Molecular and structural basis of anti-DNA antibody specificity for pyrrolated proteins.

Anti-DNA antibodies (Abs), serological hallmarks of systemic lupus erythematosus (SLE) and markers for diagnosis and disease activity, show a specificity for non-nucleic acid molecules, such as N-pyrrolated proteins (pyrP) containing Nε-pyrrole-L-lysine (pyrK) residues. However, the detailed mechanism for the binding of anti-DNA Abs to pyrP remains unknown. In the present study, to gain structural insights into the dual-specificity of anti-DNA Abs, we used phage display to obtain DNA-binding, single-chain variable fragments (scFvs) from SLE-prone mice and found that they also cross-reacted with pyrP. It was revealed that a variable heavy chain (VH) domain is sufficient for the recognition of DNA/pyrP. Identification of an antigenic sequence containing pyrK in pyrP suggested that the presence of both pyrK and multiple acidic amino acid residues plays important roles in the electrostatic interactions with the Abs. X-ray crystallography and computer-predicted simulations of the pyrK-containing peptide-scFv complexes identified key residues of Abs involved in the interaction with the antigens. These data provide a mechanistic insight into the molecular basis of the dual-specificity of the anti-DNA Abs and provide a basis for therapeutic intervention against SLE.

Procedural characteristics and cardiovascular outcomes in patients undergoing drug-coated balloon angioplasty for de novo lesions in large coronary arteries: an observational study.

Limited data exist regarding drug-coated balloon (DCB) treatment in de novo large coronary arteries. We sought to demonstrate procedural characteristics, residual stenosis, and clinical outcomes following DCB angioplasty for de novo lesions in large versus small coronary arteries. The study included 184 consecutive patients with 223 de novo coronary lesions undergoing paclitaxel DCB angioplasty between January 2019 and August 2020, who were divided according to whether the DCB diameter was ≥ 3.0 mm (large group, n = 58) or < 3.0 mm (small group, n = 125). The large group had a higher proportion of acute coronary syndrome more commonly with ostial, bifurcation, and calcified lesions in large vessels and received lesion preparation with more frequent use of scoring or cutting balloons and atherectomy devices compared to the small group. Postprocedural angiographic diameter stenosis was smaller in the large group compared to the small group (31% [22-37] vs. 35% [26-42], p = 0.032), and intravascular ultrasound revealed no significant difference in postprocedural area stenosis between the groups (66.2 ± 7.7% vs. 67.9 ± 7.8%; p = 0.26). The median follow-up duration was 995 days. The incidence of a composite of all-cause death, myocardial infarction, stroke, or target lesion revascularization was similar between the groups (log-rank p = 0.41) and was influenced by the presence of acute coronary syndrome and anemia but not by DCB diameter. The rate of cardiovascular outcomes after DCB treatment was comparable in de novo large and small coronary arteries. Notably, well-planned lesion preparation with intravascular imaging guidance was prevalent in large vessels.

Glycyrrhizin Production in Licorice Hairy Roots Based on Metabolic Redirection of Triterpenoid Biosynthetic Pathway by Genome Editing.

Glycyrrhizin, a type of the triterpenoid saponin, is a major active ingredient contained in the roots of the medicinal plant licorice (Glycyrrhiza uralensis, G. glabra and G. inflata), and is used worldwide in diverse applications, such as herbal medicines and sweeteners. The growing demand for licorice threatens wild resources and therefore a sustainable method of supplying glycyrrhizin is required. With the goal of establishing an alternative glycyrrhizin supply method not dependent on wild plants, we attempted to produce glycyrrhizin using hairy root culture. We tried to promote glycyrrhizin production by blocking competing pathways using CRISPR/Cas9-based gene editing. CYP93E3 CYP72A566 double-knockout (KO) and CYP93E3 CYP72A566 CYP716A179 LUS1 quadruple-KO variants were generated, and a substantial amount of glycyrrhizin accumulation was confirmed in both types of hairy root. Furthermore, we evaluated the potential for promoting further glycyrrhizin production by simultaneous CYP93E3 CYP72A566 double-KO and CYP88D6-overexpression. This strategy resulted in a 3-fold increase (∼1.4 mg/g) in glycyrrhizin accumulation in double-KO/CYP88D6-overexpression hairy roots, on average, compared with that of double-KO hairy roots. These findings demonstrate that the combination of blocking competing pathways and overexpression of the biosynthetic gene is important for enhancing glycyrrhizin production in G. uralensis hairy roots. Our findings provide the foundation for sustainable glycyrrhizin production using hairy root culture. Given the widespread use of genome editing technology in hairy roots, this combined with gene knockout and overexpression could be widely applied to the production of valuable substances contained in various plant roots.

Impact of COVID-19 on pediatric surgery cancellations: A retrospective study.

BACKGROUND: Prevalence trends and reasons for pediatric surgery cancellation in Japan during the coronavirus disease 2019 (COVID-19) pandemic have not previously been reported. This study aimed to compare the prevalence and reasons for cancellation of pediatric surgeries in Japan before and during the COVID-19 pandemic. METHODS: This single-center retrospective cohort study reviewed the reasons for surgery cancellations scheduled for patients aged <18 years between the prepandemic period (September 2017-December 2019) and the COVID-19 pandemic period (January 2020-April 2022). The cancellation reasons were classified into four major categories: medical, surgical, patient-related, and administrative. RESULTS: Of the 3395 and 3455 surgeries scheduled before and during the COVID-19 pandemic, 305 (9.0%) and 319 (9.2%) surgeries were canceled (p = 0.737), respectively. The proportion of cancellations due to infections or fever in medical reasons decreased from 67.9% to 56.1% (p = 0.003) and that due to patient-related reasons increased from 6.6% to 15.1% (p = 0.001). Further, the proportion of cancellations due to staff shortages in staff administrative reasons increased from 0.3% to 3.1% (p = 0.011). There was no significant difference in the proportion of surgeries canceled due to surgical reasons between the two periods. CONCLUSIONS: The proportion of cancellations due to infections or fever decreased during the COVID-19 pandemic, while that due to staff shortages increased. Infection prevention is an important measure to address the staff shortages. Implementation of national or regional policies and additional strategic interventions may be required to prepare for disasters like the COVID-19 pandemic.

Improvement in Inhalation Properties of Theophylline and Levofloxacin by Co-Amorphization and Enhancement in Its Stability by Addition of Amino Acid as a Third Component.

Co-amorphous systems are amorphous formulations stabilized by the miscible dispersion of small molecules. This study aimed to design a stable co-amorphous system for the co-delivery of two drugs to the lungs as an inhaled formulation. Theophylline (THE) and levofloxacin (LEV) were used as model drugs for treating lung infection with inflammation. Leucine (LEU) or tryptophan (TRP) was employed as the third component to improve the inhalation properties. The co-amorphous system containing THE and LEV in an equal molar ratio was successfully prepared via spray drying where reduction of the particle size and change to the spherical morphology were observed. The addition of LEU or TRP at a one-tenth molar ratio to THE-LEV did not affect the formation of the co-amorphous system, but only TRP acted as an antiplasticizer. The Fourier transform infrared spectroscopy spectra revealed intermolecular interactions between THE and LEV in the co-amorphous system that were retained after the addition of LEU or TRP. The co-amorphous THE-LEV system exhibited better in vitro aerodynamic performance than a physical mixture of these compounds and permitted the simultaneous delivery of both drugs in various stages. The co-amorphous THE-LEV system crystallized at 40 °C, and this crystallization was not prevented by LEU. However, THE-LEV-TRP maintained its amorphous state for 1 month. Thus, TRP can act as a third component to improve the physical stability of the co-amorphous THE-LEV system, while maintaining the enhanced aerodynamic properties.

Efficient and rapid linker optimization with heterodimeric coiled coils improves the response of fluorescent biosensors comprising antibodies and protein M.

We developed a coiled Q-probe (CQ-probe), a fluorescent probe containing a coiled-coil peptide pair E4/K4, to convert antibodies into biosensors for homogeneous immunoassays. This probe consists of an antibody-binding protein, protein M (PM) with the E4 peptide and the K4 peptide with a fluorescent dye. Compared to PM Q-probes, which are generated by modifying the C-terminus of PM with a fluorescent dye, CQ-probe variants with various linkers are easy to prepare and therefore enable the establishment of biosensors with a significant fluorescence response by localizing the fluorescent dye at the optimal position for quenching and antigen-dependent release. The fluorescence changes of biosensors converted from anti-BGP, anti-cortisol, and anti-testosterone antibodies using the rhodamine 6G (or TAMRA)-labeled CQ-probe upon antigen addition were 13 (or 2.6), 9.7 (or 1.5), and 2.1 (or 1.2) times larger than that of the biosensors converted using the PM Q-probe. Furthermore, the CQ-probe converted anti-digoxin IgG into a functional biosensor, whereas the PM Q-probe/antibody complex showed an insufficient response. This technology exhibits a promising capacity to convert antibodies into high-response biosensors, which are expected to be applied in a wide range of fields, including clinical diagnosis, environmental surveys, food analysis, and biological research.

Generation of a Recombinant scFv against Deoxycholic Acid and Its Conversion to a Quenchbody for One-Step Immunoassay.

Development of a rapid detection method for deoxycholic acid (DCA) is crucial for its diagnosis in the early stages of inflammation and cancer. In this study, we expressed a soluble recombinant anti-DCA single-chain variable fragment (scFv) in Escherichia coli. To convert scFv into a Quenchbody (Q-body), we labeled scFv using commercially available maleimide-linked fluorophores. The TAMRA-C5-maleimide-conjugated Q-body showed the highest response within a few minutes of DCA addition, indicating its applicability as a wash-free immunoassay probe for onsite DCA detection.

Impact of prolonged drug-coated balloon inflation on residual stenosis and clinical outcomes in coronary artery disease patients: A propensity score matched analysis.

BACKGROUND: There is a paucity of data regarding the optimal duration of drug-coated balloon (DCB) inflation for coronary lesions. We sought to explore the effect of DCB angioplasty with versus without long inflation time on residual stenosis and clinical outcomes in patients with coronary artery disease. METHODS: This study included 314 consecutive patients with 445 lesions undergoing paclitaxel DCB angioplasty using different inflation time, divided according to whether the total inflation time of the DCB was ≥180 s (prolonged group) or <180 s (standard group). The primary clinical endpoint, defined as a composite of all-cause death, myocardial infarction, stroke, or target lesion revascularization, was examined in 92 propensity score matched pairs. RESULTS: In the matched cohort, the median clinical follow-up period was 947 days. Postprocedural angiographic diameter stenosis was smaller in the prolonged group than in the standard group (30.0% [22.0-37.0] vs. 33.5% [25.5-40.5]; p = 0.042). Intravascular ultrasound measurements revealed that longer DCB inflation time resulted in smaller area stenosis (66.6 ± 7.8% vs. 69.4 ± 7.0%; p = 0.044) and a less mean increase in percent atheroma volume (-11.2 ± 7.1% vs. -7.4 ± 5.9%; p = 0.004) after angioplasty. The rate of the primary endpoint was lower in the prolonged group than in the standard group (log-rank p = 0.025). The efficacy of prolonged DCB inflation was prominent in patients with in-stent restenosis and longer lesions. CONCLUSION: Prolonged DCB inflation was associated with reduced residual stenosis and improved clinical outcomes in patients with coronary artery disease undergoing percutaneous coronary intervention. Prospective randomized trials are warranted to validate the benefits of DCB angioplasty with long inflation time.

Dissolution-permeation of hot-melt extruded amorphous solid dispersion comprising an experimental grade of HPMCAS.

Background and purpose: Physicochemical properties of an amorphous solid dispersion (ASD) comprising an experimental grade of hydroxypropyl methylcellulose acetate succinate (HPMCAS-MX) with lower glass transition temperature have been previously investigated. This study aimed to evaluate applicability of HPMCAS-MX to hot-melt extrusion (HME) and dissolution-permeation performance of prepared ASDs using MicroFLUX. Review approach: A physical mixture of indomethacin (IMC) and HPMCAS-MX or -MG (a commercial grade with higher transition temperature) at 20:80 weight ratio was hot-melt extruded to prepare an ASD (IMC-MX and IMC-MG, respectively). The dissolution-permeation performance and the stability of the ASDs were measured. Key results: A torque reduction at 120 °C implied that IMC-MX transformed into an amorphous state at this temperature, but IMC-MG required around 170 °C. This result was supported by Raman mapping of the the HME samples. IMC-MG and IMC-MX remained in an amorphous state at 40 °C for three months. The initial dissolution rate and solubility of the ASDs were higher than that of crystalline IMC. The apparent permeability of IMC from IMC-MX and IMC-MG was comparable but was approximately two-fold higher than that from crystalline IMC. Conclusion: HPMCAS-MX enabled HME process at a lower temperature and improved the dissolution-permeation performance of indomethacin.

Design of an Antigen-Triggered Nanobody-Based Fluorescence Probe for PET Immunoassay to Detect Quinalphos in Food Samples.

Photoinduced electron-transfer (PET) immunoassay based on a fluorescence site-specifically labeled nanobody, also called mini Quenchbody (Q-body), exhibits extraordinary sensitivity and saves much time in the homogeneous noncompetitive mode and is therefore regarded as a valuable method. However, limited by the efficiency of both quenching and dequenching of the fluorescence signal before and after antigen binding associated with the PET principle, not all original nanobodies can be used as candidates for mini Q-bodies. Herein, with the anti-quinalphos nanobody 11A (Nb-11A) as the model, we, for the first time, adopt a strategy by combining X-ray structural analysis with site-directed mutagenesis to design and produce a mutant Nb-R29W, and then successfully generate a mini Q-body by labeling with ATTO520 fluorescein. Based on this, a novel PET immunoassay is established, which exhibits a limit of detection of 0.007 µg/mL with a detection time of only 15 min, 25-fold improved sensitivity, and faster by 5-fold compared to the competitive immunoassay. Meanwhile, the recovery test of vegetable samples and validation by the standard ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) both demonstrated that the established PET immunoassay is a novel, sensitive, and accurate detection method for quinalphos. Ultimately, the findings of this work will provide valuable insights into the development of triggered PET fluorescence probes by using existing antibody resources.

Non-Vesicular Release of Alarmin Prothymosin α Complex Associated with Annexin-2 Flop-Out.

Nuclear protein prothymosin α (ProTα) is a unique member of damage-associated molecular patterns (DAMPs)/alarmins. ProTα prevents neuronal necrosis by causing a cell death mode switch in serum-starving or ischemic/reperfusion models in vitro and in vivo. Underlying receptor mechanisms include Toll-like receptor 4 (TLR4) and Gi-coupled receptor. Recent studies have revealed that the mode of the fatal stress-induced extracellular release of nuclear ProTα from cortical neurons in primary cultures, astrocytes and C6 glioma cells has two steps: ATP loss-induced nuclear release and the Ca2+-mediated formation of a multiple protein complex and its extracellular release. Under the serum-starving condition, ProTα is diffused from the nucleus throughout the cell due to the ATP loss-induced impairment of importin α-mediated nuclear transport. Subsequent mechanisms are all Ca2+-dependent. They include the formation of a protein complex with ProTα, S100A13, p40 Syt-1 and Annexin A2 (ANXA2); the fusion of the protein complex to the plasma membrane via p40 Syt-1-Stx-1 interaction; and TMEM16F scramblase-mediated ANXA2 flop-out. Subsequently, the protein complex is extracellularly released, leaving ANXA2 on the outer cell surface. The ANXA2 is then flipped in by a force of ATP8A2 activity, and the non-vesicular release of protein complex is repeated. Thus, the ANXA2 flop-out could play key roles in a new type of non-vesicular and non-classical release for DAMPs/alarmins, which is distinct from the modes conducted via gasdermin D or mixed-lineage kinase domain-like pseudokinase pores.

Development of green fluorescent protein-based cAMP indicators for covering a wide range of cAMP concentrations.

There is a wide range in the concentration of intracellular cyclic adenosine 3',5'-monophosphate (cAMP), which mediates specific effects as a second messenger in pathways affecting many physiological processes. Here, we developed green fluorescent cAMP indicators, named Green Falcan (Green fluorescent protein-based indicator visualizing cAMP dynamics) with various EC50 values (0.3, 1, 3, 10 µM) for covering the wide range of intracellular cAMP concentrations. The fluorescence intensity of Green Falcans increased in a cAMP dose-dependent manner, with a dynamic range of over 3-fold. Green Falcans showed a high specificity for cAMP over its structural analogues. When we expressed Green Falcans in HeLa cells, these indicators were applicable for visualization of cAMP dynamics in the low concentration range compared to the previously developed cAMP indicators, and visualized distinct kinetics of cAMP in various pathways with high spatiotemporal resolution in living cells. Furthermore, we demonstrated that Green Falcans are applicable to dual-color imaging with R-GECO, a red fluorescent Ca2+ indicator, in the cytoplasm and the nucleus. This study shows that Green Falcans open up a new avenue for understanding hierarchal and cooperative interactions with other molecules in various cAMP signaling pathways by multi-color imaging.

A Real-Time Learning Analytics Dashboard for Automatic Detection of Online Learners' Affective States.

Students' affective states describe their engagement, concentration, attitude, motivation, happiness, sadness, frustration, off-task behavior, and confusion level in learning. In online learning, students' affective states are determinative of the learning quality. However, measuring various affective states and what influences them is exceedingly challenging for the lecturer without having real interaction with the students. Existing studies primarily use self-reported data to understand students' affective states, while this paper presents a novel learning analytics system called MOEMO (Motion and Emotion) that could measure online learners' affective states of engagement and concentration using emotion data. Therefore, the novelty of this research is to visualize online learners' affective states on lecturers' screens in real-time using an automated emotion detection process. In real-time and offline, the system extracts emotion data by analyzing facial features from the lecture videos captured by the typical built-in web camera of a laptop computer. The system determines online learners' five types of engagement ("strong engagement", "high engagement", "medium engagement", "low engagement", and "disengagement") and two types of concentration levels ("focused" and "distracted"). Furthermore, the dashboard is designed to provide insight into students' emotional states, the clusters of engaged and disengaged students', assistance with intervention, create an after-class summary report, and configure the automation parameters to adapt to the study environment.

Generation of Q-bead against bone Gla protein with simplified preparation steps.

Quenchbody (Q-body)-based immunoassays enable the detection of antigen within a few minutes with high sensitivity and specificity, thereby revealing their applicability as biosensors for quantifying several biomolecules of interest; however, while producing a Q-body, it is necessary to eliminate the unconjugated dye after labeling to separate the Q-body from the capturing bead and to change the buffer using ultrafiltration, which is time-consuming and leads to yield reduction. In this study, we generated a recombinant single chain variable fragment against bone Gla protein as a model antibody. We labeled the antibody with a dye to generate a Q-body and subsequently added affinity beads to the Q-body mixture. After washing, we directly added antigen without extracting the Q-body from the bead and then measured the fluorescence intensity. As a result, the antigen-dependent fluorescence response was obtained from "Q-bead", which was almost the same as that of the Q-body generated according to the conventional method. The Q-bead was generated within only 2.5 h, thus requiring an hour and two steps less than those required for the generation of the traditional Q-body. No expensive Flag peptide was required to recover the total antibody from beads. Moreover, the ultra-filtration step was eliminated in this bead-based method, leading to improved convenience and cost- and time-saving attributes. The Q-bead-based assay can be used as a standard protocol for simple and rapid analysis of antibody-based molecular detection.

Formation of Hemihydrate Crystal form Overcomes Milling Issue Induced by Exposed Functional Groups on Cleavage Plane for a Y5 Receptor Antagonist of Neuropeptide Y.

This study aimed to investigate the crystal forms of an originally designed Y5 receptor antagonist of neuropeptide Y. Polymorphic screening was performed via solvent evaporation and slurry conversion using various solvents. The obtained crystal forms α, ß, and γ were characterized by X-ray powder diffraction analysis. Thermal analysis determined that forms α, ß, and γ were hemihydrate, metastable and stable forms, respectively; the hemihydrate and the stable forms were candidates. To arrange the particle size, forms α and γ were subjected to jet milling. However, form γ could not be milled because of powder stiction to the apparatus, whereas form α could be. To investigate this mechanism, single-crystal X-ray diffraction analysis was performed. The crystal structure of form γ was characterized by two-dimensional hydrogen bonding between neighboring molecules. This revealed that the functional groups forming hydrogen bonds were exposed on the cleavage plane of form γ. The three-dimensional hydrogen-bonding network with water stabilized the hemihydrate form, α. These results indicate that the hydrogen bondable groups exposed on the cleavage plane of form γ should result in stiction of the powder and adherence to the apparatus. It was concluded that crystal conversion is a method to overcome the milling issue.

Efficient Microfluidic Screening Method Using a Fluorescent Immunosensor for Recombinant Protein Secretions.

Microbial secretory protein expression is widely used for biopharmaceutical protein production. However, establishing genetically modified industrial strains that secrete large amounts of a protein of interest is time-consuming. In this study, a simple and versatile high-throughput screening method for protein-secreting bacterial strains is developed. Different genotype variants induced by mutagens are encapsulated in microemulsions and cultured to secrete proteins inside the emulsions. The secreted protein of interest is detected as a fluorescence signal by the fluorescent immunosensor quenchbody (Q-body), and a cell sorter is used to select emulsions containing improved protein-secreting strains based on the fluorescence intensity. The concept of the screening method is demonstrated by culturing Corynebacterium glutamicum in emulsions and detecting the secreted proteins. Finally, productive strains of fibroblast growth factor 9 (FGF9) are screened, and the FGF9 secretion increased threefold compared to that of parent strain. This screening method can be applied to a wide range of proteins by fusing a small detection tag. This is a highly simple process that requires only the addition of a Q-body to the medium and does not require the addition of any substrates or chemical treatments. Furthermore, this method shortens the development period of industrial strains for biopharmaceutical protein production.

Development of a Peptide Sensor Derived from Human ACE2 for Fluorescence Polarization Assays of the SARS-CoV-2 Receptor Binding Domain.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the continuing emergence of infectious variants have caused a serious pandemic and a global economic slump since 2019. To overcome the situation and prepare for future pandemic-prone diseases, there is a need to establish a convenient diagnostic test that is quickly adaptable to unexpected emergence of virus variants. Here we report a fluorescent peptide sensor 26-Dan and its application to the fluorescence polarization (FP) assay for the highly sensitive and convenient detection of SARS-CoV-2. The 26-Dan sensor was developed by fluorescent labeling of the 26th amino acid of a peptide derived from the N-terminal α-helix of human angiotensin-converting enzyme 2 (hACE2) receptor. The 26-Dan sensor maintained the α-helical structure and showed FP changes in a concentration-dependent manner of the receptor binding domain (RBD) of the virus. The half maximal effective concentrations (EC50's) for RBD of Wuhan-Hu-1 strain, Delta (B.1.617.2), and Omicron (BA.5) variants were 51, 5.2, and 2.2 nM, respectively, demonstrating that the 26-Dan-based FP assay can be adaptable to virus variants that evade standard diagnostic tests. The 26-Dan-based FP assay could also be applied to model screening of a small molecule that inhibits RBD binding to hACE2 and identified glycyrrhizin as a potential inhibitor. The combination of the sensor with a portable microfluidic fluorescence polarization analyzer allowed for the detection of RBD in a femtomolar range within 3 min, demonstrating the assay could be a promising step toward a rapid and convenient test for SARS-CoV-2 and other possible future pandemic-prone diseases.

A Diversifiable Synthetic Platform for the Discovery of New Carbasugar SGLT2 Inhibitors Using Azide-Alkyne Click Chemistry.

Sodium-glucose cotransporter 2 (SGLT2) inhibitors are clinically available to control blood glucose levels in diabetic patients via an insulin-independent mechanism. It was found that some carbasugar analogs of known SGLT2 inhibitors exert a high inhibiting ability toward SGLT2 and have a prolonged blood glucose lowering effect. In this study, we designed new candidates of carbasugar SGLT2 inhibitor that can be synthesized using copper-catalyzed azide-alkyne cycloaddition (CuAAC) into an aromatic ring, which is a part of the pharmacophore at the final stage in the synthetic protocol for the easier discovery of superior SGLT2 inhibitors. Based on the results of molecular docking studies, some selected compounds have been synthesized. Evaluation of these compounds using a cell-based assay revealed that the majority of these compounds had SGLT2 inhibitory activity in a dose-dependent manner. The SGLT2 inhibitory activity of 7b and 7c was almost equal to that of SGLT2 inhibitors in current use. Furthermore, molecular dynamics simulations also revealed that 7c is a promising novel SGLT2 inhibitor.

Recent progress in homogeneous immunosensors based on fluorescence or bioluminescence using antibody engineering.

Homogeneous immunosensors integrate the advantages of both biosensors and immunoassays; they include speed, high sensitivity, and accuracy. They have been developed rapidly in the past few years and offer a cost-effective alternative technology with rapidity, sensitivity, and user-friendliness, which has been applied in a wide variety of applications. This review introduces the current directions of immunosensor development, focusing on fluorescent and bioluminescent immunosensors and highlighting the advantages, improvements, and key approaches to overcome the limitations of each.