles of hexabromocyclododecanes in air, water, soil, and sediment samples in South Korea: Application of an optimized analytical method.

In this study, the levels and distribution patterns of HBCD diastereoisomers in air, water, soil, and sediment samples in South Korea were investigated after optimizing the UPLC-MS/MS analytical process. Extraction and cleanup efficiencies were tested using several different extraction solvents and adsorbents. Dichloromethane was selected as the base extraction solvent, and multi-layer silica gel (MSG) and MSG-alumina columns were selected for the removal of HBCDs from complex environmental matrices. The concentration of Æ©3 HBCDs was 22-133 pg/m3, 10-128 ng/g, 0.2-151 ng/L, and 0.5-552 ng/g dw for air, soil, water, and sediment samples, respectively. Relatively higher concentrations of Æ©3 HBCDs were observed at stations adjacent to industrial facilities (e.g., rubber and plastic, textile, chemical, fabricated metal, and wholesale trade factories) associated with the use of commercial HBCDs. The proportion of γ-HBCD in the soil (48.3-86.2%) and sediment (54.2-78.1%, except for one station) samples was similar to that found in technical and commercial HBCDs. In contrast, α-HBCD (52.3-71.2%) was dominant in all air samples, while the water samples displayed no clear trend in their diastereoisomer profiles. As the first nationwide report on HBCD diastereoisomers in the environment, this study demonstrates that most environmental compartments in South Korea are moderately contaminated with HBCDs.

Characteristics of metal contamination in paddy soils from three industrial cities in South Korea.

Paddy soil contamination is directly linked to human dietary exposure to toxic chemicals via crop consumption. In Korea, rice paddy fields are often located around industrial complexes, a major anthropogenic source of metals. In this study, rice paddy soils were collected from 50 sites in three industrial cities to investigate the contamination characteristics and ecological risk of metals in the soils. The cities studied and their major industries are as follows: Ulsan (petrochemical, nonferrous, automobile, and shipbuilding), Pohang (iron and steel), and Gwangyang (iron and steel, nonmetallic, and petrochemical). Thirteen metals (Al, As, Ba, Cd, Co, Cr, Cu, Fe, Mn, Ni, Pb, V, and Zn) were analyzed using inductively coupled plasma-optical emission spectrometry (ICP-OES). The mean concentration of Cd (1.98 mg/kg) exceeded the soil quality guideline of Canada (1.4 mg/kg), whereas concentrations of other metals were under the standards of both Korea and Canada. Generally, levels of metal concentrations decreased with increasing distance from industrial complexes. Among the three cities, Pohang showed high concentrations of Zn (142.2 mg/kg), and Ulsan and Gwangyang showed high concentrations of Cr (33.9 mg/kg) and Ba (126.4 mg/kg), respectively. These contamination patterns were influenced by the different major industries of each city, which was clearly demonstrated by the principal component analysis results. Pollution indices suggested that As, Cd, Pb, and Zn were enriched in the paddy soils via anthropogenic activities. Comprehensive potential ecological risk indices were at considerable levels for most sites, especially because of major contributions from As and Cd, which can pose potential ecological threats.

Ridge Minimization of Ablated Morphologies on ITO Thin Films Using Squared Quasi-Flat Top Beam.

In this study, we explore the improvements in pattern quality that was obtained with a femtosecond laser with quasi-flat top beam profiles at the ablated edge of indium tin oxide (ITO) thin films for the patterning of optoelectronic devices. To ablate the ITO thin films, a femtosecond laser is used that has a wavelength and pulse duration of 1030 nm and 190 fs, respectively. The squared quasi-flat top beam is obtained from a circular Gaussian beam using slits with varying x-y axes. Then, the patterned ITO thin films are measured using both scanning electron and atomic force microscopes. In the case of the Gaussian beam, the ridge height and width are approximately 39 nm and 1.1 µm, respectively, whereas, when the quasi-flat top beam is used, the ridge height and width are approximately 7 nm and 0.25 µm, respectively.

Fully Solution-Processable Fabrication of Multi-Layered Circuits on a Flexible Substrate Using Laser Processing.

The development of printing technologies has enabled the realization of electric circuit fabrication on a flexible substrate. However, the current technique remains restricted to single-layer patterning. In this paper, we demonstrate a fully solution-processable patterning approach for multi-layer circuits using a combined method of laser sintering and ablation. Selective laser sintering of silver (Ag) nanoparticle-based ink is applied to make conductive patterns on a heat-sensitive substrate and insulating layer. The laser beam path and irradiation fluence are controlled to create circuit patterns for flexible electronics. Microvia drilling using femtosecond laser through the polyvinylphenol-film insulating layer by laser ablation, as well as sequential coating of Ag ink and laser sintering, achieves an interlayer interconnection between multi-layer circuits. The dimension of microvia is determined by a sophisticated adjustment of the laser focal position and intensity. Based on these methods, a flexible electronic circuit with chip-size-package light-emitting diodes was successfully fabricated and demonstrated to have functional operations.

Vibration-Assisted Femtosecond Laser Drilling with Controllable Taper Angles for AMOLED Fine Metal Mask Fabrication.

This study investigates the effect of focal plane variation using vibration in a femtosecond laser hole drilling process on Invar alloy fabrication quality for the production of fine metal masks (FMMs). FMMs are used in the red, green, blue (RGB) evaporation process in Active Matrix Organic Light-Emitting Diode (AMOLED) manufacturing. The taper angle of the hole is adjusted by attaching the objective lens to a micro-vibrator and continuously changing the focal plane position. Eight laser pulses were used to examine how the hole characteristics vary with the first focal plane's position, where the first pulse is focused at an initial position and the focal planes of subsequent pulses move downward. The results showed that the hole taper angle can be controlled by varying the amplitude of the continuously operating vibrator during femtosecond laser hole machining. The taper angles were changed between 31.8° and 43.9° by adjusting the vibrator amplitude at a frequency of 100 Hz. Femtosecond laser hole drilling with controllable taper angles is expected to be used in the precision micro-machining of various smart devices.

Monitoring and risk assessment of polychlorinated biphenyls (PCBs) in agricultural soil from two industrialized areas.

For monitoring and risk assessment, levels and distributions of Σ29 PCBs in paddy soil samples collected from Gwangyang (10 sites) and Ulsan (20 sites), heavily industrialized cities in Korea, were investigated using high-resolution gas chromatography/high-resolution mass spectrometry. Overall, total concentrations of Σ29 PCBs in Gwangyang (216.4-978.6 pg g-1 dw) and Ulsan (273.8-1824.1 pg g-1 dw) were higher than those (106.6-222.6 pg g-1 dw) in agricultural soil from Anseong in Korea. The TEQ (toxic equivalency) values from Gwangyang (0.06-0.40 ng TEQ kg-1 dw) and Ulsan (0.06-0.22 ng TEQ kg-1 dw) were higher than those (0.04-0.11 ng TEQ kg-1 dw) in Anseong but lower than the WHO threshold level (20 ng TEQ kg-1). However, one of the most toxic congeners, PCB 126, gave the highest concentration, possibly posing a risk to the biota. Seven indicator PCB congeners contributed to 50-80% of the total concentration of Σ29 PCBs, indicating the 7 PCBs can be used as valuable indicators for monitoring. The principal component analysis and cluster analysis for the homologue profiles of PCBs indicated that all the samples from both cities had the similar PCB contamination patterns, and the major sources of the PCB contamination were most likely from the usage of Aroclor 1254 than those of Aroclors 1242 and 1260. These PCB technical mixtures were possibly significantly used by various industries including iron and steel industries in Gwangyang and petrochemical and shipbuilding industries in Ulsan.

Chemiluminometric Immunosensor for High-Sensitivity Cardiac Troponin I Employing a Polymerized Enzyme Conjugate as a Tracer.

To detect high-sensitivity cardiac troponin I (hs-cTnI; <0.01 ng/mL) at points of care, we developed a rapid immunosensor by using horseradish peroxidase polymerized in 20 molecules on average (Poly-HRP) as a tracer conjugated with streptavidin (SA-Poly-HRP). As shown in the conventional system, enhanced sensitivity could be achieved by using a sequential binding scheme for the complex formation to contain the huge molecular tracer. We used a 2-dimensional chromatographic technology to carry out the sequential bindings in cross-flow directions. After the complex formation of antigen-antibody with analyte in a vertical direction, SA-Poly-HRP was horizontally supplied across the membrane strip for additional binding via a biotin-SA linkage. The HRP substrate was subsequently supplied along the same direction to produce a chemiluminometric signal, which was measured by a cooled charge-coupled device. Hs-cTnI analysis was completed in this format within 25 min, and the results showed a high correlation with those of the CentaurXP® reference system (R(2) > 0.99). The detection limit of the rapid immunosensor was 0.003 ± 0.001 ng/mL cTnI, corresponding to a 10-fold improvement compared to results using the plain enzyme tracer. This demonstrated the measurement of hs-cTnI in a much more cost-effective manner compared to the automated versions currently available.

An innate immune system-mimicking, real-time biosensing of infectious bacteria.

An animal cell-based biosensor was investigated to monitor bacterial contamination in an unattended manner by mimicking the innate immune response. The cells (RAW 264.7 cell line) were first attached onto the solid surfaces of a 96-well microtiter plate and co-incubated in the culture medium with a sample that might contain bacterial contaminants. As Toll-like receptors were present on the cell membrane surfaces, they acted as a sentinel by binding to pathogen-associated molecular patterns (PAMPs) of any contaminant. Such biological recognition initiates signal transmission along various pathways to produce different proinflammatory mediators, one of which, tumor necrosis factor-α (TNF-α) was measured using an immunosensor. To demonstrate automated bacterium monitoring, a capture antibody specific for TNF-α was immobilized on an optical fiber sensor tip and then used to measure complex formation in a label-free sensor system (e.g., Octet Red). The sensor response time depended significantly on the degree of agitation of the culture medium, controlling the biological recognition and further autocrine/paracrine signaling by cytokines. The response, particularly under non-agitated conditions, was also influenced by the medium volume, revealing a local gradient change of the cytokine concentration and also acidity, caused by bacterial growth near the bottom surfaces. A biosensor system retaining 50 µL medium and not employing agitation could be used for the early detection of bacterial contamination. This novel biosensing model was applied to the real-time monitoring of different bacteria, Shigella sonnei, Staphylococcus aureus, and Listeria monocytogenes. They (<100 CFU mL(-1)) could be detected automatically within the working time. Such analysis was carried out without any manual handling regardless of the bacterial species, suggesting the concept of non-targeted bacterial real-time monitoring. This technique was further applied to real sample testing (e.g., with milk) to exemplify, for example, the food quality control process without using any additional sample pretreatment such as magnetic concentration.

In vitro inflammation inhibition model based on semi-continuous toll-like receptor biosensing.

A chemical inhibition model of inflammation is proposed by semi-continuous monitoring the density of toll-like receptor 1 (TLR1) expressed on mammalian cells following bacterial infection to investigate an in vivo-mimicked drug screening system. The inflammation was induced by adding bacterial lysate (e.g., Pseudomonas aeruginosa) to a mammalian cell culture (e.g., A549 cell line). The TLR1 density on the same cells was immunochemically monitored up to three cycles under optimized cyclic bacterial stimulation-and-restoration conditions. The assay was carried out by adopting a cell-compatible immunoanalytical procedure and signal generation method. Signal intensity relative to the background control obtained without stimulation was employed to plot the standard curve for inflammation. To suppress the inflammatory response, sodium salicylate, which inhibits nuclear factor-κB activity, was used to prepare the standard curve for anti-inflammation. Such measurement of differential TLR densities was used as a biosensing approach discriminating the anti-inflammatory substance from the non-effector, which was simulated by using caffeic acid phenethyl ester and acetaminophen as the two components, respectively. As the same cells exposed to repetitive bacterial stimulation were semi-continuously monitored, the efficacy and toxicity of the inhibitors may further be determined regarding persistency against time. Therefore, this semi-continuous biosensing model could be appropriate as a substitute for animal-based experimentation during drug screening prior to pre-clinical tests.

Two-dimensional paper chromatography-based fluorescent immunosensor for detecting acute myocardial infarction markers.

A novel washing scheme following antigen-antibody reactions with analyte was used during construction of a fluorescent immunosensor to resolve the background problem in the lateral flow assay with human serum. An immuno-membrane strip was devised to simultaneously measure cardiac troponin I (cTnI), creatinine kinase-MB isoform (CK-MB), and myoglobin to diagnose acute myocardial infarction. This strip was then installed within a cartridge containing a built-in washing solution tank, which was used to supply the solution across the signal generation pad of the strip after the immune reactions. Such cross-flow washing was initiated by onset-signaling from the internal control and began to run automatically upon sample addition. Under optimal conditions, the immunosensor displayed a stably suppressed background baseline, enabling us to attain a low detection limit for cTnI (0.05 ng/mL) as well as favorable reproducibility for repetitive measurements (relative standard deviation <10%). No interference was observed among the different complex formations at the respective analyte sites, and no artifacts were caused by sample matrices. We tested the performance relationship with the Pathfast reference system for positive serum samples (36 for cTnI, 58 for CK-MB, and 17 for myoglobin), and the correlation coefficients were >0.98. This result suggests that the new immunosensor system based on two-dimensional chromatography can be used for clinical testing.

Chemiluminometric immuno-analysis of innate immune response against repetitive bacterial stimulations for the same mammalian cells.

For monitoring of human cellular response to repetitive bacterial stimulations (e.g., Pseudomonas aeruginosa in a lysate form), we devised a chemiluminescent immuno-analytical system for toll-like receptor 1 (TLR1) as marker present on cell surfaces (e.g., A549). Upon stimulation, TLR1 recognizes pathogen-associated molecular patterns of the infectious agent and are then up-regulated via activation of the nuclear factor-κB (NF-κB) pathway. In this study, the receptor density was quantified by employing an antibody specific to the target receptor and by producing a chemiluminometric signal from an enzyme labeled to the binder. The activated status was then switched back to normal down-regulated stage, by changing the culture medium to one containing animal serum. The major factors affecting activation were the stimulation dose of the bacterial lysate, stimulation timing during starvation, and up- and down-regulation time intervals. Reiterative TLR regulation switching up to three times was not affected by either antibody remained after immunoassay or enzyme substrate (e.g., hydrogen peroxide) in solution. This immuno-analysis for TLRs could be unique to acquire accumulated response of the human cells to repeated stimulations and, therefore, can eventually apply to persistency testing of the cellular regulation in screening of anti-inflammatory substances.

Rapid immuno-analytical system physically integrated with lens-free CMOS image sensor for food-borne pathogens.

To realize an inexpensive, pocket-sized immunosensor system, a rapid test devise based on cross-flow immuno-chromatography was physically combined with a lens-free CMOS image sensor (CIS), which was then applied to the detection of the food-borne pathogen, Salmonella typhimurium (S. typhimurium). Two CISs, each retaining 1.3 mega pixel array, were mounted on a printed circuit board to fabricate a disposable sensing module, being connectable with a signal detection system. For the bacterial analysis, a cellulose membrane-based immunosensing platform, ELISA-on-a-chip (EOC), was employed, being integrated with the CIS module, and the antigen-antibody reaction sites were aligned with the respective sensor. In such sensor construction, the chemiluminescent signals produced from the EOC are transferred directly into the sensors and are converted to electric signals on the detector. The EOC-CIS integrated sensor was capable of detecting a traceable amount of the bacterium (4.22 × 10(3)CFU/mL), nearly comparable to that adopting a sophisticated detector such as cooled-charge-coupled device, while having greatly reduced dimensions and cost. Upon coupling with immuno-magnetic separation, the sensor showed an additional 67-fold enhancement in the detection limit. Furthermore, a real sample test was carried out for fish muscles inoculated with a sample of 3.3CFU S. typhimurium per 10 g, which was able to be detected earlier than 6h after the onset of pre-enrichment by culture.

Effects of genetic polymorphisms of OPRM1, ABCB1, CYP3A4/5 on postoperative fentanyl consumption in Korean gynecologic patients.

OBJECTIVE: Fentanyl, a µ-opioid receptor agonist, is a substrate of P-glycoprotein. Its metabolism is catalyzed by CYP3A4 and CYP3A5. The aim of this study was to investigate the association between postoperative fentanyl consumption and genetic polymorphisms of µ-opioid receptor (OPRM1), ABCB1 (gene encoding P-glycoprotein), CYP3A4 and CYP3A5 in Korean patients. METHODS: 196 female patients scheduled to undergo total abdominal hysterectomy or laparoscopic assisted vaginal hysterectomy under general anesthesia were enrolled in this study. Intravenous patient-controlled analgesia with fentanyl was provided postoperatively. Cumulative fentanyl consumption was measured during the first 48 hours postoperatively. The severity of pain at rest was assessed with the visual analogue scale. OPRM1 118A>G, ABCB1 2677G>A/T, ABCB1 3435C>T, CYP3A4*18 and CYP3A5*3 variant alleles were genotyped. The effects of genetic and non-genetic factors on fentanyl requirements were evaluated with multiple linear regression analysis. RESULTS: The 24-hour cumulative fentanyl doses were significantly associated with pain core, weight and type of surgery (p < 0.05). The 48-hour cumulative fentanyl doses were significantly associated with pain score, type of surgery and history of PONV or motion sickness (p < 0.05). Genetic polymorphisms were not associated with fentanyl requirements. CONCLUSION: In Korean gynecologic patients, no association was found between genetic factors and postoperative fentanyl consumption.

Label-free, needle-type biosensor for continuous glucose monitoring based on competitive binding.

With the goal of developing a method for the continuous monitoring of blood glucose, an implantable sensor was developed by placing an optical fiber probe within the internal hollow space of a syringe needle. A glucose binder, concanavalin A (Con A), was immobilized on the probe tip and a protein (e.g., bovine serum albumin) chemically coupled with a sugar ligand (e.g., mannose) was loaded as a solution inside of the needle, which were then closed using a semi-permeable membrane. Upon immersion in the glucose sample, small molecules were able to freely pass through the membrane and compete with the ligand conjugate for Con A binding. This changed the molecular layer thickness on the probe surfaces depending on the glucose concentration, which shifted the wavelength of the guided light along the fiber. Such interference in the wavelength pattern was measured using a commercial sensor system, Octet, without employing a label. Using this analytical approach, two major steps controlling the performance of glucose detection were overcome: permeation of glucose (optimum with 50 nm-porous polycarbonate membrane under the experimental conditioned used) and molecular diffusion of the ligand conjugate within the sensor compartment (19 gauge-needle, offering minimal demensions for the probe). Under optimal conditions, the sensor was able to monitor glucose fluctuations, even in serum medium, with a response time of less than 15 min in a range 10-500 mg/dL. This, however, could be further shortened down to about 5 min in principle by miniaturizing the sensor dimensions.

Toll-like receptor-based immuno-analysis of pathogenic microorganisms.

In this study, a novel mammalian cell receptor-based immuno-analytical method was developed for the detection of food-poisoning microorganisms by employing toll-like receptors (TLRs) as sensing elements. Upon infection with bacterium, the host cells respond by expressing TLRs, particularly TLR1, TLR2, and TLR4, on the outer membrane surfaces. To demonstrate the potential of using this method for detection of foodborne bacteria, we initially selected two model sensing systems, expression of TLR1 on a cell line, A549, for Escherichia coli and TLR2 on a cell line, RAW264.7, for Shigella sonnei (S. sonnei). Each TLR was detected using antibodies specific to the respective marker. We also found that the addition of immunoassay for the pathogen captured by the TLRs on the mammalian cells significantly enhanced the detection capability. A dual-analytical system for S. sonnei was constructed and successfully detected an extremely low number (about 3.2 CFU per well) of the pathogenic bacterium 5.1 h after infection. This detection time was 2.5 h earlier than the time required for detection using the conventional immunoassay. To endow the specificity of detection, the target bacterium was immuno-magnetically concentrated by a factor of 50 prior to infection. This further shortened the response to approximately 3.4 h, which was less than half of the time needed when the conventional method was used. Such enhanced performance could basically result from synergistic effects of bacterial dose increase and subsequent autocrine signaling on TLRs' up-regulation upon infection with live bacterium. This TLR-based immuno-sensing approach may also be expanded to monitor infection of the body, provided scanning of the signal is feasible.

Performance characteristics of monoclonal antibodies as recyclable binders to cardiac troponin I.

Acute myocardial infarction is a typical disorder that requires continuous monitoring for early detection of potential life-threatening situations. To this end, we used different methods to screen for rapidly reversible antibodies, among 22 hybridoma clones, against cardiac troponin I (cTnI), which is a specific marker indicating the disease. The dissociation rates of antibodies were underestimated by up to a factor of 1000 because of bivalent binding when tested with the antigen immobilized on solid surfaces. This effect was also observed in a sandwich immunoassay, in which the detection antibody cross-linked with various antigen molecules already bound to the capture antibody. Although multiple binding events contributed to enhanced detection capability, it was difficult to recycle the immunosensor. We then devised a screening system by arranging the test antibody for the capture binder immobilized on a label-free sensor. This enabled us to select fast reactive antibodies of which one (clone 24) was shown to be recyclable, even in serum-containing medium. Using this antibody, repetitive detection of cTnI with a rapid response time (half-life of dissociation: about 4min on average) and high detection capability (0.1ng/ml) was achieved, which is very important for detection in a clinical setting.