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.

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.

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.

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.

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).

Using Synthetic Biology to Engineer Living Cells That Interface with Programmable Materials.

We have developed an abiotic-biotic interface that allows engineered cells to control the material properties of a functionalized surface. This system is made by creating two modules: a synthetically engineered strain of E. coli cells and a functionalized material interface. Within this paper, we detail a protocol for genetically engineering selected behaviors within a strain of E. coli using molecular cloning strategies. Once developed, this strain produces elevated levels of biotin when exposed to a chemical inducer. Additionally, we detail protocols for creating two different functionalized surfaces, each of which is able to respond to cell-synthesized biotin. Taken together, we present a methodology for creating a linked, abiotic-biotic system that allows engineered cells to control material composition and assembly on nonliving substrates.

Programming Surface Chemistry with Engineered Cells.

We have developed synthetic gene networks that enable engineered cells to selectively program surface chemistry. E. coli were engineered to upregulate biotin synthase, and therefore biotin synthesis, upon biochemical induction. Additionally, two different functionalized surfaces were developed that utilized binding between biotin and streptavidin to regulate enzyme assembly on programmable surfaces. When combined, the interactions between engineered cells and surfaces demonstrated that synthetic biology can be used to engineer cells that selectively control and modify molecular assembly by exploiting surface chemistry. Our system is highly modular and has the potential to influence fields ranging from tissue engineering to drug development and delivery.

Conformation-sensitive antibody-based point-of-care immunosensor for serum Ca(2+) using two-dimensional sequential binding reactions.

To assess the homeostasis of Ca(2+) metabolism, we have developed a rapid immunosensor for ionic calcium using a membrane chromatographic technique. As calcium-binding protein (CBP) is available for the recognition and undergone conformation change upon Ca(2+) binding, a monoclonal antibody sensitive to the altered structure of CBP has been employed. The sequential binding scheme was mathematically simulated and shown to match with the experimental results. At the initial stage, the rapid analytical system using lateral flow was constructed by immobilizing the antibody on the immuno-strip nitrocellulose membrane and labeling CBP with colloidal gold as a tracer. A major problem with this system in measuring ionic calcium levels was retarded migration of the gold tracer along the immuno-strip. It was conceivable that the divalent cation at a high concentration caused a change in the physical properties of the tracer, resulting in a non-specific interaction with the membrane surface. This problem was circumvented by first eluting a sample containing biotinylated CBP along the immuno-strip and then supplying the gold coupled to streptavidin across the signal generation pad of the strip. The color signal was then generated via biotin-SA linkage and measured using a smartphone-based detector developed in our laboratory. This two-dimensional chromatographic format completed the Ca(2+) analysis within 15min, the analytical performance covered the clinical dynamic range (0.25-2.5mM) and highly correlated with that of the reference system, i-STAT. These results inspired us to eventually investigate a dual-immunoassay system that measures simultaneously ionic calcium and parathyroid hormone, which regulates the ionic calcium level in serum. This will significantly simplify the current diagnostic protocols, which involve separate devices.

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.

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.