CRISPR-Cas12a-Based Nucleic Acid Amplification-Free DNA Biosensor via Au Nanoparticle-Assisted Metal-Enhanced Fluorescence and Colorimetric Analysis.

Cell-free DNA (cfDNA) has attracted significant attention due to its high potential to diagnose diseases, such as cancer. Still, its detection by amplification method has limitations because of false-positive signals and difficulty in designing target-specific primers. CRISPR-Cas-based fluorescent biosensors have been developed but also need the amplification step for the detection. In this study, for the first time CRISPR-Cas12a based nucleic acid amplification-free fluorescent biosensor was developed to detect cfDNA by a metal-enhanced fluorescence (MEF) using DNA-functionalized Au nanoparticle (AuNP). Upon activating the CRISPR-Cas12a complex by the target cfDNA and subsequent single-strand DNA (ssDNA) degradation between AuNP and fluorophore, MEF occurred with color changes from purple to red-purple. Using this system, breast cancer gene-1 (BRCA-1) can be detected with very high sensitivity in 30 min. This rapid and highly selective sensor can be applied to measure other nucleic acid biomarkers such as viral DNA in field-deployable and point-of-care testing (POCT) platform.

Construction of an antimyoglobin single-chain variable fragment with rapid reaction kinetics.

Antibodies with rapid reaction kinetics (high association and dissociation rates), named reversible antibodies, are used to perform continuous monitoring of sensitive disease biomarkers. In cases of acute myocardial infarction (AMI), continuous monitoring and early diagnosis are important. Human myoglobin (Myo) is a useful biomarker for AMI during the early stage after the onset of symptoms. In this study, a single-chain variable fragment (scFv) specific to Myo was derived from an IgG antibody that has rapid reaction kinetics. Enzyme-linked immunosorbent assay revealed that recombinant scFv exhibited 3.8-fold reduced affinity compared with the parent IgG antibody based on the antibody concentration necessary for 50% of the maximum signal. The scFv retained the rapid reaction kinetic mode with average kon and koff of 2.63 × 10(5) M(-1) Sec(-1) and 3.25 × 10(-3) Sec(-1) , respectively, which were reduced to 10- and 2.3-fold compared with those of the parent antibody. The equilibrium constant for the association of the scFv (KA = 8.09 × 10(7) M(-1) ) was 4.6-fold lower than that of its parent IgG antibody. This scFv may be a starting point for further mutagenesis/kinetic and structural analyses providing valuable insight into the mechanism of reversible antibodies.

A Human Serum-Based Enzyme-Free Continuous Glucose Monitoring Technique Using a Needle-Type Bio-Layer Interference Sensor.

The incidence of diabetes is continually increasing, and by 2030, it is expected to have increased by 69% and 20% in underdeveloped and developed countries, respectively. Therefore, glucose sensors are likely to remain in high demand in medical device markets. For the current study, we developed a needle-type bio-layer interference (BLI) sensor that can continuously monitor glucose levels. Using dialysis procedures, we were able to obtain hypoglycemic samples from commercial human serum. These dialysis-derived samples, alongside samples of normal human serum were used to evaluate the utility of the sensor for the detection of the clinical interest range of glucose concentrations (70-200 mg/dL), revealing high system performance for a wide glycemic state range (45-500 mg/dL). Reversibility and reproducibility were also tested over a range of time spans. Combined with existing BLI system technology, this sensor holds great promise for use as a wearable online continuous glucose monitoring system for patients in a hospital setting.

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.

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.

Purification and characterization of Fab fragments with rapid reaction kinetics against myoglobin.

Myoglobin is an early biomarker for acute myocardial infarction. Recently, we isolated the antibody IgG-Myo2-7ds, which exhibits unique rapid reaction kinetics toward human myoglobin antigen. Antibodies with rapid dissociation kinetics are thought to be premature IgG forms that are produced during the early stage of in vivo immunization. In the present study, we identified the epitope region of the IgG-Myo2-7ds antibody to be the C-terminal region of myoglobin, which corresponds to 144-154 aa. The Fab fragment was directly purified by papain cleavage and protein G affinity chromatography and demonstrated kinetics of an association constant of 4.02 × 10(4) M(-1) s(-1) and a dissociation constant of 2.28 × 10(-2) s(-1), which retained the unique reaction kinetics of intact IgG-Myo2-7ds antibodies. Because a rapid dissociation antibody can be utilized for antibody recycling, the results from this study would provide a platform for the development of antibody engineering in potential diagnostic areas such as a continuous monitoring system for heart disease.

Nucleoside diphosphate kinase and flagellin from Pseudomonas aeruginosa induce interleukin 1 expression via the Akt/NF-κB signaling pathways.

Inflammatory responses are a first line of host defense against a range of invading pathogens, consisting of the release of proinflammatory cytokines followed by attraction of polymorphonuclear neutrophils (PMNs) to the site of inflammation. Among the many virulence factors that contribute to the pathogenesis of infections, nucleoside diphosphate kinase (Ndk) mediates bacterially induced toxicity against eukaryotic cells. However, no study has examined how Ndk affects inflammatory responses. The present study examined the mechanisms by which Pseudomonas aeruginosa activates inflammatory responses upon infection of cells. The results showed that bacterial Ndk, with the aid of an additional bacterial factor, flagellin, induced expression of the proinflammatory cytokines interleukin-1α (IL-1α) and IL-1ß. Cytokine induction appeared to be dependent on the kinase activity of Ndk and was mediated via the NF-κB signaling pathway. Notably, Ndk activated the Akt signaling pathway, which acts upstream of NF-κB, as well as caspase-1, which is a key component of inflammasome. Thus, this study demonstrated that P. aeruginosa, through the combined effects of Ndk and flagellin, upregulates the expression of proinflammatory cytokines via the Akt/NF-κB signaling pathways.

A novel Pseudomonas aeruginosa-derived effector cooperates with flagella to mediate the upregulation of interleukin 8 in human epithelial cells.

Infection with Pseudomonas aeruginosa results in a massive accumulation of neutrophils in response to prolonged and sustained expression of inflammatory mediators. The major chemokine associated with this excessive neutrophil recruitment is IL-8, the accumulation of which is a hallmark of cornea and cystic fibrosis airway inflammation. To date, several P. aeruginosa-associated and extracellular factors required for the stimulation of IL-8 expression have been identified. Here, we report a novel effector molecule, nucleoside diphosphate kinase (Ndk), which increases the expression of IL-8 by translocating into host cells. The induction appears to be dependent on both the kinase activity of Ndk and an additional bacterial factor, flagellin, via an NF-κB signaling pathway. This study demonstrates the role of a novel effector, Ndk, which is capable of inducing prominent inflammatory chemokine IL-8 expression with the aid of flagellin during P. aeruginosa infection.

Semi-continuous, label-free immunosensing approach for Ca2+-based conformation change of a calcium-binding protein.

A label-free immunosensing method based on the conformational change of calcium-binding protein (CBP) depending on analyte concentration was explored for semi-continuous analysis of free Ca(2+). Glucose-galactose-binding protein as a CBP and produced as a recombinant protein by Escherichia coli was used as the immunogen to produce monoclonal antibodies by hybridoma technology. We finally screened the 3-6F cell clone, which produced the desired antibody specific to a particular structural conformation of the protein that occurred only upon CBP-calcium complex formation. To construct an immunosensor, the antibody was immobilized via a secondary antibody on an Octet Red optical fiber-based label-free sensor. Calcium analysis was conducted on the sensor in combination with CBP previously added to the aqueous sample, which distinguished the sensor signal according to the analyte concentration. The immunosensor produced a signal in real time with a response time of approximately 15 min and could be reused for analyses of different samples in a semi-continuous manner. The minimum detection limit of the analyte under optimal conditions was 0.09 mM and the upper limit was about 5 mM (log-logit transformed standard curve linearity: R(2) > 98%). In sample tests with milk, the analytical performance of the sensor was highly correlated (R(2) > 99%) with that of the reference system based on the KMnO4 titration method (ISO 12081). Although the sensor showed cross-reactivity at high concentrations (>1 mM) of cations including zinc, iron, manganese, and copper, these ionic components were not traceable (<0.01 mM) in milk.

Refolded scFv antibody fragment against myoglobin shows rapid reaction kinetics.

Myoglobin is one of the early biomarkers for acute myocardial infarction. Recently, we have screened an antibody with unique rapid reaction kinetics toward human myoglobin antigen. Antibodies with rapid reaction kinetics are thought to be an early IgG form produced during early stage of in vivo immunization. We produced a recombinant scFv fragment for the premature antibody from Escherichia coli using refolding technology. The scFv gene was constructed by connection of the V(H)-V(L) sequence with a (Gly4Ser)3 linker. The scFv fragment without the pelB leader sequence was expressed at a high level, but the solubility was extremely low. A high concentration of 8 M urea was used for denaturation. The dilution refolding process in the presence of arginine and the redox reagents GSH and GSSH successfully produced a soluble scFv protein. The resultant refolded scFv protein showed association and dissociation values of 9.32 × 10⁻4 M⁻¹·s⁻¹ and 6.29 × 10⁻³ s⁻¹, respectively, with an affinity value exceeding 107 M⁻¹ (k(on)/k(off)), maintaining the original rapid reaction kinetics of the premature antibody. The refolded scFv could provide a platform for protein engineering for the clinical application for diagnosis of heart disease and the development of a continuous biosensor.

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.

Premature antibodies with rapid reaction kinetics and their characterization for diagnostic applications.

In this study, rapidly reversible antibodies were produced and the binding kinetics, stability, and utility as an analytical binder were evaluated. The number of times the animals were immunized with the antigen (myoglobin as marker for acute myocardial infarction [AMI]) was limited to two, increasing the chances of producing premature antibodies that rapidly reacted with the binding partner in both association and dissociation. The rate constants were higher than 1×10(6)M(-1)s(-1) and 1×10(-3)s(-1), respectively, and the affinity exceeded 10(8)M(-1). They responded to an abrupt environmental change (acidic pH in this study) where the reaction kinetics was changed to slow binding, particularly for dissociation, resulting in a 10-fold increase in affinity. The binding characteristic before and after the transition were stable at 37°C for longer than 1 month, suggesting that the rapidly reversible antibody was the intermediate of the slow binder. The rapid kinetic antibody was used as the primary binder in the conventional competitive immunoassay, which displayed a lower sensitivity than the transformed antibody due to its lower affinity. We further demonstrated that, on combination with a microfluidic label-free sensor, the reaction could be continuously monitored in serum medium by recycling the same antibody without employing the regeneration step.

Detecting early-stage malignant melanoma using a calcium switch-enriched exosome subpopulation containing tumor markers as a sample.

An exosome species containing CD63 as a marker of melanoma was isolated from bulk exosome population and used as a sample for detecting malignant melanoma. A calcium binding protein (CBP) was produced and then used to raise monoclonal antibody. The antibody was sensitive to a conformational change of CBP caused by Ca2+ binding. Immuno-magnetic beads were prepared by immobilizing the conformation-sensitive binder and subsequent binding of CBP conjugated with the capture antibody specific to CD63. These immuno-beads were used to isolate CD63-positive exosome from a bulk exosome sample (normal or melanoma) based on the 'calcium switch-on/off' mechanism through magnetic separation. After recovery, the subpopulation sample was analyzed by immunoassays for cavelion1 (Cav1), CD81, and CD9 as sub-subpopulation markers. Normalized signals of Cav1 and/or CD81 over CD9 were higher in melanoma samples than in normal samples, depending on clinical stages (I, II, and IV) of patients. This was in contrast to assay results for the bulk exosome population that showed a completely mixed state of melanoma and normal samples. These results showed that an exosome subpopulation sample prepared using a 'Ca2+-dependent switch' technology might be useful for diagnosing malignant melanoma at an early stage to increase 5-year survival rates.

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.

Modeling Hypoxic Stress In Vitro Using Human Embryonic Stem Cells Derived Cardiomyocytes Matured by FGF4 and Ascorbic Acid Treatment.

Mature cardiomyocytes (CMs) obtained from human pluripotent stem cells (hPSCs) have been required for more accurate in vitro modeling of adult-onset cardiac disease and drug discovery. Here, we found that FGF4 and ascorbic acid (AA) induce differentiation of BG01 human embryonic stem cell-cardiogenic mesoderm cells (hESC-CMCs) into mature and ventricular CMs. Co-treatment of BG01 hESC-CMCs with FGF4+AA synergistically induced differentiation into mature and ventricular CMs. FGF4+AA-treated BG01 hESC-CMs robustly released acute myocardial infarction (AMI) biomarkers (cTnI, CK-MB, and myoglobin) into culture medium in response to hypoxic injury. Hypoxia-responsive genes and potential cardiac biomarkers proved in the diagnosis and prognosis of coronary artery diseases were induced in FGF4+AA-treated BG01 hESC-CMs in response to hypoxia based on transcriptome analyses. This study demonstrates that it is feasible to model hypoxic stress in vitro using hESC-CMs matured by soluble factors.

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.

Normal-incidence type solution immersed silicon (SIS) biosensor for ultra-sensitive, label-free detection of cardiac troponin I.

Early diagnosis of acute myocardial infarction (AMI) significantly reduce the mortality rate and can be achieved via high-sensitive detection of AMI specific cardiac troponin I (cTnI) biomarker. Here, we present normal-incident type solution-immersed silicon (NI-SIS) ellipsometric biosensor, designed for ultra-high sensitive, high-throughput, label-free detection of the target protein. The NI-SIS sensors are equipped with a specially designed prism that maintains the angle of incidence close to the Brewster angle during operation, which significantly reduces SIS noise signals induced by the refractive index fluctuations of the surrounding medium, improves the signal-to-noise ratio, in-results lowers the detection limit. We applied NI-SIS biosensor for ultra-sensitive detection of cTnI biomarkers in human serum. The optimized sensor chip fabrication and detection operation procedures are proposed. The wide linear concentration ranges of fg/mL to ng/mL is achieved with the detection limit of 22.0 fg/mL of cTnI. The analytical correlation was assessed by linear regression analysis with the results of the Pathfast reference system. These impressive biosensing capabilities of NI-SIS technology have huge potentials for accurate detection of target species in different application areas, such as diagnosis, drug discovery, and food contaminations.