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In this study, a homogeneous one-step immunoassay based on switching peptides is presented for the detection of influenza viruses A and B (Inf-A and Inf-B, respectively). The one-step immunoassay represents an immunoassay method that does not involve any washing steps, only treatment of the sample. In this method, fluorescence-labeled switching peptides quantitatively dissociate from the antigen-binding site of immunoglobulin G (IgG). In particular, the one-step immunoassay based on soluble detection antibodies with switching peptides is called a homogeneous one-step immunoassay. The immunoassay developed uses switching peptides labeled with two types of fluorescence dyes (FAM and TAMRA) and detection antibodies labeled with two types of fluorescence quenchers (TQ2 for FAM and TQ3 for TAMRA). The optimal switching peptides for the detection of Inf-A and Inf-B have been selected as L1-peptide and H2-peptide. The interactions between the four kinds of switching peptides and IgG have been analyzed using computational docking simulation and SPR biosensor. The location of labeling for the fluorescence quenchers has been determined based on the distance between the fluorescence dyes of the switching peptides and the fluorescence quenchers, calculated on the basis of the efficiency of fluorescence quenching, using the Förster equation. To demonstrate the feasibility of the one-step immunoassay, binding constants (

Switching peptides were designed to bind reversibly to the binding pocket of antibodies (IgG) by interacting with frame regions (FRs). These peptides can be quantitatively released when antigens bind to IgG. As FRs have conserved amino acid sequences, switching peptides can be used as antibodies for different antigens and different source animals. In this study, an electrochemical one-step immunoassay was conducted using switching peptides labeled with ferrocene for the quantitative measurement of analytes. For the effective amperometry of the switching peptides labeled with ferrocene, a pyrolyzed carbon electrode was prepared by pyrolysis of the parylene-C film. The feasibility of the pyrolyzed carbon electrode for the electrochemical one-step immunoassay was determined by analyzing its electrochemical properties, such as its low double-layer capacitance (

As the shelf life of platelets collected from donated blood is very short, approximately 5 days, the determination of bacterial contamination in platelets has become necessary. In this study, rapid analysis of Gram-positive and Gram-negative bacterial contamination in platelet samples was presented without pre-enrichment using pig serum-derived antibodies against the outer membrane proteins (OMP) of Gram-negative bacteria and antibodies against lipoteichoic acid (LTA) on the surface of Gram-positive bacteria. The anti-OMP antibodies against Gram-negative bacteria were isolated using sequential incubation with (1) the modified Gram-negative bacteria ClearColi, which lacks lipopolysaccharide (LPS) on the outer membrane, and (2) the Gram-positive bacteria

A one-step immunoassay was developed for five types of food-poisoning-related bacteria using a switching peptide and antibodies isolated from unimmunized horse serum. The one-step immunoassay involves mixing samples and reagents in a homogeneous solution without any washing steps. In this work, a one-step immunoassay configuration was developed using isolated antibodies labelled with an organic fluorescence quencher and a switching-peptide labelled with a fluorescent dye. The fluorescence-labelled switching-peptide was bound to the antigen-binding site of the isolated antibodies before binding to the bacteria (no fluorescence signal), and the switching-peptide dissociated from the antibodies as soon as they bound to the bacteria (fluorescence signal turns on). By quantifying the generated fluorescence signal, the one-step immunoassay presented here allows microbial detection without any washing step.

Several endemic corona viruses (eCoVs) have been reported to be the most common etiologic agents for the seasonal common cold and also cause pneumonia. These eCoVs share extensive sequence homology with SARS-CoV-2, and immune responses to eCoVs can cross-react with SARS-CoV-2 antigens. Based on such cross-reactivity of antigens among eCoVs, the IgG antibodies against the spike protein (SP) of severe acute respiratory syndrome coronavirus (SARS-CoV) were isolated from pig serum using magnetic beads immobilized with SARS-CoV SP and a protein-A column. The selectivity of the isolated antibodies was tested using different types of antigens, such as SARS-CoV-2 nucleoprotein (NP), influenza A virus (Beijing type), influenza B virus (Tokio and Florida types), human hepatitis B virus surface antigen (HBsAg), and bovine serum albumin (BSA). From the selectivity test, the anti-SP antibodies isolated from pig serum had sufficient selectivity to other kinds of viral antigens, and the apparent binding constant of the isolated antibodies was approximately 1.5 × 10–8 M from the surface plasmon resonance (SPR) measurements. Finally, the isolated anti-SP antibodies were applied to the immunoassay of SP using competitive immunoassay configuration. The feasibility of the detection as well as the quantitative analysis of the SARS-CoV viral culture fluid was determined using four viral culture samples, namely, SARS-CoV, SARS-CoV-2, MERS-CoV, and CoV-229E.

In this study, parylene-C films from plasma deposition as well as thermal deposition were pyrolyzed to prepare a carbon electrode for application in electrochemical immunoassays. Plasma deposition could prepare parylene-C in a faster deposition rate and more precise control over the thickness in comparison with the conventional thermal deposition. To analyze the influence of the deposition method, the crystal and electronic structures of the pyrolyzed parylene-C films obtained

Chemiluminescence immunoassays have been widely employed for diagnosing various diseases. However, because of the extremely low intensity chemiluminescence signals, highly sensitive transducers, such as photomultiplier tubes and image sensors with cooling devices, are required to overcome this drawback. In this study, a hypersensitive photosensor was developed based on cesium lead bromide (CsPbBr3) perovskite quantum dots (QDs) with sufficient high sensitivity for chemiluminescence immunoassays. First, CsPbBr3 QDs with a highly uniform size, that is, 5 nm, were synthesized under thermodynamic control to achieve a high size confinement effect. For the fabrication of the photosensor, MoS2 nanoflakes were used as an electron transfer layer and heat-treated at an optimum temperature. Additionally, a parylene-C film was used as a passivation layer to improve the physical stability and sensitivity of the photosensor. In particular, the trap states on the CsPbBr3 QDs were reduced by the passivation layer, and the sensitivity was increased. Finally, a photosensor based on CsPbBr3 QDs was employed in chemiluminescence immunoassays for the detection of human hepatitis B surface antigen, human immunodeficiency virus antibody, and alpha-fetoprotein (AFP, a cancer biomarker). When compared with the conventionally used equipment, the photosensor was determined to be feasible for application in chemiluminescence immunoassays.

Antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleoprotein (NP) were purified from pig serum through two steps: (1) isolation of anti-NP IgG antibodies using magnetic beads with immobilized human SARS-CoV-2 NP and (2) filtration of anti-spike protein (SP) IgG antibodies using magnetic beads with immobilized human SARS-CoV SP. The enhanced specificity of the purified antibodies to the NP of SARS-CoV-2 was demonstrated using an immunoassay with anti-NP IgG antibodies after the isolation and filtration steps. The binding constants (Kd) of the purified anti-NP IgG antibodies to the NP of SARS-CoV-2 and the SP of SARS-CoV were estimated using a surface plasmon resonance biosensor (SPR). A competitive assay using the two-step purified anti-NP IgG antibodies from pig serum demonstrated (a) the detection of SARS-CoV-2 in viral fluid and (b) the discrimination of SARS-CoV-2 from SARS-CoV, MERS-CoV, and CoV strain 229E in viral fluids.

Anti-coronavirusdisease-2019 (COVID-19; anti-severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2)) antibodies against nucleoprotein (NP) were purified from pig sera. Following the separation of the antibody fraction using a protein-A column, the final yield of the purified antibodies against SARS-CoV-2 NPs was estimated to be 0.26 ± 0.05 % (absolute amount of 143.4 ± 25.2 ng, n=5) from 1 mL of pig sera. The binding activities of the isolated antibodies were confirmed using immunoassay and immunostaining. Based on the specific binding activity to NPs, a quantitative assay was performed using a surface plasmon resonance (SPR) biosensor. From the doseresponse curve, the binding constant (Kd) was calculated to be 185 pM and the limit of detection was estimated to be 1.02 pM. The SPR biosensor with the isolated antibodies against SARS-CoV-2 NPs was applied for the detection of SARS-CoV-2, MERS-CoV, and CoV strain 229E in culture fluid.

The passivation layers of an in situ-synthesized cadmium sulfide (CdS) nanowire (NW) photosensor were prepared for two reasons: (1) to improve the physical stability of the NW on an interdigitated electrode and (2) to enhance the immobilization efficiency of proteins for the on-chip immunoassay. The passivation layer was estimated to have suitable optical properties, and the photoresponse of photosensor was increased after the formation of passivation layer. The immobilization efficiency of a parylene-H film through covalent bonding was compared with the immobilization of Z-domains through physical adsorption. Finally, on-chip chemiluminescent immunoassay of bacteria was carried out by immobilizing antibodies against Escherichia coli through Z-domains for the orientation control of antibodies. The limit of detection was determined to be less than 104E. coli/mL (n=3), and the sensitivity of bacterial detection was estimated to be 0.339 pA/E. coli/mL (n=3) with a linearity factor (R2) of 0.999. These results showed that the on-chip chemiluminescent immunoassay for bacterial detection could be performed using passivation layer coated CdS NW photosensor.

A novel method for quantifying galactose was developed to serve as a newborn screening test for galactosemia using laser desorption/ionization time-of-flight (LDI-TOF) mass spectrometry (MS) with a TiO2 nanowire chip. Herein, phosphate citrate buffer, serum, and dried blood spot (DBS) were employed for the quantitative analysis of galactose. To quantitatively analyze galactose, its reduction potential was used to oxidize o-phenylene diamine (OPD) into 2,3-diaminophenazine (DA), which were both detected using LDI-TOF MS with a TiO2 nanowire chip according to the concentration of galactose. The reproducibility and the interference of glucose were determined to demonstrate the applicability of this method. Moreover, mixtures of galactose, phenylalanine, and 17 α-OHP were analyzed to determine the interference induced by other biomarkers of metabolic disorders. The OPD oxidation of galactose was found to be selectively achieved under high-glucose conditions, similar to human blood, thereby showing good reproducibility. The intensities of the mass peaks of OPD and DA based on LDI-TOF MS with a TiO2 nanowire chip were linearly correlated in the galactose concentration range of 57.2–220.0 μg/mL (r2 = 0.999 and 0.950, respectively) for serum samples and 52.5–220.0 μg/mL (r2 = 0.993 and 0.985, respectively) for DBS after methanol precipitation/extraction. The enzyme immunoassay and LDI-TOF MS analysis results were statistically analyzed, and a mixture of phenylalanine, 17 α-OHP, and galactose was simultaneously investigated quantitatively at the cutoff level.

One-step immunoassay detects a target analyte simply by mixing a sample with a reagent solution without any washing steps. Herein, we present a one-step immunoassay that uses a peptide mimicking a target analyte (mimotope). The key idea of this strategy is that the mimotopes are screened from an autodisplayed F

Highly N-doped SiC was presented as an optimal electrode for electrochemical immunoassays with a far higher sensitivity than chemiluminescence detection. As the first step, the electrochemical properties of highly N-doped SiC, such as the double-layer capacitance (Cdl), rate constant for electron transfer (kapp) and ideal polarizable potential range (electrochemical window) were analyzed and compared with those of Au, Pt, and graphite electrodes. The highly N-doped SiC electrode was used for the quantification of oxidized 3,3′,5,5′-tetramethylbenzidine (TMB) which was widely used as chromogenic substrate for commercialized immunoassay kits. In order to enhance the sensitivity for the quantification of the oxidized TMB the chronoamperometry was applied to avoid the background current of i-V measurement. Finally, the chronoamperometry based on the highly N-doped SiC electrode was applied to commercial immunoassay kits for the medical diagnosis of the human immunodeficiency virus (HIV) and the human hepatitis B surface antigen (hHBsAg). The chronoamperometric measurement based on the highly N-doped SiC electrode was proved to detect at far lower limits in comparison with the conventional optical density measurement as well as the chemiluminescence assay based on luminol as a chemiluminescent probe.

Chemiluminescence immunoassays have been widely employed for diagnosing various diseases. However, because of the extremely low intensity chemiluminescence signals, highly sensitive transducers, such as photomultiplier tubes and image sensors with cooling devices, are required to overcome this drawback. In this study, a hypersensitive photosensor was developed based on cesium lead bromide (CsPbBr

Vertically paired electrodes (VPEs) with multiple electrode pairs were developed for the enhancement of capacitive measurements by optimizing the electrode gap and number of electrode pairs. The electrode was fabricated using a conductive polymer layer of PEDOT:PSS instead of Ag and Pt metal electrodes to increase the VPE fabrication yield because the PEDOT:PSS layer could be effectively etched using a reactive dry etching process. In this study, sensitivity enhancement was realized by decreasing the electrode gap and increasing the number of VPE electrode pairs. Such an increase in sensitivity according to the electrode gap and the number of electrode pairs was estimated using a model analyte for an immunoassay. Additionally, a computer simulation was performed using VPEs with different electrode gaps and numbers of VPE electrode pairs. Finally, VPEs with multiple electrode pairs were applied for SARS-CoV-2 nucleoprotein (NP) detection. The capacitive biosensor based on the VPE with immobilized anti-SARS-CoV-2 NP was applied for the specific detection of SARS-CoV-2 in viral cultures. Using viral cultures of SARS-CoV-2, SARS-CoV, MERS-CoV, and CoV-strain 229E, the limit of detection (LOD) was estimated to satisfy the cutoff value (dilution factor of 1/800) for the medical diagnosis of COVID-19, and the assay results from the capacitive biosensor were compared with commercial rapid kit based on a lateral flow immunoassay.

Herein, we present switching-peptides for a one-step immunoassay, without the need for additional antibody treatment or washing steps to detect antigen–antibody interactions. Fluorescently labeled switching-peptides were dissociated from the immobilized antibody soon after the antigens were bound to the binding pockets. In this study, four different parts of the antibody (IgG) frame regions were chemically synthesized, and these peptides were bound to immobilized antibodies as switching-peptides. We presented the design principle of switching-peptides and used Pymol software, based on the changes in thermodynamic parameters, to study the interaction between antibodies and switching-peptides. The binding properties of switching-peptides were analyzed based on Forster resonance energy transfer between switching-peptides as well as between switching-peptides and antibodies (IgGs) isolated from different animals. The binding constants of the four switching-peptides to antibodies were estimated to be in the range of 1.48–3.29 μM. Finally, the feasibility of using switching-peptides for the quantitative one-step immunoassay was demonstrated by human hepatitis B surface antigen (hHBsAg) detection and statistical comparison of the assay results with those of conventional ELISA. The limit of detection for HBsAg was determined to be 56 ng/mL, and the dynamic range was estimated to be 136 ng/mL–33 μg/mL. These results demonstrate the feasibility of the one-step immunoassay for HBsAg.

Immunoassays are used to detect a target analyte by using specific interaction with detection antibodies which form antigen-antibody complexes. In thermophoretic immunoassays the antigen-antibody complexes make slow thermophoresis (movement by thermal stimulation) in comparison with unbound antigens and antibodies. In this work, a sensitive thermophoretic immunoassay was demonstrated by mixing outer membrane (OM) particles of E. coli with autodisplayed Z-domains. In order to compare the influence of OM particles with autodisplayed Z-domains on the sensitivity of thermophoretic immunoassay, the conventional method only with target analyte and detection antibodies was compared with the hyper sensitive thermophoretic immunoassay by mixing OM particles with autodisplayed Z-domains, and the affinity constant was calculated. Finally, the diagnosis of inflammation by detection of a biomarker called C-reactive protein (CRP) was carried out by demonstrating the thermophoretic immunoassay for CRP based on autodisplayedZ-domains could be applied for the medical diagnosis.

In this study, a single-domain antibody against negative regulatory factor (anti-NEF scFv) was autodisplayed on the outer membrane of Escherichia coli and used to detect NEF in an immunoassay based on fluorescence-activated cell sorting, enzyme-linked immunosorbent assay, and surface plasmon resonance biosensors. Next, the autodisplayed single-domain antibody was oxidized to form disulfide bonds by using glutathione, and the change in NEF-binding activity of anti-NEF scFv was analyzed by fluorescence-activated cell sorting-based immunoassay, chromogenic immunoassay, and surface plasmon resonance biosensor. For each type of immunoassays the anti-NEF scFv on the isolated outer membrane showed more NEF binding activity after the disulfide bond formation by glutathione. To determine the role of cysteines in anti-NEF scFv, three mutants were prepared, and the NEF binding activity of mutants was compared with that of wild-type anti-NEF scFv in a competitive immunoassay based on FACS. In these mutant studies, the refolding process of autodisplayed anti-NEF scFv by following oxidation via GSH/GSSG revealed that disulfide bonds formed and increased NEF binding activity.

In this work, the chronoamperometry-based redox cycling of 3,3',5,5'-tetramethylbenzidine (TMB) was performed by using interdigitated electrode (IDE). The signal was obtained from two sequential chronoamperometric profiles: (1) with the generator at the oxidative potential of TMB and the collector at the reductive potential of TMB, and (2) with the generator at the reductive potential of TMB and the collector at the oxidative potential of TMB. The chronoamperometry-based redox cycling (dual mode) showed a sensitivity of 1.49 μA/OD, and the redox cycling efficiency was estimated to be 94% (n = 10). The sensitivities of conventional redox cycling with the same interdigitated electrode and chronoamperometry using a single working electrode (single mode) were estimated to be 0.67 μA/OD and 0.18 μA/OD, respectively. These results showed that the chronoamperometry-based redox cycling (dual mode) could be more effectively used to quantify the oxidized TMB than other amperometric methods. The chronoamperometry-based redox cycling (dual mode) was applied to immunoassays using a commercial ELISA kit for medical diagnosis of the human hepatitis B virus surface antigen (hHBsAg). Finally, the chronoamperometry-based redox cycling (dual mode) provided more than a 10-fold higher sensitivity than conventional chronoamperometry using a single working electrode (single mode) when applied to a commercial ELISA kit for medical diagnosis of hHBsAg.

Many conventional optical biosensing systems use a single responsive signal in the visible light region. This limits their practical applications, as the signal can be readily perturbed by various external environmental factors. Herein, a near-infrared (NIR)-based self-calibrating luminescence resonance energy transfer (LRET) system was developed for background-free detection of analytes in homogeneous sandwich-immunoassays. The inorganic LRET pair was comprised of NIR dual-emitting lanthanide-doped nanoparticles (LnNPs) as donors and NIR-absorbing LnNPs as acceptors, which showed a narrow absorption peak (800 nm) and long-term stability, enabling stable LRET with a built-in self-calibrating signal. Screened single-chain variable fragments (scFvs) were used as target avian influenza virus (AIV)-binding antibodies to increase the LRET efficiency in sandwich-immunoassays. The compact sensor platform successfully detected AIV nucleoproteins with a 0.38 pM limit of detection in buffer solution and 64 clinical samples. Hence, inorganic LnNP pairs may be effective for self-calibrating LRET systems in the background-free NIR region.

The direct in situ synthesis of cadmium sulfide (CdS) nanowires (NWs) was presented by direct synthesis of CdS NWs on the gold surface of an interdigitated electrode (IDE). In this work, we investigated the effect of a strong oxidant on the surfaces of the CdS NWs using X-ray photoelectron spectroscopy, transmission electron microscopy, and time-of-flight secondary ion mass spectrometry. We also fabricated a parylene-C film as a surface passivation layer for in situ-synthesized CdS NW photosensors and investigated the influence of the parylene-C passivation layer on the photoresponse during the coating of parylene-C under vacuum using a quartz crystal microbalance and a photoanalyzer. Finally, we used the in situ-synthesized CdS NW photosensor with the parylene-C passivation layer to detect the chemiluminescence of horseradish peroxidase and luminol and applied it to medical detection of carcinoembryonic antigen.

Highly sensitive in situ-synthesized cadmium sulfide (CdS) nanowires (NWs) for the detection of chemiluminescence in immunoassays with a photoresist (PR) layer to stabilize the CdS NWs before and after coating with a parylene film were developed. The thickness of the PR layer was controlled by adjusting the viscosity of the PR solution used for spin-coating. PR2005 was the optimal PR for passivation of the NW surface. After the addition of a parylene coating on the CdS NWs, the photocurrent increased by as much as 50% over a broad range of light intensities, and the additional PR layer increased the photoresponse over the whole range of light intensities. When the photoresponses of the CdS NWs with and without the parylene film were compared after the addition of a PR layer, significant differences were observed in the photocurrent behavior after the incident light was turned off. For the CdS NWs with a parylene film and PR layer, the photocurrent reached the baseline within milliseconds of the incident light being turned off. However, the CdS NWs without a parylene film but with a PR layer required >60 s to reach the baseline level. This difference was due to the capacitance arising from the contact between the NWs. The in situ-synthesized CdS NW photosensor passivated by the parylene film and a PR layer was used in a chemiluminescence-based immunoassay. Finally, the detection of human immunodeficiency virus antibodies was demonstrated via a chemiluminescent enzyme-linked immunosorbent assay based on the CdS NW photosensor in comparison with the optical-density measurement for the chromogenic reaction of TMB(3,3′,5,5′-Tetramethylbenzidine).

Escherichia coli cells with autodisplayed Z-domains have been used for immunoassays of specific target analytes. In this study, a magnetite suspension was used for the washing step in immunoassays of E. coli cells with autodisplayed Z-domains. This approach enhanced the washing conditions for these immunoassays by determining (1) the optimal concentration of the magnetite suspension, (2) the capacity of the magnetite suspension-based washing method to recover E. coli cells, and (3) the level at which the activity of autodisplayed Z-domains is maintained. In immunoassays of C-reactive protein (CRP), the immunoassay incorporating the magnetite suspension-based washing method showed a sensitivity and limit of detection considerably higher than those of the conventional centrifugation-based washing method. The results indicated that immunoassays incorporating the magnetite suspension-based washing method are effective for medical diagnoses based on CRP assay.

A band-type microelectrode was made using a parylene-N film as a passivation layer. A circular-type, mm-scale electrode with the same diameter as the band-type microelectrode was also made with an electrode area that was 5000 times larger than the band-type microelectrode. By comparing the amperometric signals of 3,5,3',5'-tetramethylbenzidine (TMB) samples at different optical density (OD) values, the band-type microelectrode was determined to be 9 times more sensitive than the circular-type electrode. The properties of the circular-type and the band-type electrodes (e.g., the shape of their cyclic voltammograms, the type of diffusion layer used, and the diffusion layer thickness per unit electrode area) were characterized according to their electrode area using the COMSOL Multiphysics software. From these simulations, the band-type electrode was estimated to have the conventional microelectrode properties, even when the electrode area was 100 times larger than a conventional circular-type electrode. These results show that both the geometry and the area of an electrode can influence the properties of the electrode. Finally, amperometric analysis based on a band-type electrode was applied to commercial ELISA kits to analyze human hepatitis B surface antigen (hHBsAg) and human immunodeficiency virus (HIV) antibodies.

The Z-domain has the potential to control the orientation of immobilized antibodies because of its binding affinity to the Fc regions of antibodies (IgGs). In this work, Z-domains were autodisplayed on the outer membrane (OM) of Escherichia coli. OM particles were isolated and coated onto microbeads with positive, neutral, or negative surface charges. Other conditions such as incubation time and initial OM concentration were also optimized for the OM coating to obtain maximum antibody-binding. Using three kinds of model proteins with different isoelectric points (pI), streptavidin (pI = 5, negative charge at pH 7), horseradish peroxidase (pI = 7, neutral charge at pH 7), and avidin (pI = 10, positive charge at pH 7), protein immobilization onto the microbeads was carried out through physical adsorption and electrostatic interactions. Using fluorescently labeled antibodies and fluorescence-activated cell sorting, it was determined that the neutral and the positively charged microbeads effectively bound antibodies while minimizing non-specific protein binding. The OM-coated microbeads with autodisplayed Z-domains were applied to C-reactive protein immunoassay. This immunoassay achieved 5-fold improved sensitivity compared to conventional immunoassay based on physical adsorption of antibodies at the cutoff concentration of medical diagnosis of inflammatory diseases (1000 ng/ml) and cardiovascular diseases (200 ng/ml).

A one-step immunoassay for influenza A virus detection was developed using two different microbeads and a filter-inserted bottle. Two bead types with diameters of 15 (capture bead) and 3 (detection bead) μm were prepared to specifically detect influenza A virus. Anti-influenza A virus antibodies were coated on both bead types, whereas urease was immobilized only on the detection bead. An influenza A-positive sample could form a sandwich complex with the capture and detection beads; this complex would not pass through the filter, which had a controlled pore size. As the detection bead was used at a limiting concentration, it would be prevented from crossing the filter; thus, it would further react with the substrate urea and consequently increase the pH. An influenza A-negative sample would fail to form the sandwich complex in the presence of the capture and detection beads. Accordingly, the detection bead would pass through the filter into the urea buffer and increase the pH. The pH change in the urease reaction could be quantitatively measured by an indicator such as phenol red or using ion-selective field-effect transistor (ISFET). This one-step immunoassay was used for the detection of influenza A virus in real samples. The receiver operating characteristic (ROC) plot analysis showed an area under the curve (AUC) value of 0.931; the sensitivity and specificity of the assay was 80% and 90%, respectively, at a cutoff value of 0.9986. These results demonstrate that the one-step immunoassay could increase the sensitivity of influenza A virus detection in real samples.

Thermophoresis is the physical diffusion of molecules from hot to cold induced by a thermal gradient. Thermophoresis has been used to evaluate the interaction of biomolecules in solution. In this study, the outer membrane from E. coli was isolated and used to produce OM particles with a diameter of approximately 100 nm. These prepared OM particles were applied in a thermophoretic immunoassay. First, outer membrane (OM) particles with lipopolysaccharides (LPS) and anti-LPS antibodies were used as a model to demonstrate proof of concept and the difference in E. coli thermophoresis was explained by the changes in the molecular surface area (A) and effective charge (σeff). The hydrodynamic size of the molecules was measured as a changing parameter, molecular surface area (A), by dynamic laser scattering (DLS), and the zeta potential was measured as a changing parameter of effective charge (σeff) and then evaluated by the Soret equation. Using the hydrodynamic size and zeta potential values, the interaction between the antigen (OM particle with LPS) and antibody (anti-LPS antibodies) could be monitored and the results were fitted to the thermophoretic immunoassay using the Soret coefficient and equation. Finally, this OM-based immunoassay was applied to the medical diagnosis of systemic lupus erythematosus. Here, OM particles with Ro and La proteins were used to analyze the autoantibodies in patient and control sera. Thermophoretic immunoassay results were also compared to the fitted analysis using hydrodynamic size and zeta potential values and the Soret coefficient and equation.

Upconversion nanoparticles (UCNPs) are promising materials for biological applications based on luminescence resonance energy transfer (LRET). In contrast to classical RET donors such as quantum dots, gold nanoparticles, UCNPs can emit near-infrared (NIR) upon the NIR irradiation, which provides enhanced signal-to-noise due to strong penetration and low autofluorescence in the NIR region known as the diagnostic window. Here we report the first efficient NIR-to-NIR signal-based LRET system for the detection of progesterone, chosen as a proof-of-concept target, via homogeneous competitive immunoassay. To enhance the efficiency of LRET, we constructed inert-core/active-shell/inert-ultrathin shell UCNPs (NaYF4@NaYF4:Yb,Tm@NaYF4) as an LRET donor and a compact progesterone/horseradish peroxidase/IRdyeQC-1 (P-HRP-dyes) complex as an LRET acceptor. The designed donor and acceptor showed significantly improved LRET efficiencies (95% and 85% for donor and acceptor, respectively) compared with conventional donor and acceptor (70% and 50%, respectively). Using the developed NIR-to-NIR LRET system, progesterone was successfully detected with a background-free signal and low limit of detection (1.36 pg/ml in ten-fold diluted human serum). We believe that the efficient NIR-to-NIR signal-based LRET system developed here has potential as a simple probe for homogeneous competitive immunoassay, with the ability to rapidly detect biomarkers.

Through orientation control of antibodies, Z-domains autodisplaying Escherichia coli outer cell membrane (OM) may be utilized to improve the sensitivity and limit of detection (LOD) of immunoassays and immunosensors. A regenerative immunoaffinity layer based on Z-domains autodisplaying E. coli OM was developed for the surface plasmon resonance (SPR) biosensor. Regeneration conditions for the Z-domains autodisplaying E. coli OM-based immunoassays and immunosensors were optimized by varying pH and detergent concentration. An E. coli cell-based HRP immunoassay was tested and validated in three sequential regenerative immunoassays under optimal conditions. The OM of Z-domains autodisplaying E. coli was isolated and coated on the two-dimensional substrate (microplate). The OM-based HRP immunoassay was tested and validated in four regenerative immunoassays. This regenerative OM layer was applied to the SPR biosensor. Z-domains autodisplaying OM layered onto the gold surface of SPR biosensors was developed, and the OM-based regenerative immunoaffinity layer with orientation control was tested using CRP analyte. The SPR biosensor regenerative immunoaffinity layer demonstrated that CRP biosensing was repeated for five regeneration cycles with less than 2% signal difference. Therefore, the newly developed regenerative immunoaffinity layer with antibody orientation control may improve biosensing sensitivity and reduce the cost of medical diagnosis.

Through orientation control of antibodies, Z-domains autodisplaying Escherichia coli outer cell membrane (OM) may be utilized to improve the sensitivity and limit of detection (LOD) of immunoassays and immunosensors. A regenerative immunoaffinity layer based on Z-domains autodisplaying E. coli OM was developed for the surface plasmon resonance (SPR) biosensor. Regeneration conditions for the Z-domains autodisplaying E. coli OM-based immunoassays and immunosensors were optimized by varying pH and detergent concentration. An E. coli cell-based HRP immunoassay was tested and validated in three sequential regenerative immunoassays under optimal conditions. The OM of Z-domains autodisplaying E. coli was isolated and coated on the two-dimensional substrate (microplate). The OM-based HRP immunoassay was tested and validated in four regenerative immunoassays. This regenerative OM layer was applied to the SPR biosensor. Z-domains autodisplaying OM layered onto the gold surface of SPR biosensors was developed, and the OM-based regenerative immunoaffinity layer with orientation control was tested using CRP analyte. The SPR biosensor regenerative immunoaffinity layer demonstrated that CRP biosensing was repeated for five regeneration cycles with less than 2% signal difference. Therefore, the newly developed regenerative immunoaffinity layer with antibody orientation control may improve biosensing sensitivity and reduce the cost of medical diagnosis.

In this work, the gram-negative bacterium Escherichia coli strain BL21(DE3) (with lipopolysaccharide (LPS) in its outer membrane) and its modified ClearColi™ strain (lacking LPS) were used for the separation of anti-LPS antibodies from human serum by the following steps: (1) binding of the serum proteins to BL21(DE3); (2) dissociation of the bound proteins (including anti-LPS antibodies) from BL21(DE3) with acid; (3) filtering of the dissociated proteins using ClearColi to remove unwanted proteins; and (4) separation of the antibody fraction by protein-A column chromatography. The binding properties of the separated antibodies were analyzed by fluorescence-activated cell sorting to confirm their selective binding to LPS on the outer membrane of BL21(DE3), and by thermophoretic immunoassay to estimate their dissociation constant. The in vitro applicability of the separated anti-LPS antibodies was demonstrated through a fluorescence assay of BL21(DE3), after immobilizing the antibodies onto a modified microplate surface. The electrochemical detection of BL21(DE3) was also achieved after immobilizing the anti-LPS antibodies onto a gold electrode.

In this work, two proteins, Z-domains and bovine casein, were auto-displayed on the outer membrane of the same Escherichia coli cells by co-transformation of two different auto-display vectors. On the basis of SDS-PAGE densitometry, Z-domains and bovine casein were expressed at 3.12 × 10⁵ and 1.55 × 10⁵ proteins/E. coli cell, respectively. The co-auto-displayed Z-domains had antibody-binding activity and the bovine casein had adhesive properties. E. coli with co-auto-displayed proteins were analyzed by fluorescence assisted cell sorting (FACS). E. coli with co-auto-displayed Z-domains and bovine casein aggregated due to hydrophobic interaction. For application to immunoassays, the Z-domain activity was estimated after (1) immobilizing the E. coli and (2) forming an OM layer. E. coli with co-auto-displayed two proteins that were immobilized on a polystyrene microplate had the same antibody-binding activity as did E. coli with auto-displayed Z-domains only. The OM layer from the co-transformed E. coli had Z-domains and bovine casein expressed at a 1:2 ratio from antibody-binding activity measurements.

A lateral flow immunoassay (LF-immunoassay) with an enhanced sensitivity and thermostability was developed by using Pt nanoparticles with a peroxidase activity. The Pt nanoparticles were synthesized by citrate reduction method, and the peroxidase activity of Pt nanoparticles was optimized by adjusting reaction conditions. The peroxidase activity was estimated by using Michaelis-Menten kinetics model with TMB as a chromogenic substrate. The kinetics parameters of KM and Vmax were calculated and compared with horseradish peroxidase (HRP). The thermal stability of the Pt nanoparticles was compared with horseradish peroxidase (HRP) according to the storage temperature and long-term storage period. The feasibility of lateral flow immunoassay with a chemiluminescent signal band was demonstrated by the detection of human chorionic gonadotropin (hCG) as a model analyte, and the sensitivity was determined to be improved by as much as 1000-fold compared to the conventional rapid test based on colored gold-colloids.

Escherichia coli cells that autodisplay Z-domains have been used to improve the sensitivity and limit of detection (LOD) of immunoassays by controlling antibody orientation. In this study, a rapid, wash-free immunoassay was developed using E. coli cells with autodisplayed Z-domains and flow cytometry. The fluorescence signal from a single E. coli cell could be identified and background noise from non-bound detection antibodies was minimal. Flow cytometric measurement was demonstrated to be more effective than microscopy or fluorescence photometry. The concentrations of the capture and detection antibodies were optimized by testing various concentrations; the incubation time was optimized in the same way. The wash-free immunoassay was used to quantify the C-reactive protein (CRP) and the results were compared with those of an established assay method. The results of the wash-free immunoassay were significantly correlated with the results of the washing method, but showed an improved LOD and dynamic range. Thus, the wash-free immunoassay based on E. coli cells with autodisplayed Z-domains and flow cytometry developed here was confirmed to be feasible for use in medical diagnosis.

A microarray-based immunoassay for the detection of autoantibodies against Ro protein was developed using Escherichia coli with autodisplayed Ro proteins (Ro(+)-E. coli). Patient serum usually contains various antibodies against the outer membrane components of E. coli as well as autoantibodies against the Ro protein. Therefore, the conventional immunoassay based on Ro(+)-E. coli requires both wild type E. coli (blank test) and Ro(+)-E. coli, and both strains of E. coli must be prepared in situ for each individual test serum. In this study, we tested the feasibility of using several types of animal sera as a replacement for individual human sera. An immunoassay without the blank test was developed using Ro(+)-E. coli by (1) blocking with rabbit serum, and (2) cleaving the Fc region from antibodies using papain. Modified E. coli with autodisplayed Ro protein was immobilized to a surface-modified microplate and the applicability of the immunoassay without the blank test was demonstrated using sera from patients with systemic lupus erythematosus (SLE). Using this approach, a microarray-based fluorescence immunoassay with immobilized Ro(+)-E. coli was able to detect anti-Ro autoantibodies in SLE patient sera with high specificity and selectivity and improved efficiency.

Fluorescence-activated cell sorter (FACS)-based immunoassays using E. coli cells with autodisplayed Z-domains were performed to (1) improve the sensitivity of the immunoassay through orientation control of antibodies with autodisplayed Z-domains and (2) minimize the required amount of analyte by using FACS to measure the fluorescent signal from individual E. coli cells. The expression (autodisplay) of Z-domains on the outer membrane of E. coli was confirmed by fluorescence image analysis, and the homogeneous distribution of autodisplayed Z-domains was presented by SEM image analysis after treatment with antibodies labeled with gold nanoparticles (diameter of 15 nm). As FACS measures the fluorescent signal from individual E. coli cells, the optimal FACS parameters for the effective detection of fluorescently labeled E. coli cells were determined, and the minimum amount of analyte required for the E. coli cell-based immunoassay was identified using two model immunoassay configurations. Finally, the medical diagnosis of heart infarction with a biomarker called troponin-I using the E. coli cell-based immunoassay with FACS analysis was demonstrated.

Recently, we reported a highly sensitive immunoassay using Escherichia coli cells with autodisplayed Z-domains. In this work, E. coli cells with autodisplayed Z-domains were applied to the flow-cytometry-based simultaneous detection of multiple analytes. The E. coli cells were doubly transfected to express a fluorescent protein (tdTomato) in the cytosol and the autodisplayed Z-domains on the outer membrane. By using E. coli cells with only the autodisplayed Z-domains, immunoassay of multiple analytes could be performed simultaneously on the same sample. Flow cytometry can be used to identify the immunoassay type by simultaneously detecting the fluorescence signal from the cytosol (tdTomato) and the fluorescence from the outer membrane, enabling the quantification of bound analytes after treatment with additional fluorescently labeled antibodies. To demonstrate the immunoassay of multiple analytes by using flow cytometry, human hepatitis B virus surface antigen (HBsAg) and C-reactive protein (CRP), a broad spectrum inflammation marker, were used as model analytes.

Escherichia coli cells with autodisplayed Z-domains could increase the sensitivity of immunoassays by immobilizing antibodies in a controlled orientation. In the work presented here, E. coli cells with autodisplayed Z-domains were immobilized to magnetic beads for subsequent immunoassay. In comparing conventional immunoassay using the E. coli cells with autodisplayed Z-domains, the magnetic-bead-based immunoassay improved immunoassay efficiency by minimizing the loss of E. coli cells during repeated centrifugation steps during washing. For the immobilization of E. coli cells to magnetic beads, the magnetic beads were modified with poly-l-lysine to bind to negatively charged E. coli cells. During the surface modification process, physical parameters such as the surface charge and size of the magnetic beads were analyzed to confirm the formation of E. coli-magnetic bead complexes. To test the feasibility of the magnetic-bead-based immunoassay, horseradish peroxidase (HRP) was used as a model analyte, and a biomarker for inflammatory diseases, C-reactive protein (CRP), was used for a demonstration of an application in medical diagnosis.

We developed a fluorescence-activated cell sorter (FACS)-based immunoassay using Escherichia coli with autodisplayed Z-domains as a solid support for detection antibodies as well as a method to optimize the signal-to-noise ratio of fluorescence signals from E. coli. To improve the sensitivity of FACS measurements by eliminating artifactual signals from unrelated small particles in the sample mixture, E. coli with autodisplayed Z-domains was modified to express enhanced green fluorescence protein (eGFP) in the cytosol. By selectively gating the measurement channel for eGFP in the cytosol, we were able to discriminate between green fluorescent E. coli and artifact particles. We confirmed the efficacy of this optimization method for FACS-based immunoassay using several model reactions. Finally, as proof of concept, we used the FACS-based immunoassay and our optimization method for the medical diagnosis of human hepatitis B virus surface antigen.

A capacitive biosensor was developed by using a vertically paired ring-electrode for the non-labeled immunoassay. The vertically paired ring-electrode was prepared by sequential deposition and etching processes. Two electrodes were layered on glass substrate by sputtering of gold layers with thicknesses of 50 nm and 100 nm, and a parylene-C film with a thickness of 550 nm was positioned between the electrode layers as a dielectric material by thermal deposition. The top layer was made by spin coating of SU-8. And then, the ring-electrodes were exposed at the wall of the layered structure by sequential etching processes. The fabricated electrodes were characterized by cyclic voltammetry of a well-known redox couple of 3,3',5,5'-tetramethylbenzidine. The non-labeled detection of antigen–antibody interaction was demonstrated by using anti-horseradish peroxidase (HRP) antibodies and C-reactive protein (CRP) as model analytes. When the model analytes were bound to the vertically paired ring-electrode, the impedance change was measured during the immunoassay steps, and the measured impedance was analyzed by using a model circuit of the ring-electrode, and the capacitance was estimated to be dependent on the adsorption of analytes between the ring-electrodes.

Redox cycling based on an interdigitated electrode (IDE) was used as a highly sensitive immunoassay for carcinogenic embryonic antigen (CEA) through the quantification of 3,3′,5,5′-tetramethylbenzidine (TMB). For the redox cycling process, one pair of interdigitated finger electrodes was used as the first working electrode (generator) for cyclic voltammetry of TMB, and another pair of interdigitated finger electrodes was used as the second working electrode (collector) for sequential application of potentials for reduction and oxidation of TMB. The reduction (and oxidation) products of TMB at the collector were supplied to the generator, and following sequential oxidization (and reduction) at the generator, again supplied to the collector. Such redox recycling processes between the generator and collector allowed signal amplification. In this work, the influences of the following factors on the redox cycling of TMB were analyzed: (1) the redox potential at the collector, (2) the gap between the interdigitated finger electrodes, and (3) the scan rate of the generator. The redox potential and electrode gap influences were simulated with COMSOL software and compared with empirical results. At the optimum redox potentials and electrode gap, redox cycling was estimated to be five-fold more sensitive for the quantification of TMB than conventional cyclic voltammetry using one pair of interdigitated finger electrodes as the working electrode. Finally, redox cycling was applied to a commercial immunoassay for CEA, and the sensitivity of redox cycling was three-fold higher than that of conventional cyclic voltammetry using a single set of interdigitated finger electrodes as the working electrode.

The objective of this study was to present an immunoassay for the diagnosis of Sjogren's syndrome based on the autodisplayed La/SSB protein on the outer membrane of intact E. coli (strain UT-5600) and LPS-free E. coli (ClearColi™). As the first step, an autodisplay vector (pCK002) was transfected into intact E. coli and LPS-free E. coli for comparison of efficiency of autdisplay of La/SSB. The maximal level of La/SSB expression was estimated to be similar for LPS-free E. coli and intact E. coli at different optimal induction periods. Intact E. coli was found to grow twofold faster than LPS-free E. coli, and the maximal level of expression for LPS-free E. coli was obtained with a longer induction period. When the zeta potential was measured, both intact E. coli and LPS-free E. coli showed negative values, and the autodisplay of negatively charged La/SSB protein (pI

A hypersensitive CdS nanowire (NW) photosensor was fabricated by an in-situ synthesis process that involved the direct synthesis of CdS NWs on an interdigitated electrode (IDE). Analysis of the photoresponse properties showed that the newly synthesized photosensor had enhanced sensitivity and a highly reproducible photoresponse compared to photosensors prepared from CdS NW suspensions. The NW photosensor was applied to measure the chemiluminescence of luminol, and the sensitivity was compared to a commercial photosensing system. Finally, the feasibility of the CdS NW photosensor for the application to the medical diagnosis of the human hepatitis B surface antigen (hHBsAg) was demonstrated using a lateral-flow immunoassay with a chemiluminescent signal band.

Immunoassay has been widely used for the detection of target analytes in complex mixtures such as blood by using highly specific antigen-antibody interactions. In this work, antibodies against a target analyte was immobilized to magnetic beads and the change of surface charge of a magnetic bead was measured after the binding of a target protein by using a zeta potential analyzer. For the feasibility test of the zeta potential analysis for immunoassays, horseradish peroxidase (HRP) was used as a model target analyte. The limit of detection (LOD) of HRP from zeta potential analysis was estimated to be less than 1 ng/mL and the dynamic range was from less than 1 ng/mL to more than 1 μg/mL. The application to medical diagnosis was demonstrated by detection of C-reactive protein (CRP) which is known to be a biomarker for inflammatory diseases. In the case of CRP samples in human serum, the limit of detection was estimated to be around 10 ng/mL and the response curve shows also a linear correlation at the concentration range from 10 ng/mL to 1 μg/mL. From these results, the the immunoassay based on zeta potential analysis was considered to be feasible for medical diagnosis of CRP in serum.

Recently, we reported autodisplayed Z-domains on Escherichia coli can highly improve the sensitivity of immunoassays through the orientation control of detection antibodies. In this work, the amperometric analysis was applied to the immunoassay based on E. coli cells with autodisplayed Z-domains. For amperometric analysis, the reduction current of 3,5,3′,5′-tetramethylbenzidine (TMB) was measured at the potential of −50 mV and the correlation between the reduction current and the OD value was estimated to be 28 nA/OD and the limit of detection was calculated to be 0.07 in OD unit. In this work, the feasibility of the immunoassay based on amperometric analysis was presented by the quantification of HRP as a model analyte. For the demonstration of medical diagnosis, C-reactive protein (CRP) was detected by using electrochemical ELISA based on E. coli with autodisplayed Z-domains.

The Z-domains of protein A was expressed as a fusion protein at the outer membrane of E.coli by using the autodisplay technology. Because of the specific affinity towards the Fc region of immunoglobulins (IgG’s), the Z-domains have been used for the orientation control of antibodies in order to improve the sensitivity of immunoassays. In this work, the E.coli with autodispalyed Z-domains was immobilized to the SPR biosensor by the charge interaction. The surface modification was carried out by covalent layering of the poly-L-lysine with amino groups to the parylene-H film with formyl groups. And then, the negatively charged E.coli cells were immobilized by charge interaction with the positivley charged of poly-L-lysine. The effectiveness of this layer for the immobilization of E.coli was estimated by counting the number of E.coli cells in comparison with the bare gold surface and the poly-L-lysine coated gold surface. For the test of feasibility of the immobilized E.coli cells to SPR biosensor, the stability of immobilized E.coli cells was estimated by treatment of salt solutions at the known concentrations to the immobilized E.coli cells which were bound through the charge interaction. From this test, the E.coli cells immobilized to the parylene-H film with poly-L-lysine coating were determined to be stable at the salt concentration of human serum. Then, the applicability of the immobilized E.coli cells with autodisplayed Z-domains was demonstrated by detection of C-reactive protein (CRP). The effect of orientation control with autodisplayed Z-domains was estimated by comparing the sensivities by immobilization through the physical adsorption and charge interaction to poly-L-lysine coated layer.

An immunoassay based on stick-type solid support (immunostick assay) was developed. To demonstrate the medical diagnosis of rheumatoid arthritis (RA), cyclic-citrullinated peptide (CCP), one of specific antigens against RA autoantibodies, was immobilized on the surface of the immunostick, and a color index table was prepared for the determination of CCP-positivity of the test sera. The positivity of RA-positive (n = 31) and RA-negative (n = 20) sera was tested using the immunostick assay and a conventional enzyme-linked immunosorbent assay (ELISA). The statistical agreement of the test results from both methods was analyzed using inter-rater coefficient kappa and Bland-Altman test. The immunostick assay with a color index table was determined to have a high statistical correlation to the conventional ELISA method.

Escherichia coli with autodisplayed Z-domains was reported to improve the sensitivity of immunoassays by the orientation control of antibodies. In this work, a sensitive microplate-based immunoassay is presented by immobilizing E. coli cells to a surface-modified microplate. The microplate was prepared by coating parylene-H film with formyl groups, and then covalently coupling poly-L-lysine to the parylene-H film. The E. coli cells were bound to the microplate by charge interactions between the negatively charged E. coli outer membrane and the positively charged microplate surface. In this work, the preparation of the microplate coated with poly-L-lysine is presented. The immobilization efficiency of E. coli to the modified surface was estimated to be far higher than non-specific interaction by fluorescence microscope and the optical transmittance of the modified microplate was measured to be feasible for immunoassay. The microplate-based immunoassay is demonstrated to be feasible for medical diagnosis of inflammatory diseases by using C-reactive protein as a target analyte for the medical diagnosis of inflammatory diseases.

In this work, the Z-domain of protein A with IgG-binding activity was expressed on the outer membrane (OM) of E. coli as a fusion protein of AIDA-1 by using “autodisplay technology”. The OM of E. coli with the autodisplayed Z-domains was isolated and then the OM with the Z-domains was layered on the gold surface of SPR biosensor in order to prepare the orientation-controlled antibody layer. The properties of the OM layer were analyzed by cyclic voltammetry, impedance spectroscopy and AFM. The immunoassay by using the OM layer with Z-domains showed that the limit of detection was improved as much as 50-folds by the orientation-control effect of the Z-domains. The sensitivity of assay (y-axis) at the each analyte concentration (x-axis) was also observed to be far improved by the orientation-control. The OM layer was then prepared on the SPR biosensor, and the far improved sensitivity from the orientation-control effect by the Z-domains was presented in comparison to the randomly oriented antibody layer by using hIgG as an analyte. For the regeneration of the SPR biosensor with the antibody layer immobilized to the Z-domains of OM layer, a cross-linker called glutaraldehyde was used for the covalent immobilization of the antibodies, and the feasibility of this method was demonstrated by performing repeated regeneration processes.

Recently, we reported autodisplayed Z-domains on E.coli can highly improve the sensitivity of immunoassays through the orientation control of detection antibodies [1–3]. In this work, the amperometric analysis was applied to the immunoassay based on E.coli cells with autodisplayed Z-domains. For amperometric analaysis, the reduction current of 3,5,3’,5’-tetramethylbenzidine (TMB) was measured at the potential of -50mV and the correlation between the reduction current and the OD value was estimated to be 28 nA/OD and the limit of detection was calculated to be 0.07 in OD unit. In this work, an immunoassay was prepared by immobilizing anti-HRP antibodies to E.coli cells with autodisplayed Z-domains and the amperometric analysis was demonstrated the quantification of bound HRP. For the demonstration of medical diagnosis, C-reactive protein (CRP) was detected by using electrochemical ELISA based on E.coli with autodisplayed Z-domains.