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.

Current Technologies of Electrochemical Immunosensors: Perspective on Signal Amplification.

An electrochemical immunosensor employs antibodies as capture and detection means to produce electrical charges for the quantitative analysis of target molecules. This sensor type can be utilized as a miniaturized device for the detection of point-of-care testing (POCT). Achieving high-performance analysis regarding sensitivity has been one of the key issues with developing this type of biosensor system. Many modern nanotechnology efforts allowed for the development of innovative electrochemical biosensors with high sensitivity by employing various nanomaterials that facilitate the electron transfer and carrying capacity of signal tracers in combination with surface modification and bioconjugation techniques. In this review, we introduce novel nanomaterials (e.g., carbon nanotube, graphene, indium tin oxide, nanowire and metallic nanoparticles) in order to construct a high-performance electrode. Also, we describe how to increase the number of signal tracers by employing nanomaterials as carriers and making the polymeric enzyme complex associated with redox cycling for signal amplification. The pros and cons of each method are considered throughout this review. We expect that these reviewed strategies for signal enhancement will be applied to the next versions of lateral-flow paper chromatography and microfluidic immunosensor, which are considered the most practical POCT biosensor platforms.

Current Technical Approaches for the Early Detection of Foodborne Pathogens: Challenges and Opportunities.

The development of novel and high-tech solutions for rapid, accurate, and non-laborious microbial detection methods is imperative to improve the global food supply. Such solutions have begun to address the need for microbial detection that is faster and more sensitive than existing methodologies (e.g., classic culture enrichment methods). Multiple reviews report the technical functions and structures of conventional microbial detection tools. These tools, used to detect pathogens in food and food homogenates, were designed via qualitative analysis methods. The inherent disadvantage of these analytical methods is the necessity for specimen preparation, which is a time-consuming process. While some literature describes the challenges and opportunities to overcome the technical issues related to food industry legal guidelines, there is a lack of reviews of the current trials to overcome technological limitations related to sample preparation and microbial detection via nano and micro technologies. In this review, we primarily explore current analytical technologies, including metallic and magnetic nanomaterials, optics, electrochemistry, and spectroscopy. These techniques rely on the early detection of pathogens via enhanced analytical sensitivity and specificity. In order to introduce the potential combination and comparative analysis of various advanced methods, we also reference a novel sample preparation protocol that uses microbial concentration and recovery technologies. This technology has the potential to expedite the pre-enrichment step that precedes the detection process.

A High-Performance Fluorescence Immunoassay Based on the Relaxation of Quenching, Exemplified by Detection of Cardiac Troponin I.

The intramolecular fluorescence self-quenching phenomenon is a major drawback in developing high-performance fluorometric biosensors which use common fluorophores as signal generators. We propose two strategies involving liberation of the fluorescent molecules by means of enzymatic fragmentation of protein or dehybridization of double-stranded DNA. In the former, bovine serum albumin (BSA) was coupled with the fluorescent BODIPY dye (Red BSA), and then immobilized on a solid surface. When the insolubilized Red BSA was treated with proteinase K (10 units/mL) for 30 min, the fluorescent signal was significantly increased (3.5-fold) compared to the untreated control. In the second case, fluorophore-tagged DNA probes were linked to gold nanoparticles by hybridization with capture DNA strands densely immobilized on the surface. The quenched fluorescence signal was recovered (3.7-fold) by thermal dehybridization, which was induced with light of a specific wavelength (e.g., 530 nm) for less than 1 min. We next applied the Red BSA self-quenching relaxation technique employing enzymatic fragmentation to a high-performance immunoassay of cardiac troponin I (cTnI) in a microtiter plate format. The detection limit was 0.19 ng/mL cTnI, and the fluorescent signal was enhanced approximately 4.1-fold compared with the conventional method of direct measurement of the fluorescent signal from a non-fragmented fluorophore-labeled antibody.

Evidence Supporting Oral Hygiene Management by Owners through a Genetic Analysis of Dental Plaque Bacteria in Dogs.

With the increase in the number of households raising dogs and the reports of human-to-dog transmission of oral bacteria, concerns about dogs' oral health and the need for oral hygiene management are increasing. In this study, the owners' perceptions about their dogs' oral health and the frequency of oral hygiene were determined along with the analysis of dog dental plaque bacteria through metagenomic amplicon sequencing so as to support the need for oral hygiene management for dogs. Although the perception of 63.2% of the owners about their dogs' oral health was consistent with the veterinarian's diagnosis, the owners' oral hygiene practices regarding their dogs were very poor. The calculi index (CI) and gingiva index (GI) were lower in dogs who had their teeth brushed more than once a week (57.89%) than in dogs brushed less than once a month (42.10%); however, the difference was nonsignificant (CI: p = 0.479, GI: p = 0.840). Genomic DNA was extracted from dental plaque bacteria removed during dog teeth scaling, and metagenomic amplicons were sequenced. The 16S amplicons of 73 species were identified from among the plaque bacteria of the dogs. These amplicons were of oral disease-causing bacteria in humans and dogs. The 16S amplicon of Streptococcus mutans matched that of the human S. mutans, with type c identified as the main serotype. This result suggests that human oral bacteria can be transmitted to dogs. Therefore, considering the high frequency of contact between dogs and humans because of communal living and the current poor oral health of dogs, owners must improve the oral hygiene management of their dogs.

Lateral-flow enzyme immunoconcentration for rapid detection of Listeria monocytogenes.

Lateral-flow enzyme immunochromatography coupled with an immunomagnetic step was developed for rapid detection of Listeria monocytogenes in food matrices. The target bacteria was first separated and concentrated by magnetic nanoparticles containing the enzyme and directly applied to the assay system to induce an antigen-antibody reaction without any additional steps. The color signals produced by an enzyme-substrate reaction at a specific site on the immunostrip were found to be directly proportional to the concentration of L. monocytogenes in the sample. The detection concept was demonstrated by performing an enzyme immunoassay on a microtiter well prior to applying it to the lateral-flow assay. Results of the chromatographic analysis yield a limit of detection of 95 and 97 ± 19.5 CFU/mL in buffer solution and 2 % milk sample, respectively. In addition to the high sensitivity, it was also possible to shorten the separation and detection time to within 2 h. The system also showed no cross-reactivity with other bacteria (e.g., Escherichia coli O157:H7, Salmonella typhimurium, and Salmonella enteritidis). The analytical procedure developed will enable us to not only utilize the assay in the field where fast screening for pathogenic agents is required but also as a preventive measure to contain disease outbreak.

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.

Biophysical characterization of the molecular orientation of an antibody-immobilized layer using secondary ion mass spectrometry.

The molecular orientation of antibody layers formed on separate solid matrices (e.g., gold-coated glass substrate) was characterized by means of time-of-flight secondary ion mass spectrometry (ToF-SIMS) in static mode. For comparison, three different antibody species, IgG, F(ab')(2), and Fab, were prepared, biotinylated in random and site-directed fashions, and immobilized on distinct streptavidin-coated surfaces. ToF-SIMS analyses of each antibody layer revealed that the secondary ion intensity peaks measured at the mass-to-charge (m/z) ratio 253, 325, and 647 were unique to the site-directly immobilized antibodies. The ions in the three peaks were detected neither from the streptavidin layer nor from the randomly prepared antibody, indicating that the insolubilized antibody layers constructed in the two different manners had distinct molecular arrangements. The antibody preparations were further tested for their binding characteristics in sandwich-type immunoassays, which showed that the site-directed antibodies consistently enhanced the detection capability comparing to those randomly prepared. Based on the analytical results of both the ToF-SIMS analysis and sandwich-type immunoassays, the site-directed antibody species were immobilized on the surfaces in a more orientated manner, with their antigen binding sites exposed to the bulk solution, than when random immobilization was used.

Production of rapidly reversible antibody and its performance characterization as binder for continuous glucose monitoring.

To effectively control diabetes, a method to reliably measure glucose fluctuations in the body over given time periods needs to be developed. Current glucose monitoring systems depend on the substrate decomposition by an enzyme to detect the product; however, the enzyme activity significantly decays over time, which complicates analysis. In this study, we investigated an alternative method of glucose analysis based on antigen-antibody binding, which may be active over an extended period of time. To produce monoclonal antibodies, mice were immunized with molecular weight (M(W)) 10K dextran chemically conjugated with keyhole limpet hemocyanin. Since dextran contains glucose molecules polymerized via a 1,6-linkage, the produced antibodies had a binding selectivity that could discriminate biological glucose compounds with a 1,4-linkage. Three antibody clones with different affinities were screened using the M(W) 1K dextran-bovine serum albumin conjugates as the capture ligand. Among the antibodies tested, the antibody clone Glu 26 had the lowest affinity (K(A) = 3.56 × 10(6) M(-1)) and the most rapid dissociation (k(d) = 1.17 × 10(-2) s(-1)) with the polysaccharide immobilized on the solid surfaces. When glucose was added to the medium, the sensor signal was inversely proportional to the glucose concentration in a range between 10 and 1000 mg dL(-1), which covered the clinical range. Under the optimal conditions, the response time was about 3 min for association and 8 min for dissociation based on a 95% recovery of the final equilibrium.

Minimum-step immuno-analysis based on continuous recycling of the capture antibody.

Most immuno-analytical systems employ antibodies that do not readily dissociate upon binding to its partner antigen (i.e., target analyte; α2-macroglobulin as a model) and, thus, either need to be disposed of after one-time use or be reused after binding has been reset. To achieve a minimum-step analysis, an antibody that is capable of rapidly reversible binding with high affinity to an antigen was investigated in this study. This antibody was immobilized on the surface of a label-free sensor, which was combined with microfluidic channels, to demonstrate its applicability. The antibody was successively reused without a regeneration step under physiological conditions, offered specific analysis in the serum medium, and detected the analyte at concentrations as low as 0.1 ng mL(-1), which could further be enhanced by 100-fold. The sensor response reached 95% equilibrium after 8.3 and 14.9 min in average on each dose level for the concentration increase and decrease, respectively. The dynamic range covered a 5 logarithmic analyte concentration. Since the sampling size was in the nanolitre to millilitre range per day under the conditions used and the sensor may retain a long shelf-life, it could potentially be used in a clinical setting for long-term, on-line monitoring of diseases.

Identification of galectin­10 as a biomarker for periodontitis based on proteomic analysis of gingival crevicular fluid.

Periodontitis is a chronic inflammatory disease caused by the gradual breakdown of tissues surrounding the teeth due to various factors. The disease has been frequently noted in dental outpatients for a number of years. Improvements are required to current diagnostic methods, which have limitations in assessing the condition and progression of periodontitis. The development of diagnostic biomarkers for periodontitis to increase the sensitivity and accuracy of diagnosis is important for the management of periodontitis. In the present study, whole gingival crevicular fluid (GCF) from patients with periodontitis and healthy individuals was characterized via liquid chromatography with tandem mass spectrometry. Label­free quantification was used to identify the differentially abundant protein biomarkers. A total of 1,295 proteins were identified from the whole GCF of patients with periodontitis and healthy individuals via proteomic analysis. When analyzing biological processes, 'metabolic process' and 'cell organization and biogenes' were identified to play important roles in GCF under periodontitis conditions according to Gene Ontology. When analyzing molecular functions, 'catalytic activity' and 'protein binding' were the terms most enriched with differentially abundant proteins under periodontitis conditions. Galectin­10 (Gal­10) was one of the most upregulated proteins in the GCF of patients with periodontitis. The levels of prostaglandin E2 were increased in oral keratinocytes and gingival fibroblasts treated with recombinant (r)Gal­10. The levels of interleukin­8, matrix metalloproteinase 9 and C­reactive protein were increased in the conditioned media (CM) of rGal­10­treated gingival fibroblasts. In addition, the CM of rGal­10­treated gingival fibroblasts induced osteoclast differentiation. These results suggested that Gal­10 expression was increased in the GCF of patients with periodontitis and contributed to the process of osteoclastogenesis. Therefore, Gal­10 may be a candidate biomarker for periodontitis.

An ELISA-on-a-chip biosensor system coupled with immunomagnetic separation for the detection of Vibrio parahaemolyticus within a single working day.

In this study, we constructed a rapid detection system for a foodborne pathogen, Vibrio parahaemolyticus, by using enzyme-linked immunosorbent assay (ELISA)-on-a-chip (EOC) biosensor technology to minimize the risk of infection by the microorganism. The EOC results showed a detection capability of approximately 6.2x10(5) cells per ml, which was significantly higher than that of the conventional rapid test kit. However, this high level of sensitivity required cultivation of the pathogen prior to analysis, which typically exceeded a day. To shorten the test period, we combined the EOC technology with immunomagnetic separation (IMS), which could enhance the sensitivity of the biosensor. IMS was carried out with magnetic particles coated with a monoclonal antibody specific to the microbe. To test the performance of the IMS-EOC method, fish intestine samples were prepared by artificially inoculating less than 1 or 5 CFU/10 g, allowing for enrichment over predetermined times, and analyzing the sample by using the EOC sensor after concentrating the culture 86-fold via IMS. Using this approach, the bacterium was detected after (at most) 9 h, which approximately corresponds to standard working hours. Thus, the IMS-EOC method allowed for the rapid detection of V. parahaemolyticus, which is responsible for foodborne diseases, and this method could be used for early isolation of contaminated foods before distribution.

Electrochemical biosensors: perspective on functional nanomaterials for on-site analysis.

BACKGROUND: The electrochemical biosensor is one of the typical sensing devices based on transducing the biochemical events to electrical signals. In this type of sensor, an electrode is a key component that is employed as a solid support for immobilization of biomolecules and electron movement. Thanks to numerous nanomaterials that possess the large surface area, synergic effects are enabled by improving loading capacity and the mass transport of reactants for achieving high performance in terms of analytical sensitivity. MAIN BODY: We categorized the current electrochemical biosensors into two groups, carbon-based (carbon nanotubes and graphene) and non-carbon-based nanomaterials (metallic and silica nanoparticles, nanowire, and indium tin oxide, organic materials). The carbon allotropes can be employed as an electrode and supporting scaffolds due to their large active surface area as well as an effective electron transfer rate. We also discussed the non-carbon nanomaterials that are used as alternative supporting components of the electrode for improving the electrochemical properties of biosensors. CONCLUSION: Although several functional nanomaterials have provided the innovative solid substrate for high performances, developing on-site version of biosensor that meets enough sensitivity along with high reproducibility still remains a challenge. In particular, the matrix interference from real samples which seriously affects the biomolecular interaction still remains the most critical issues that need to be solved for practical aspect in the electrochemical biosensor.

Characterization for binding complex formation with site-directly immobilized antibodies enhancing detection capability of cardiac troponin I.

The enhanced analytical performances of immunoassays that employed site-directly immobilized antibodies as the capture binders have been functionally characterized in terms of antigen-antibody complex formation on solid surfaces. Three antibody species specific to cardiac troponin I, immunoglobulin G (IgG), Fab, and F(ab')(2) were site-directly biotinylated within the hinge region and then immobilized via a streptavidin-biotin linkage. The new binders were more efficient capture antibodies in the immunoassays compared to randomly bound IgG, particularly, in the low antibody density range. The observed improvements could have resulted from controlled molecular orientation and also from flexibility, offering conditions suitable for binding complex formations.

Gemcitabine-incorporated polyurethane films for controlled release of an anticancer drug.

BACKGROUND: Local delivery of anti-cancer drugs through a stent is a very promising and anticipated treatment modality for patients who have obstructions in their gastrointestinal tract with malignant tumors. Anticancer drug release via stents, however, needs to be optimized with respect to drug delivery behavior for the stents to be effective for prolonged containment of tumor proliferation and stent re-obstruction. Local stent-based drug delivery has been tested using an effective anti-cancer drug, gemcitabine, but the release from the stent-coated polyurethane films is often too fast and the drug is depleted from the coated film virtually in a day. METHODS: To moderate the drug release from a polyurethane film, a gemcitabine-incorporated polyurethane film was enveloped with a pure polyurethane film, with no drug loading, and with a silicone film by solution casting after activation of the silicone film surface with plasma treatment. RESULTS: The pure polyurethane barrier film was effective; the interface of the two were indistinguishable on scanning electron microscopy, and the initial burst, i.e., the cumulative release in a day, decreased from 90 to 26%. The silicone film barrier, on the other hand, was defective as voids were seen using a scanning electron microscope, and micro-separation of the two layers was observed after the film was immersed in phosphate-buffered saline for 1 day during the in vitro drug release study. CONCLUSIONS: Enveloping a gemcitabine-releasing polyurethane film with a homo-polymer barrier film was quite effective for moderating the initial burst of gemcitabine, thus, prolonging the release time of the drug. Enveloping the polyurethane film with a silicone film was also possible after plasma treatment of the silicone film surface, but the two films eventually separated in the aqueous environment. More studies are needed to tune the drug release behavior of gemcitabine from the stent covering film before attempting a clinical application of an anti-cancer drug releasing stent.

Fascin is involved in cancer cell invasion and is regulated by stromal factors.

The tumor microenvironment plays an important role in cancer growth, invasion and metastasis. The stroma surrounding a tumor is known to contain a variety of factors that can increase angiogenesis, cancer growth and tumor progression. The aim of the present study was to determine the role of fascin in cancer growth and invasion and identify stromal factors involved in cancer progression. A fascin­depleted cell line (fascindep) was used to observe the role of fascin in cancer invasion. Compared with wild­type Mock cells, cancer cell invasion in Matrigel­coated Transwell and three­dimensional (3D) culture system were reduced by fascin depletion. Tumor cell growth in vivo was also significantly reduced in mice injected with fascindep cells. Notably, fascin expression was increased during Transwell invasion with Matrigel compared to Transwell invasion without Matrigel. TGF­ß1, EGF and IL­1ß significantly stimulated fascin expression. Such increased expression of fascin was also observed in cultured cells using conditioned media (CM) from cancer­associated fibroblasts (CAFs). However, no significant change in fascin expression was observed using CM from normal fibroblasts (NFs). Stimulated expression of fascin by Matrigel and CAFs was reduced by biological specific inhibitor of TGF­ß1, EGF and IL­1ß. Compared with wild­type Mock cells, the fascindep cell line showed low RhoA and NF­κB activity, suggesting that RhoA and NF­κB signals are involved in fascin expression. In conclusion, stromal factors are involved in cancer invasion and progression by activating intracellular signaling of cancer cells to increase fascin expression.

Age- and sex-dependence of five major elements in the development of human scalp hair.

BACKGROUND: Human scalp hair is composed of bio-synthesized protein that stores and excretes excess elements from the body. Thus, the concentration of major and trace elements in the hair may provide insight into both the physiology and health status of humans. Monitoring of health status by hair analysis is limited by the uncertainty surrounding natural changes in composition based on age and sex parameters. METHODS: A total of 322 hair samples from subjects aged 0-89 years were collected and analyzed to determine their sulfur, calcium, magnesium, zinc, and copper concentrations by inductively coupled plasma mass spectrometry. The age- and sex-dependence of the concentrations of these elements within scalp hair was evaluated. Age-dependence was analyzed by least squares fitting of the data from young people (up to 25 years old). Sex-dependence was evaluated by comparing the average element concentrations of males and females in each age groups. RESULTS: The concentration of mineral elements increased with age up to 25 years old. Calcium and magnesium contents were strongly affected by age, whereas the effects were weaker for zinc and copper. In participants over 20 years old, sex and the concentrations of calcium and magnesium were significantly associated. The concentrations of these elements were higher in most of the subgroups of adult women. The concentrations of sulfur, zinc, and copper were not significantly associated with age or sex. CONCLUSIONS: The concentrations of major inorganic elements in scalp hair showed an increasing trend up to 25 years of age, and a strong sex dependence of calcium and magnesium concentrations in the subjects older than 20 years. More research is needed to understand the physiology of calcium and magnesium excretion though scalp hair.

Association of the Anxiety/Depression with Nutrition Intake in Stroke Patients.

Stroke patients often experience a walking dysfunction caused by decreased mobility, weakened muscular strength, abnormal posture control, and cognitive dysfunction. Anxiety/depression is the most important and prevalent neuropsychiatric complication of stroke survivors. Brain injury and the presence of malnutrition after stroke contribute to metabolic status and clinical outcome of patients. We examined the level of nutrition intake in stroke patients according to their degree of anxiety/depression. The data were obtained from 2013 to 2015 through the Korea National Health and Nutrition Examination Survey (KNHANES). Study subjects were categorized to either a group having no problem of anxiety/depression (n = 274) or a group having a problem of anxiety or depression (n = 104). The EuroQoL-5 Dimensions Health Questionnaire (EQ-5D) index score was derived from the first description of an individual health status based on the EQ-5D classification system, including mobility, self-care, usual daily activities, pain/discomfort, and anxiety/depression. The mean age was 67.4 years in the normal group and 68.0 years in the anxiety or depression group. In the anxiety or depression group, 39.4% were men vs. 53.3% in the normal group. The total energy intake (p = 0.013), riboflavin (p = 0.041), and niacin (p = 0.038) was significantly higher in stroke patients with no anxiety/depression than those in stroke patients with having an anxiety/depression. The group having no problem of anxiety/depression had significantly higher EQ-5D index compared to the group having a problem of anxiety/depression group (p < 0.001) had. The results suggest the association between nutrition intake, usual activities and pain/discomfort status in the stroke patients with having an anxiety/depression.

Technical advances in global DNA methylation analysis in human cancers.

Prototypical abnormalities of genome-wide DNA methylation constitute the most widely investigated epigenetic mechanism in human cancers. Errors in the cellular machinery to faithfully replicate the global 5-methylcytosine (5mC) patterns, commonly observed during tumorigenesis, give rise to misregulated biological pathways beneficial to the rapidly propagating tumor mass but deleterious to the healthy tissues of the affected individual. A growing body of evidence suggests that the global DNA methylation levels could serve as utilitarian biomarkers in certain cancer types. Important breakthroughs in the recent years have uncovered further oxidized derivatives of 5mC - 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC), thereby expanding our understanding of the DNA methylation dynamics. While the biological roles of these epigenetic derivatives are being extensively characterized, this review presents a perspective on the opportunity of innovation in the global methylation analysis platforms. While multiple methods for global analysis of 5mC in clinical samples exist and have been reviewed elsewhere, two of the established methods - Liquid Chromatography coupled with mass spectrometry (LC-MS/MS) and Immunoquantification have successfully evolved to include the quantitation of 5hmC, 5fC and 5caC. Although the analytical performance of LC-MS/MS is superior, the simplicity afforded by the experimental procedure of immunoquantitation ensures it's near ubiquity in clinical applications. Recent developments in spectroscopy, nanotechnology and sequencing also provide immense promise for future evaluations and are discussed briefly. Finally, we provide a perspective on the current scenario of global DNA methylation analysis tools and present suggestions to develop the next generation toolset.

Semi-continuous, real-time monitoring of protein biomarker using a recyclable surface plasmon resonance sensor.

Although label-free immunosensors based on, for example, surface plasmon resonance (SPR) provide advantages of real-time monitoring of the analyte concentration, its application to routine clinical analysis in a semi-continuous manner is problematic because of the high cost of the sensor chip. The sensor chip is in most cases regenerated by employing an acidic pH. However, this causes gradual deterioration of the activity of the capture antibody immobilized on the sensor surface. To use sensor chips repeatedly, we investigated a novel surface modification method that enables regeneration of the sensor surface under mild conditions. We introduced a monoclonal antibody (anti-CBP Ab) that detects the conformational change in calcium binding protein (CBP) upon Ca2+ binding (>1mM). To construct a regenerable SPR-based immunosensor, anti-CBP Ab was first immobilized on the sensor surface, and CBP conjugated to the capture antibody (specific for creatine kinase-MB isoform (CK-MB); CBP-CAb) then bound in the presence of Ca2+. A serum sample was mixed with the detection antibody to CK-MB, which generated an SPR signal proportional to the analyte concentration. After each analysis, the sensor surface was regenerated using medium (pH 7) without Ca2+, and then adding fresh CBP-CAb in the presence of Ca2+ for the subsequent analysis. Analysis of multiple samples using the same sensor was reproducible at a rate >98.7%. The dose-response curve was linear for 1.75-500.75ng/mL CK-MB, with an acceptable coefficient of variation of <8.8%. The performance of the immunosensor showed a strong correlation with that of the Pathfast reference system (R2>96%), and exhibited analytical stability for 1 month. To our knowledge, this is the first report of a renewal of a sensor surface with fresh antibody after each analysis, providing high consistency in the assay during a long-term use (e.g., a month at least).