Ultrasensitive colorimetric detection of Staphylococcus aureus using wheat germ agglutinin and IgY as a dual-recognition strategy.

A novel colorimetric platform was designed for the determination of S. aureus by utilizing a dual-recognition strategy, where wheat germ agglutinin (WGA)-functionalized magnetic beads were served as separation elements to capture and enrich S. aureus efficiently from the matrix. Horseradish peroxidase (HRP) labeled chicken anti-protein A IgY (HRP-IgY) was used to label the captured S. aureus. A chicken IgY was introduced as a signal tracer to bind with staphylococcal protein A (SPA) on the surface of S. aureus, which can circumvent the interference from protein G-producing Streptococcus. Subsequently, the colorimetric signal was achieved by an HRP-catalyzed reaction, which was amplified by HRP-IgY bound by approximately 80,000 SPA molecules on one S. aureus. The entire detection process could be accomplished within 90 min. Under optimal conditions, the linear response of different S. aureus concentrations ranged from 7.8 × 102 to 2.0 × 105 CFU/mL and the limit of detection reached down to 3.9 × 102 CFU/mL. Some common non-target bacteria yielded negative results, indicating the excellent specificity of the method. The developed strategy was successfully applied to the determination of S. aureus in various types of samples with satisfactory recoveries. Therefore, the novel dual-recognition strategy possessed the advantages of high sensitivity, specificity, and low cost and exhibited considerable potential as a promising tool to defend public health.

Effects of Fatigue and Unanticipated Factors on Knee Joint Biomechanics in Female Basketball Players during Cutting.

(1) This study examined the impact of fatigue and unanticipated factors on knee biomechanics during sidestep cutting and lateral shuffling in female basketball players, assessing the potential for non-contact anterior cruciate ligament (ACL) injuries. (2) Twenty-four female basketball players underwent fatigue induction and unanticipated change of direction tests, and kinematic and kinetic parameters were collected before and after fatigue with a Vicon motion capture system and Kistler ground reaction force (GRF) sensor. (3) Analysis using two-way repeated-measures ANOVA showed no significant interaction between fatigue and unanticipated factors on joint kinematics and kinetics. Unanticipated conditions significantly increased the knee joint flexion and extension angle (p < 0.01), decreased the knee flexion moment under anticipated conditions, and increased the knee valgus moment after fatigue (p ≤ 0.05). One-dimensional statistical parametric mapping (SPM1d) results indicated significant differences in GRF during sidestep cutting and knee inversion and rotation moments during lateral shuffling post-fatigue. (4) Unanticipated factors had a greater impact on knee load patterns, raising ACL injury risk. Fatigue and unanticipated factors were independent risk factors and should be considered separately in training programs to prevent lower limb injuries.

Taurine protects R28 cells from hypoxia/re-oxygenation-induced damage via regulation of mitochondrial energy metabolism.

Oxidative-induced damage and hypoxia/re-oxygenation (H/R) injury are common causes of irreversible visual impairment. The goals of this study were to explore the effects of taurine on R28 cells under the two damage models and the underlying mechanisms. Low doses of taurine supplementation promoted cell viability, mitochondrial membrane potential (MMP), SOD levels, ATP contents and attenuated cytotoxicity and intracellular ROS generation of the R28 cells under the two kinds of damage. The expression level of GTPBP3, a mitochondrial-tRNA (mt-tRNA) modification enzyme that catalyzes the taurine involved modification, was decreased under the two damage and taurine could reverse the reduction. After knocking down GTPBP3, the R28 cells become vulnerable to damage. The viability, cytotoxicity, MMP and intracellular ROS level of knockdown cells changed more obviously under the H/R injury than those of control cell. We also found that knockdown of GTPBP3 significantly decreased mitochondrial energy metabolism by measuring the oxidative respiration rate by the Seahorse XFe24 extracellular flux analyzer. The protection of low doses of taurine disappeared on knockdown R28 cells, indicating that GTPBP3 is crucial in the protection mechanisms of taurine. However, the impacts of the reduction of GTPBP3 level can be reversed by relatively high doses of taurine, implying the protection effects of taurine were dose-dependent, and there were more complicated mechanisms remain to be explored. This study explored a new mechanism of the neuroprotective effects of taurine, which depend on the GTPBP3-mediated taurine modification of mt-tRNAs and the promotion of mitochondrial energy metabolism.

Development of a Fluorescently Labeled Aptamer Structure-Switching Assay for Sensitive and Rapid Detection of Gliotoxin.

Gliotoxin, one of the most toxic metabolites produced during the growth of Aspergillus fumigatus, can cause direct damage to the immune system and results in infection and spread of Aspergillus, or even leads to invasive aspergillosis. Accurate, rapid, and sensitive detection of the disease-specific marker gliotoxin, particularly in serum, urine, or other body fluids, is therefore an important approach to achieving early and rapid diagnosis of Invasive Aspergillus Fumigatus Infection (IAFI). In this study, aptamers that specifically bind to gliotoxin were successfully obtained using immobilization-free GO-SELEX technology. Furthermore, the performance of the aptamer, including binding affinity, targeting specificity, and structural stability, was further improved by optimizing through truncation and mutation. Finally, the optimized aptamer APT8T1M was used to develop a novel fluorescently labeled aptamer structure-switching assay (FLASSA) for the detection of gliotoxin. The method exhibited a good linear range from 0.1 nM to 100 nM of gliotoxin, with a lower detection limit of 0.05 nM. Moreover, FLASSA was applied to the detection of gliotoxin in spiked serum and urine samples. A good mean recovery of 98.76-110.85% and a low coefficient of variation (5.45-14.59%) were obtained, indicating a high degree of selectivity for gliotoxin, good reproducibility, and stability. These results show that the developed FLASSA has significant potential and offers an alternative to the traditional analytical methods for the rapid, sensitive, and efficient detection of gliotoxin, thus, providing an effective tool for the early and rapid diagnosis of IAFI.

Post-SELEX optimization of aptamers.

Aptamers are functional single-stranded DNA or RNA oligonucleotides, selected in vitro by SELEX (Systematic Evolution of Ligands by Exponential Enrichment), which can fold into stable unique three-dimensional structures that bind their target ligands with high affinity and specificity. Although aptamers show a number of favorable advantages such as better stability and easier modification when compared with the properties of antibodies, only a handful of aptamers have entered clinical trials and only one, pegaptanib, has received US Food and Drug Administration approval for clinical use. The main reasons that limit the practical application of aptamers are insufficient nuclease stability, bioavailability, thermal stability, or even affinity. Some aptamers obtained from modified libraries show better properties; however, polymerase amplification of nucleic acids containing non-natural bases is currently a primary drawback of the SELEX process. This review focuses on several post-SELEX optimization strategies of aptamers identified in recent years. We describe four common methods in detail: truncation, chemical modification, bivalent or multivalent aptamer construction, and mutagenesis. We believe that these optimization strategies should improve one or more specific properties of aptamers, and the type of feature(s) selected for improvement will be dependent on the application purpose.

Study of the binding mechanism between aptamer GO18-T-d and gonyautoxin 1/4 by molecular simulation.

GTX1/4 can induce the formation of an antiparallel G-quadruplex structure in aptamer GO18-T-d and combine steadily in the groove at the top of the G-quadruplex structure. The complex structures and special induced fit mechanism between aptamer and small molecules provide a reference for aptamer development in molecular diagnostics and therapeutic application.

Escin Increases the Survival Rate of LPS-Induced Septic Mice Through Inhibition of HMGB1 Release from Macrophages.

BACKGROUND: Previous studies have described the effects of Escin on improving the survival rate of endotoxemic animals. The purpose of this study was to explore the molecular mechanisms of this potentially beneficial treatment. METHODS: First, the survival rate of endotoxemic mice was monitored for up to 2 weeks after Escin pretreatment, Escin post-treatment, or Escin post-treatment + rHMGB1. The effects of Escin on the release of pro-inflammatory cytokines such as TNF-α, IL-1ß, IL-6 and HMGB1 in the serum of endotoxemic mice and LPS-induced macrophages were evaluated by ELISA. Furthermore, the mRNA and protein levels of HMGB1 in LPS-induced macrophages were measured by qRT-PCR and Western blot, respectively. Additionally, the release of pro-inflammatory cytokines such as TNF-α, IL-1ß, IL-6 was evaluated by ELISA in rHMGB1-induced macrophages. Finally, the protein levels and the activity of NF-κB in macrophages were checked by Western blot and ELISA, respectively. RESULTS: Both pretreatment and post-treatment with Escin could improve the survival rate of endotoxemic mice, while exogenous rHMGB1 reversed this effect. In addition, Escin decreased the level of the pro-inflammatory cytokinesTNF-α,IL-1ß, IL-6 and HMGB1 in endotoxemic mice and in LPS-induced macrophages. Escin could also inhibit the mRNA levels and activity of HMGB1. The release of the pro-inflammatory cytokinesTNF-α,IL-1ß, IL-6 could be suppressed in rHMGB1-induced macrophages by Escin. Finally, Escin could suppress the activation of NF- κB in LPS-induced macrophages. CONCLUSION: Escin could improve the survival of mice with LPS-induced endotoxemia. This effect maybe meditated by reducing the release of HMGB1, resulting in the suppression of the release of pro-inflammatory cytokines.

A triple read-out visible biosensing platform based on multifunctional nanozyme and bipolar electrode for multi-mode detection and imaging of CEA.

In this paper, a proposal of closed bipolar electrode (BPE) and nanozyme based multi-mode biosensing platform is first presented. As a novel integrated chip, multi-mode-BPE (MMBPE) combines enzyme-linked immunoassay (ELISA), electrochemiluminescence (ECL), ECL imaging and light emitting diode (LED) imaging, enabling highly sensitive triple read-out visible detection of cancer embryonic antigen (CEA). The ECL probe Ab2@Au@Co3O4/CoFe2O4 hollow nanocubes (HNCs) with excellent peroxidase (POD) activity is introduced into the BPE cathode through immune adsorption. The Au@Co3O4/CoFe2O4 HNCs can increase the rate of hydrogen peroxide oxidation of TMB, thus promoting the reaction, and can be used for ELISA detection of CEA at different concentrations. The modification of the BPE sensing interface and reporting interface involved the introduction of the luminescent reagent Ru(bpy)32+ to the BPE anode. The decomposition rate of H2O2 increased under the catalytic action of Au@Co3O4/CoFe2O4 HNCs nanozyme, leading to an accelerated electron transfer rate in the MMBPE system and an enhanced ECL signal from Ru(bpy)32+. The LED imaging technology further provides a convenient and visible approach for CEA imaging in which no additional chemicals are needed. The integration of nanoenzymes as the catalytic core in MMBPE system provides impetus, while the combination of nanozymes with BPE expands the application of nanoenzymes in the field of biological analysis. The integration of intelligent chips with multiple modes of detection shows portable, miniaturized, and integrated excellent properties which meets the requirements of modern detection devices and thus offers a flexible approach for determination of nucleic acids, proteins, and cells.

Effect of orthopedic insoles on lower limb motion kinematics and kinetics in adults with flat foot: a systematic review.

Flatfoot is characterized by the collapse of the medial longitudinal arch, eversion of the rearfoot and abduction of the loaded forefoot. Orthopedic insoles are the frequently recommended treatment to support the arch of the foot, adjust the structure of the foot, reduce pain, improve stability and new techniques have been applied to the design of orthopedic insoles in recent years. However, the effectiveness of orthopedic insoles in different motions is still debated from the perspective of biomechanics. Therefore, this study aimed to explore the impact of orthopedic insoles on the kinematics and kinetics of lower limb motion, and to verify effectiveness and propose possible future research directions. We conducted a literature search across three databases employing Boolean operations and filtered results based on eligibility criteria. A total of 671 relevant literature were searched in this review, and 19 literature meeting the requirements were finally included. The results showed that: 1) orthopedic insoles were effective when patients walk, run and jump from the perspective of biomechanics; 2) orthopedic insoles had different result on the change of ankle sagittal angle, moment and peak pressure in the metatarsal region; 3) Whether the effect of insoles, which uses new techniques such as different 3D printed technologies and adds various accessories, can be further improved remains to be further studied; 4) Follow-up studies can pay more attention to the differences between diverse populations, increase the breadth of running and jumping and other movements research and long-term intervention.

Metabolomics identifies and validates serum androstenedione as novel biomarker for diagnosing primary angle closure glaucoma and predicting the visual field progression.

Background: Primary angle closure glaucoma (PACG) is the leading cause of irreversible blindness in Asia, and no reliable, effective diagnostic, and predictive biomarkers are used in clinical routines. A growing body of evidence shows metabolic alterations in patients with glaucoma. We aimed to develop and validate potential metabolite biomarkers to diagnose and predict the visual field progression of PACG. Methods: Here, we used a five-phase (discovery phase, validation phase 1, validation phase 2, supplementary phase, and cohort phase) multicenter (EENT hospital, Shanghai Xuhui Central Hospital), cross-sectional, prospective cohort study designed to perform widely targeted metabolomics and chemiluminescence immunoassay to determine candidate biomarkers. Five machine learning (random forest, support vector machine, lasso, K-nearest neighbor, and GaussianNaive Bayes [NB]) approaches were used to identify an optimal algorithm. The discrimination ability was evaluated using the area under the receiver operating characteristic curve (AUC). Calibration was assessed by Hosmer-Lemeshow tests and calibration plots. Results: Studied serum samples were collected from 616 participants, and 1464 metabolites were identified. Machine learning algorithm determines that androstenedione exhibited excellent discrimination and acceptable calibration in discriminating PACG across the discovery phase (discovery set 1, AUCs=1.0 [95% CI, 1.00-1.00]; discovery set 2, AUCs = 0.85 [95% CI, 0.80-0.90]) and validation phases (internal validation, AUCs = 0.86 [95% CI, 0.81-0.91]; external validation, AUCs = 0.87 [95% CI, 0.80-0.95]). Androstenedione also exhibited a higher AUC (0.92-0.98) to discriminate the severity of PACG. In the supplemental phase, serum androstenedione levels were consistent with those in aqueous humor (r=0.82, p=0.038) and significantly (p=0.021) decreased after treatment. Further, cohort phase demonstrates that higher baseline androstenedione levels (hazard ratio = 2.71 [95% CI: 1.199-6.104], p=0.017) were associated with faster visual field progression. Conclusions: Our study identifies serum androstenedione as a potential biomarker for diagnosing PACG and indicating visual field progression. Funding: This work was supported by Youth Medical Talents - Clinical Laboratory Practitioner Program (2022-65), the National Natural Science Foundation of China (82302582), Shanghai Municipal Health Commission Project (20224Y0317), and Higher Education Industry-Academic-Research Innovation Fund of China (2023JQ006).

Curved carbon-plated shoe may further reduce forefoot loads compared to flat plate during running.

Using a curved carbon-fiber plate (CFP) in running shoes may offer notable performance benefit over flat plates, yet there is a lack of research exploring the influence of CFP geometry on internal foot loading during running. The objective of this study was to investigate the effects of CFP mechanical characteristics on forefoot biomechanics in terms of plantar pressure, bone stress distribution, and contact force transmission during a simulated impact peak moment in forefoot strike running. We employed a finite element model of the foot-shoe system, wherein various CFP configurations, including three stiffnesses (stiff, stiffer, and stiffest) and two shapes (flat plate (FCFP) and curved plate (CCFP)), were integrated into the shoe sole. Comparing the shoes with no CFP (NCFP) to those with CFP, we consistently observed a reduction in peak forefoot plantar pressure with increasing CFP stiffness. This decrease in pressure was even more notable in a CCFP demonstrating a further reduction in peak pressure ranging from 5.51 to 12.62%, compared to FCFP models. Both FCFP and CCFP designs had a negligible impact on reducing the maximum stress experienced by the 2nd and 3rd metatarsals. However, they greatly influenced the stress distribution in other metatarsal bones. These CFP designs seem to optimize the load transfer pathway, enabling a more uniform force transmission by mainly reducing contact force on the medial columns (the first three rays, measuring 0.333 times body weight for FCFP and 0.335 for CCFP in stiffest condition, compared to 0.373 in NCFP). We concluded that employing a curved CFP in running shoes could be more beneficial from an injury prevention perspective by inducing less peak pressure under the metatarsal heads while not worsening their stress state compared to flat plates.

One-pot assay using a target-driven split aptamer recognition and assembly strategy for convenient and rapid detection of gliotoxin.

An aptamer targeting gliotoxin (GTX) was optimized to increase the binding affinity by approximately 20 times and achieve higher structural stability and targeting specificity. Molecular dynamics simulations were used to explore the molecular mechanism and key action sites underlying the recognition of GTX by the optimized aptamer. Subsequently, the optimized aptamer was split into two fragments and a convenient and rapid one-pot assay for GTX detection was successfully established using a target-driven split aptamer recognition and assembly strategy. The method exhibited a good linear range of 0.128 nM to 2 µM, a low detection limit of 0.07 nM, and excellent selectivity for GTX. Furthermore, the method had good accuracy and stability in real sample analysis. Therefore, the developed one-pot method provides a reliable, convenient, and cost-effective approach for the widespread application of GTX detection.

A novel biosensing platform for detection of glaucoma biomarker GDF15 via an integrated BLI-ELASA strategy.

Glaucoma is a leading cause of irreversible blindness worldwide. Early discovery and prioritized intervention significantly impact its prognosis. Precise monitoring of the biomarker GDF15 contributes towards effective diagnosis and assessment of glaucoma. In this study, we demonstrate that GDF15 monitoring can also aid screening for glaucoma risk and early diagnosis. We obtained an aptamer (APT2TM) with high affinity, high specificity, and high stability for binding to both human-derived and rat-derived GDF15. Simulation results showed that the binding capabilities of APT2TM are mainly affected by the interplay between van der Waals forces and polar solvation energy, and that salt bridges and hydrogen bonds play critical roles. We then integrated an enzyme-linked aptamer sandwich assay (ELASA) into a biolayer interferometry (BLI) system to develop an automated, high-throughput, real-time monitoring BLI-ELASA biosensing platform. This platform exhibited a wide linear detection window (10-810 pg/mL range) and high sensitivity for GDF15 (detection limit of 5-6 pg/mL). Moreover, we confirmed its excellent performance when applied to GDF15 quantification in real samples from glaucomatous rats and clinical patients. We believe that this technology represents a robust, convenient, and cost-effective approach for risk screening, early diagnosis, and animal modeling evaluation of glaucoma in the near future.

Combined Therapy of Experimental Autoimmune Uveitis by a Dual-Drug Nanocomposite Formulation with Berberine and Dexamethasone.

Purpose: Autoimmune uveitis is a kind of sight-threatening ocular and systemic disorders. Recent treatments on autoimmune uveitis still remain many limitations due to extreme complexity and undetermined pathogenesis. In this study, a novel dual-drug nanocomposite formulation is developed to treat experimental autoimmune uveitis by a combined and sustained therapy method. Methods: The dual-drug nanocomposite formulation is constructed by integrating berberine (BBR)-loaded mesoporous silica nanoparticles (MSNs) into dexamethasone (DEX)-loaded thermogel (BBR@MSN-DEX@Gel). The BBR@MSN-DEX@Gel is characterized by transmission electron microscopy, dynamic light scattering, Fourier transform infrared spectrometer and rheometer. The in vitro drug release profile, cytotoxicity and anti-inflammation effectiveness of BBR@MSN-DEX@Gel on lipopolysaccharide-stimulated human conjunctival epithelial cells are investigated. After the in vivo drug release profile and biosafety of the dual-drug nanocomposite formulation are confirmed, its treatment effectiveness is fully assessed based on the induced experimental autoimmune uveitis (EAU) Lewis rat's model. Results: The dual-drug nanocomposite formulation has good injectability and thermosensitivity, suitable for administration by an intravitreal injection. The BBR@MSN-DEX@Gel has been found to sustainably release both drugs for up to 4 weeks. The carrier materials have minimal in vitro cytotoxicity and high in vivo biosafety. BBR@MSN-DEX@Gel presents obviously anti-inflammatory effectiveness in vitro. After administration of BBR@MSN-DEX@Gel into Lewis rat's eye with EAU by an intravitreal injection, the nanocomposite formulation significantly suppresses inflammatory reaction of autoimmune uveitis via a dual-drug combined and sustained therapy method, compared with the equivalent dose of single-component formulations. Conclusion: BBR@MSN-DEX@Gel serves as a promising dual-drug nanocomposite formulation for future treatment of autoimmune uveitis.

Sustained release of brimonidine from polydimethylsiloxane-coating silicone rubber implant to reduce intraocular pressure in glaucoma.

Glaucoma is the leading cause of irreversible blindness, affecting 111 million people by 2040 worldwide. Intraocular pressure (IOP) is the only controllable risk factor for the disease and current treatment options seek to reduce IOP via daily taking eye drops. However, shortcomings of eye drops, such as poor bioavailability and unsatisfied therapeutic effects, may lead to inadequate patient compliance. In this study, an effective brimonidine (BRI)-loaded silicone rubber (SR) implant coated with polydimethylsiloxane (BRI@SR@PDMS) is designed and fully investigated for IOP reduction treatment. The in vitro BRI release from BRI@SR@PDMS implant reveals a more sustainable trend lasting over 1 month, with a gradually declined immediate drug concentration. The carrier materials show no cytotoxicity on human corneal epithelial cells and mice corneal epithelial cells in vitro. After administrated into rabbit's conjunctival sac, the BRI@SR@PDMS implant releases BRI in a sustained fashion and effectively reduces IOP for 18 days with great biosafety. In contrast, BRI eye drops only maintain IOP-lowering effect for 6 h. Therefore, as a substitute of eye drops, the BRI@SR@PDMS implant can be applied as a promising non-invasive platform to achieve long-term IOP-lowering in patients suffering from ocular hypertension or glaucoma.

Site-specific detection of circulating tumor DNA methylation in biological samples utilizing phosphorothioated primer-based loop-mediated isothermal amplification.

DNA methylation, a kind of epigenetic alteration, plays a vital role in tumorigenesis and offers a new class of targets for cancer treatment. DNA hypermethylation at the E-Box site (CACGTG, -288 bp) in the SLC22A2 promoter was related to multidrug resistance of renal cell carcinoma (RCC), which can provide the target for both treatment and monitoring. Herein, we developed a novel phosphorothioated primer based loop-mediated isothermal amplification (PS-LAMP) assay to detect circulating tumor DNA (ctDNA) methylation levels in E-Box sites in tumor tissue, urine, and plasma samples from patients with RCC. Bisulfite treatment converted methylated/unmethylated discrepancy to a single base discrepancy (C/U). PS-LAMP amplified the templates to a tremendous amount. One-step strand displacement (OSD) probe provided single base resolution in amplified products and finally realized the specific site methylation detection. Our proposed method provided a linear range from 0% to 100% for methylation levels and was available in samples at low concentrations (102 copies/µL). Visually observable colorimetric detection can be achieved by incorporating the OSD probe with gold nanoparticles (AuNP). Our assay performed better than traditional methods in biological samples with low ctDNA concentration. Further, we found a potential consistency of methylation levels between tumor tissue and plasma sample from the same patient (Spearman's ρ = 0.886, P = 0.019, n = 6). In general, this work provides a PS-LAMP assay combining OSD probes for site-specific methylation detection in various biological samples, offering a method for noninvasive detection.

One-step high-throughput detection of low-abundance biomarker BDNF using a biolayer interferometry-based 3D aptasensor.

Although biosensors for signal monitoring have been extensively developed, their application in one-step high-throughput detection of low-abundance disease biomarkers remains challenging. This study presents a 3D aptasensor based on a biolayer interferometry (BLI) technique, followed by the sensitive and rapid detection of the specific biomarker brain-derived neurotrophic factor (BDNF) for early screening of glaucoma, an irreversible disease that causes blindness. The developed 3D aptasensor enabled one-step batch conversion of the low-abundance biomarker BDNF binding into optical interference signal, which was mainly attributed to the following factors: (1) A dimeric aptamer with extremely high targeting affinity was constructed as a biorecognition molecule, (2) highly sensitive 3D matrix sensors were integrated as signal transduction elements, and (3) the BLI Octet system with automated, high-throughput, and real-time online monitoring capabilities was used for reporting. The 3D aptasensor exhibited a broad detection window from 0.41 to 250 ng/mL BDNF, with a limit of detection of 0.2 ng/mL. Furthermore, detection of BDNF in glaucoma patient serum using the aptasensor showed good agreement with ELISA findings as well as the clinical diagnosis of the patient, demonstrating the feasibility of the system as a screening tool for glaucoma. This one-step high-throughput screening approach provides a valuable solution for the early diagnosis of glaucoma and may reduce the risk of blindness in visually impaired people.

A Fluorescent Aptasensor Based on Assembled G-Quadruplex and Thioflavin T for the Detection of Biomarker VEGF165.

VEGF165, a regulator of angiogenesis, has been widely used as a serum biomarker for a number of human diseases, including cancer, rheumatoid arthritis, bronchial asthma, and diabetic eye disease. The rapid, accurate, and convenient detection of VEGF165 is a crucial step in effective healthcare monitoring, disease diagnosis, and prognosis assessment. In this study, a fluorescent aptasensor based on an assembled G-quadruplex and the signal molecule ThT was developed for VEGF165 detection. First, G-rich DNA fragments were assembled at both ends of the anti-VEGF165 aptamer, and the B-DNA form was converted into a G-quadruplex structure aptamer (G4-Apt). Then, ThT was introduced, and the G-quadruplex significantly enhanced the fluorescence intensity of the bound ThT. When VEGF165 was present, the higher affinity of the aptamer to the target protein allowed the G4-Apt/VEGF165 complex to form and release ThT, which emitted only weak fluorescence in the free state. Therefore, the aptasensor exhibited a good linear detection window from 1.56 to 25 nM VEGF165, with a limit of detection of 0.138 nM. In addition, the aptasensor was applied to detect VEGF165 in clinical serum samples, showing good accuracy, reproducibility, and stability. These results indicate that our developed fluorescent aptasensor can potentially be a reliable, convenient, and cost-effective approach for the sensitive, specific, and rapid detection of the VEGF165 biomarker.

Recent Advances in Aptamer-Based Biosensors for Detection of Pseudomonas aeruginosa.

Increasing concerns about nosocomial infection, food and environmental safety have prompted the development of rapid, accurate, specific and ultrasensitive methods for the early detection of critical pathogens. Pseudomonas aeruginosa is one of the most common pathogens that cause infection. It is ubiquitous in nature, being found in water, soil, and food, and poses a great threat to public health. The conventional detection technologies are either time consuming or readily produce false positive/negative results, which makes them unsuitable for early diagnosis and spot detection of P. aeruginosa. To circumvent these drawbacks, many efforts have been made to develop biosensors using aptamers as bio-recognition elements. Various aptamer-based biosensors for clinical diagnostics, food, and environmental monitoring of P. aeruginosa have been developed in recent years. In this review, we focus on the latest advances in aptamer-based biosensors for detection of P. aeruginosa. Representative biosensors are outlined according to their sensing mechanisms, which include optical, electrochemical and other signal transduction methods. Possible future trends in aptamer biosensors for pathogen detection are also outlined.

Fluorescent aptasensor based on G-quadruplex-assisted structural transformation for the detection of biomarker lipocalin 1.

Diabetic retinopathy (DR) is the leading global cause of blindness in the working-age population. Early diagnosis and intervention can effectively reduce the risk for blindness. However, the current diagnostic methods in clinical practice remain constrained by nonquantitative examinations and individual ophthalmologists' experiences. Sensitive, specific and accurate detection of DR-specific biomarkers is an important approach to achieve its early and rapid diagnosis. In this study, a high-affinity aptamer APT12TM that specifically binds to the tear-derived DR biomarker lipocalin 1 was obtained. The aptamer APT12TM can be folded into a stable B-DNA structure, and its strong interaction with LCN 1, including hydrogen bonding and hydrophobic interactions, is an important factor for targeted recognition and high-affinity binding. A G-rich DNA fragment was further assembled at both ends of the aptamer APT12TM, and the B-DNA form was successfully converted into a parallel G-quadruplex. Most importantly, LCN 1 could induce further transformation of the G-quadruplex structure. Therefore, a fluorescent aptasensor based on G-quadruplex-assisted structural transformation was developed through the Thioflavin T mediator. The aptasensor exhibited a broad detection window from 0.25 to 1000 nM LCN 1, with a limit of detection of 0.2 nM. Furthermore, the aptasensor was applied to LCN 1 detection in artificial tear samples and displayed good reproducibility and stability. These results show that the developed aptasensor has significant potential for sensitive, specific and convenient detection of the DR-specific biomarker LCN 1.