Search Results (20)

Lateral flow immunoassay (LFIA) is a promising tool for rapid detection in the field of agricultural product analysis due to its advantages of cost-effectiveness and operational simplicity. In this work, Eu metal–organic frameworks (MOFs) were introduced to LFIA as a rapid detection method characterized by high stability and low interference. Key research objectives included strong fluorescence, ease of labeling, and the utilization of fluorescent probes. Eu-MOFs were synthesized in one step via the hydrothermal method, exhibiting a fluorescence lifetime of 163 μs and spherical particles with diameters ranging from 250 to 400 nm. These conditions fulfill the characteristics and requirements of LFIA. Eu-MOFs exploit the porous nature of MOFs to mitigate the drawbacks associated with complex crosslinking agents. This enables antibody proteins to be cross-linked merely upon contact, thereby simplifying the detection process. A time-resolved LFIA method was developed utilizing Eu-MOFs for the detection of aflatoxin B1 (AFB1) in corn, achieving a limit of detection (LOD, IC10) of 0.149 ng/mL. The accuracy and reliability of the Eu-MOFs-LFIA method were validated through comparisons with spiked concentrations during spiking and blind sample analyses, with verification conducted using ultra-high-performance liquid chromatography mass spectrometry (UPLC-MS). Furthermore, testing of real samples demonstrated that the Eu-MOFs-LFIA method can effectively facilitate rapid detection of AFB1 in corn. Full article

Background/Objectives: In Sudan, oral cancer is one of the top ten most common cancers, with OSCC representing the majority of the cases. To date, despite the fact that saliva can be collected simply and non-invasively, there is no approved salivary tumor marker for OSCC. This study aimed to investigate the reliability of salivary CA-125 as a tumor marker for OSCC by measuring and comparing its level among OSCC and healthy individuals as well as its level across different histopathological grades. Methods: A total of 100 subjects were enrolled; 50 were patients with OSCC, while the other 50 were matched healthy individuals. Non-stimulated whole saliva was collected before the administration of definitive treatment, and the concentration of salivary CA-125 was quantified using an automated immunoassay analyzer that employs a one-step sandwich fluorescent enzyme immunoassay (FEIA). Results: The level of salivary CA-125 was 342.65 U/mL in the cases group, which was significantly increased compared with 203.65 U/mL in the healthy controls (p = 0.017). Statistically significant differences in the level of salivary CA-125 among different histopathological grades were observed (p = 0.014). The sensitivity, specificity, accuracy, and positive and negative predictive values were 48%, 78%, 63%, 68.6%, and 60%, respectively. Conclusions: This study suggests that salivary CA-125 could serve as a potential tumor marker for OSCC. However, its clinical application requires further validation. Full article

Lateral flow immunoassays (LFIAs) were integrated into microfluidic chips and tested to enhance point-of-care testing (POCT), with the aim of improving sensitivity and expanding the range of CRP detection. The microfluidic approach improves upon traditional methods by precisely controlling fluid speed, thus enhancing sensitivity and accuracy in CRP measurements. The microfluidic approach also enables a one-step detection system, eliminating the need for buffer solution steps and reducing the nitrocellulose (NC) pad area to just the detection test line. This approach minimizes the non-specific binding of conjugated antibodies to unwanted areas of the NC pad, eliminating the need to block those areas, which enhances the sensitivity of detection. The gold nanoparticle method detects CRP in the high-sensitivity range of 1–10 $\mu$g/mL, which is suitable for chronic disease monitoring. To broaden the CRP detection range, including infection levels beyond 10 $\mu$g/mL, fluorescent labels were introduced, extending the measuring range from 1 to 70 $\mu$g/mL. Experimental results demonstrate that integrating microfluidic technology significantly enhances operational efficiency by precisely controlling the flow rate and optimizing the mixing efficiency while reducing fabrication resources by eliminating the need for separate pads, making these methods suitable for resource-limited settings. Microfluidics also provides greater control over fluid dynamics compared to traditional LFIA methods, which contributes to enhanced detection sensitivity even with lower sample volumes and no buffer solution, helping to enhance the usability of POCT. These findings highlight the potential to develop accessible, accurate, and cost-effective diagnostic tools essential for timely medical interventions at the POC. Full article

3D-printed microdevices have become increasingly important to the advancement of point-of-care (POC) immunoassays. Despite its great potential, using 3D-printed surfaces on the solid support for immunorecognition has been limited due to the non-ideal adsorption properties for many photocurable resins. In this work, we report a simple surface modification protocol that works for diverse commercial photocurable resins, improving ELISAs performed directly on 3D-printed devices. This surface modification strategy involves surface activation via air plasma followed by the one-step incubation of GLYMO-labeled streptavidin. We successfully immobilized biotinylated anti-activin A antibodies on the 3D-printed surfaces and performed the complete ELISA protocol on the 3D-printed surfaces. We demonstrated that this protocol achieved an improved performance over passive adsorption for ELISAs. The present method is also compatible with diverse commercial resins and works with both microwells and microchannels. Finally, this method demonstrated a comparable limit of detection to the ELISA performed using commercial microwells. We believe the simplicity and broad compatibility of the present surface modification strategy will facilitate the development of 3D-printed POC ELISA devices. Full article

Total testosterone (TT) and free testosterone (FT) are important biochemical markers for anabolism of the human body, and can also serve as early screening indicators for overtraining syndrome (OTS). Presently, there is no fast and reliable serum TT and FT determination method in the field of sport science that can meet the requirements of sports research. Thus, a rapid and accurate determination method for serum TT and FT to fill the gap is needed urgently in sports training. Herein, a simple and reliable liquid chromatography–tandem mass spectrometry (LC-MS/MS) method for the simultaneous determination of TT and FT in serum was developed and fully validated, followed by the application of professional athletes in training monitoring. Efficient pretreatments based on only one-step liquid–liquid extraction (LLE) for TT and one-step LLE after a 20 min ultrafiltration for FT were adopted in this study, and the isotope internal standard of testosterone-13C3 was used to ensure the reliability of the whole procedure. A linear range of four orders of magnitude with 0.02–100 ng/mL can meet the concentration range requirement between a higher limit for male TT and a lower limit for female FT. The accuracy, precision, stability, and matrix effect were all within the limits of the guidelines. The serum TT and FT levels of 200 professional athletes (98 male athletes and 102 female athletes) were investigated by this method. Serum TT, FT, and FT/TT levels of professional athletes were significantly higher than the general population, and serum TT levels were significantly higher by LC-MS/MS than by a chemiluminescence immunoassay. In conclusion, the LC-MS/MS method for TT and FT measurement developed in this study is time-saving and easy to operate, which can be used as a reliable method for the determination of serum TT and FT in sports training, offering valuable information for monitoring anabolism of athletes and screening OTS in the early stage. Full article

Although vitamin D insufficiency has been correlated with an increased risk of cardiovascular disease (CVD), there are few data on the association between 25-hydroxyvitamin D (25(OH)D) and atherogenic indices predictive of CVD. This study investigated the relationship of vitamin D status with lipid profile and atherogenic indices in adult women in Morocco. Three hundred women aged 18 to 50 years from Meknes were included. Fasting 25(OH)D and lipid concentrations were assayed by a one-step electrochemiluminescence-based immunoassay and an enzymatic method, respectively. Atherogenic indices (atherogenic index of plasma (AIP), atherogenic coefficient (AC), non-HDL cholesterol (non-HDL-C), Castelli risk indices I and II (CRI-I and II), and CHOLIndex (CI)) were calculated using conventional lipid parameters. Logistic regression models and operating characteristic curve (ROC) analysis were used to assess the relationship of the variables and estimate the threshold of 25(OH)D levels associated with high atherogenic indices. 25(OH) D below 20 ng/mL was significantly associated with an enhanced risk of hypertriglyceridemia and elevated values of AIP, AC, non-HDL-C, and CRI-I with an OR (95% CI) of 4.904 (1.856–12.959), 3.637 (2.149–6.158), 3.589 (1.673–7.700), 2.074 (1.215–3.540), and 2.481 (1.481–4.123), respectively. According to the ROC analysis, the likelihood of hypertriglyceridemia and high values of AIP, AC, non-HDL-C, and CRI-I were associated with 25(OH)D thresholds ≤15.15 ng/mL, ≤17.5 ng/mL, ≤19.8 ng/mL, ≤20.1 ng/mL, and ≤19.5 ng/mL, respectively, all p < 0.01. Based on the atherogenic indices, this study indicates that vitamin D below 20 ng/mL may increase the risk of cardiovascular disease in adult women. Additional health measures are essential to raise awareness among women and health professionals of preventing and controlling cardiovascular risk factors, particularly among young individuals. Full article

Vancomycin (VAN) is an effective antibiotic against Gram-positive bacteria and the first-line therapy to prevent and treat methicillin-resistant Staphylococcus aureus (MRSA) and severe infections. However, low concentrations of VAN can result in resistant strains. High doses of VAN can cause nephrotoxicity and ototoxicity; thus, VAN is a representative drug for which drug monitoring is recommended. Several methods have been proposed to detect VAN. Among them, lateral flow immunoassays (LFIAs) have advantages, such as simple and user-friendly operation, low sample volume requirement, and cost effectiveness. In this study, we developed an LFIA capable of rapid on-site detection such that the VAN concentration in plasma could be monitored within 20 min by a one-step detection process using whole blood without plasma separation. VAN can be detected in whole blood over a wide range of concentrations (20−10,000 ng/mL), and the LFIA reported here has a detection limit of 18 ng/mL. The applicability of the developed LFIA compared to the results of measuring VAN with a commercial enzyme-linked immunosorbent assay kit showed a satisfactory correlation (Spearman’s rho, ρ = 0.891). Therefore, the developed LFIA enables rapid and wide-range VAN detection in whole blood and can aid in drug monitoring to evaluate patients’ responses to treatment. Full article

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. Full article

Forchlorfenuron (CPPU) is a widely used plant growth regulator in agriculture, and CPPU residue in food can cause harm to human health. Thus, it is necessary to develop a rapid and sensitive detection method for CPPU monitoring. In this study, a new monoclonal antibody (mAb) against CPPU with high affinity was prepared by a hybridoma technique, and a magnetic bead (MB)-based analytical method was established for the determination of CPPU by a one-step procedure. Under optimized conditions, the detection limit of the MB-based immunoassay was as low as 0.0004 ng/mL, which was five times more sensitive than the traditional indirect competitive ELISA (icELISA). In addition, the detection procedure took less than 35 min, a significant improvement over the 135 min required for icELISA. The selectivity test of the MB-based assay also showed negligible cross-reactivity with five analogues. Furthermore, the accuracy of the developed assay was assessed by the analysis of spiked samples, and the results agreed well with those obtained by HPLC. The excellent analytical performance of the proposed assay suggests its great potential for routine screening of CPPU, and it provides a basis for promoting the application of more immunosensors in the quantitative detection of low concentrations of small organic molecules in food. Full article

The common mycotoxins in polluted grains are aflatoxin B1(AFB1), zearalenone (ZEN) and deoxynivalenol (DON). Because of the potential threat to humans and animals, it is necessary to detect mycotoxin contaminants rapidly. At present, later flow immunoassay (LFIA) is one of the most frequently used methods for rapid analysis. However, multistep sample pretreatment processes and organic solvents are also required to extract mycotoxins from grains. In this study, we developed a one-step and “green” sample pretreatment method without using organic solvents. By combining with LFIA test strips and a handheld detection device, an on-site method for the rapid detection of AFB1, ZEN and DON was developed. The LODs for AFB1, ZEN and DON in corn are 0.90 μg/kg, 7.11 μg/kg and 10.6 μg/kg, respectively, and the working ranges are from 1.25 μg/kg to 40 μg/kg, 20 μg/kg to 2000 μg/kg and 35 μg/kg to 1500 μg/kg, respectively. This method has been successfully applied to the detection of AFB1, ZEN and DON in corn, rice and peanut, with recoveries of 89 ± 3%–106 ± 3%, 86 ± 2%–108 ± 7% and 90 ± 2%–106 ± 10%, respectively. The detection results for the AFB1, ZEN and DON residues in certified reference materials by this method were in good agreement with their certificate values. Full article

Low-cost diagnostic tools for point-of-care immunoassays, such as the paper-based enzyme-linked immunoassay (ELISA), have become increasingly important, especially so in the recent COVID-19 pandemic. ELISA is the gold-standard antibody/antigen sensing method. This paper reports an easy-to-fabricate nitrocellulose (NC) paper plate, coupled with a desktop scanner for ELISA, which provides a higher protein immobilization efficiency than the conventional cellulose paper-based ELISA platforms. The experiments were performed using spiked samples for the direct ELISA of rabbit IgG with a limit of detection (LOD) of 1.016 μg/mL, in a measurement range of 10 ng/mL to 1 mg/mL, and for the sandwich ELISA of sperm protein (SP-10) with an LOD of 88.8 ng/mL, in a measurement range of 1 ng/mL to 100 μg/mL. The described fabrication method, based on laser-cutting, is a highly flexible one-step laser micromachining process, which enables the rapid production of low-cost NC paper-based multi-well plates with different sizes for the ELISA measurements. Full article

The paper presents development and characterization of a new bioanalytical test system for rapid detection of lipopolysaccharide (LPS) and whole cells of Francisella tularensis, a causative agent of tularemia, in water samples. Gold nanoparticles (AuNPs) coated by the obtained anti-LPS monoclonal antibodies were used for the assay. Their contact with antigen in tested samples leads to aggregation with a shift of absorption spectra from red to blue. Photometric measurements at 530 nm indicated the analyte presence. Three preparations of AuNPs with different diameters were compared, and the AuNPs having average diameter of 34 nm were found to be optimal. The assay is implemented in 20 min and is characterized by detection limits equal to 40 ng/mL for LPS and 3 × 10⁴ CFU/mL for whole cells of F. tularensis. Thus, the proposed simple one-step assay integrates sensitivity comparable with other immunoassay of microorganisms and rapidity. Selectivity of the assay for different strains of F. tularensis was tested and the possibility to choose its variants with the use of different antibodies to distinguish virulent and non-virulent strains or to detect both kinds of F. tularensis was found. The test system has been successfully implemented to reveal the analyte in natural and tap water samples without the loss of sensitivity. Full article

Steroid analysis in clinical laboratories is dominated by immunoassays (IAs) that have a high sample turnover but are inherently limited in trueness, precision, and sensitivity. Liquid chromatography coupled to mass spectrometry (LC-MS/MS) has proved to be a far more capable tool, delivering better sensitivity, specificity, and the possibility of parallel analysis of multiple steroids and metabolites, providing the endocrinologist with more reliable and comprehensive diagnostic information. An LC-MS/MS assay with gradient elution over less than eight minutes and a one-step sample preparation combining protein precipitation with phospholipid removal of off-line solid-phase extraction was developed and validated. It allowed the quantification of 11-deoxycorticosterone (11-DOC), 11-deoxycortisol (11-DF), 17-OH-progesterone (17P), 21-deoxycortisol (21-DF), androstenedione (ANDRO), aldosterone (ALDO), corticosterone (CC), cortisol (CL), cortisone (CN), dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate (DHEAS), dihydrotestosterone (DHT), estradiol (E2), progesterone (PROG), and testosterone (TES) in human serum. Interday imprecision was generally better than 15%, trueness was proven by recovery experiments with ISO 17034-certified reference materials, proficiency testing (UK NEQAS), and measuring serum reference standards. In-house comparison against IVD-CE-certified immunoassays (IA) for 17P, ANDRO, CL, DHEAS, E2, PROG, and TES was conducted by assessing leftover routine patient samples and purpose-built patient serum pools. None of the compared routine IAs were meeting the standards of the LC-MS/MS. Insufficient overall comparability was found for ANDRO and 17P (mean bias > +65%). Accuracy limitations at lower concentrations were present in IAs for PROG, E2, and TES. Full article

Neutralizing antibody (NAb) is a family of antibodies with special functions, which afford a degree of protection against infection and/or reduce the risk of clinically severe infection. Receptor binding domain (RBD) in the spike protein of SARS-CoV-2, a portion of the S1 subunit, can stimulate the immune system to produce NAb after infection and vaccination. The detection of NAb against SARS-CoV-2 is a simple and direct approach for evaluating a vaccine’s effectiveness. In this study, a direct, rapid, and point-of-care bicolor lateral flow immunoassay (LFIA) was developed for NAb against SARS-CoV-2 detection without sample pretreatment, and which was based on the principle of NAb-mediated blockage of the interaction between RBD and angiotensin-converting enzyme 2. In the bicolor LFIA, red and blue latex microspheres (LMs) were used to locate the test and control lines, leading to avoidance of erroneous interpretations of one-colored line results. Under the optimal conditions, NAb against SARS-CoV-2 detection carried out using the bicolor LFIA could be completed within 9 min, and the visible limit of detection was about 48 ng/mL. Thirteen serum samples were analyzed, and the results showed that the NAb levels in three positive serum samples were equal to, or higher than, 736 ng/mL. The LM-based bicolor LFIA allows one-step, rapid, convenient, inexpensive, and user-friendly determination of NAb against SARS-CoV-2 in serum. Full article

Plasma thrombopoietin (TPO) measurements help distinguish between different types of thrombocytopenia but are not feasible in routine clinical practice. We developed a fully automated quantitative chemiluminescent enzyme immunoassay (CLEIA) for measuring TPO (TPO-CLEIA), which is a one-step sandwich-type assay. This assay utilizes a mouse monoclonal capture antibody, which has the neutralizing epitope of the interaction between TPO and the TPO receptor, and a newly generated rabbit monoclonal detector antibody. In analytical performance studies, this assay showed good linearity over the measuring range and high sensitivity. The limit of quantification (LoQ) of this assay was 3.4 pg/mL; low TPO concentration values of almost all healthy individuals exceeded the LoQ value. In clinical validation studies, TPO levels obtained from patients with aplastic anemia (AA) significantly increased, whereas those of patients with immune thrombocytopenia (ITP) were normal or slightly increased. The cutoff value for TPO-CLEIA corresponding to the previously reported values was useful for distinguishing between ITP and AA. These results suggest that TPO-CLEIA can quantify human plasma TPO levels with high accuracy and sensitivity and has the potential to facilitate routine clinical measurement of TPO in patients with various types of thrombocytopenia. Full article