Colorimetric Enzyme-linked Immunosorbent Assay Research Articles

Dual improved enzyme-linked immunosorbent assay based on multivalent bifunctional nanobody and polymerized horseradish peroxide for the ultrasensitive detection of Ochratoxin A in coffee samples

Enzyme-linked immunosorbent assay (ELISA) is favored for its high throughput, low cost, and simplicity, but the limited sensitivity of the traditional colorimetric ELISA restricts its wider application. Therefore, developing improvement strategies to enhance its sensitivity is a current research hotspot. In this work, we developed a multivalent bifunctional nanobody (Nb28-C4-SBP) by fusing a nanobody gene (Nb28) against ochratoxin A (OTA) with a self-assembling peptide (C4) and a streptavidin-conjugated peptide (SBP), which can introduce streptavidin-conjugated polymerized horseradish peroxidase (SA-PolyHRP) as a probe into the detection system. Subsequently, a dual improved ELISA (DI-ELISA) was developed using the higher affinity multivalent bifunctional nanobody (Nb28-C4-SBP) and the more enzyme-loaded probe (SA-PolyHRP) for the ultrasensitive detection of OTA in coffee samples. The DI-ELISA demonstrated a detection limit of 1 pg/mL, which was about 20-fold lower than that of the Nb28-C4-SBP and SA-HRP based ELISA and 266-fold lower than that of the Nb28-SBP and SA-PolyHRP based ELISA, respectively. In summary, we developed an ultrasensitive, rapid, and simple immunoassay for detecting OTA in coffee samples. Additionally, the proposed improvement strategy based on multivalent nanobody and polymerized horseradish peroxide is straightforward to operate and holds great potential for enhancing the sensitivity in various immunoassays.

Concave single-atom Co nanozymes with densely edge-hosted active sites for highly sensitive immunoassay

Regulating the geometric and electronic structures of single-atom catalysts could promote the potential to improve their enzyme-like catalytic performance. Herein, we demonstrate that concave single-atom Co catalysts with edge-hosted and dense active Co-N4 sites incorporated in nitrogen-doped hierarchical porous carbon (H-Co SACs) can act as highly efficient oxidase mimics. In particular, the engineered H-Co SACs with dense active Co-N4 moieties in the edge sites were found to display 3.1 times higher catalytic activity than that of traditional intact Co single-atom catalysts (L-Co SACs). Combined experimental and theoretical simulations reveal that the geometric structure and the interactions between adjacent edge-hosted CoN4 sites synergistically affect the electronic structure of the Co single-atom nanozymes, resulting in strong oxygen adsorption/activation and low energy barrier of the rate-determining step (RDS) in the reaction process, which are demonstrated be more beneficial for the oxidase-like catalytic performance. As a proof-of-concept application, the H-Co SACs were applied to the colorimetric enzyme-linked immunosorbent assay (ELISA) of neuron specific enolase (NSE) with a detection limit of 5.19 pg/ mL, outperforming both the intact Co single-atom catalysts and standard commercial ELISA kits. The present study could not only highlight the significance of geometric structure and active sites density effect on their enzyme-like catalytic efficiency, but also broaden their practical clinical application range of nanozymes at the atomic level.

Parkinson’s disease with anxiety: clinical characteristics and their correlation with oxidative stress, inflammation, and pathological proteins

ObjectiveThis study was performed to explore the differences in the clinical characteristics and oxidative stress indicators, inflammatory factors, and pathological proteins in serum between Parkinson’s disease (PD) with anxiety (PD-A) and with no anxiety (PD-NA) patients, and further correlations among clinical characteristics and above variables were analyzed in PD-A and PD-NA groups.MethodsA total of 121 patients with PD were enrolled in this study and assessed by the Hamilton Anxiety Scale (14 items) (HAMA-14). These patients were divided into PD-A and PD-NA groups according to a cut-off point of 7 of HAMA-14. Demographic variables were collected, and clinical symptoms were assessed by multiple rating scales. The levels of free radicals, inflammatory factors, and pathological proteins in serum were measured by chemical colorimetric method and enzyme-linked immunosorbent assay (ELISA). The differences of above variables were compared between PD-A and PD-NA groups, and the correlations of clinical symptoms with the abovevariables were analyzed in PD-A and PD-NA groups.ResultsThe frequency of PD-A was 62.81%. PD-A group exhibited significantly impaired motor dysfunction and multiple non-motor symptoms, including fatigue, sleep behavior disorder, restless leg syndrome and autonomic dysfunction, and dramatically compromised activities of daily living compard with PD-NA group. PD-A group displayed prominently increasedlevels of hydroxyl radical (·OH) and tumor necrosis factor (TNF)-α, and a decreased nitric oxide (NO) level in serum compared with PD-NA group (P<0.001, P = 0.001, P= 0.027, respectively). ·OH, NO, and TNF-α were identified as the risk factors of PD-A (OR = 1.005, P = 0.036; OR = 0.956, P = 0.017; OR = 1.039, P = 0.033, respectively). In PD patients, HAMA-14 score was significantly and positively correlated with the levels of ·OH and TNF-α in serum (P<0.001, P = 0.002, respectively). In PD-A group, ·OH level was significantly and negatively correlated with Aβ1−42 level, while TNF-α level was significantly and positively correlated with P-tau (S396) level in serum.ConclusionsThe frequency of PD-A is high. PD-A patients present more severe motor dysfunction and multiple non-motor symptoms, and poorer activities of daily living. The increased levels of ·OH and TNF-α levels and the decreased NO level in serum are all associated with more severe anxiety in PD patients.Findings from this study may provide in-depth insights into the clinical characteristics, underlying mechanisms of PD-A, and potential correlations among anxiety, oxidative stress, inflammation, and cognitive decline in PD patients.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) and clinical outcomes in dialysis patients.

Proprotein convertase subtilisin/kexin type 9 (PCSK9), a factor accelerating the degradation of LDL receptors, was associated with a gender-dependent risk for cardiovascular (CV) events in the general population and with all-cause and CV mortality in two relatively small studies in black Africans and South Korean haemodialysis patients. The effect modification by gender was untested in these studies. The study enrolled 1188 dialysis patients from the Prospective Registry of The Working Group of Epidemiology of Dialysis Region Calabria (PROGREDIRE) cohort. PCSK9 was measured by colorimetric enzyme-linked immunosorbent assay. The primary outcomes were all-cause and CV mortality. Statistical analysis included Cox regression analysis and effect modification analysis. During a median 2.9-year follow-up, out of 494 deaths, 278 were CV-related. In unadjusted analyses, PCSK9 levels correlated with increased all-cause (HRfor1ln unit increase: 1.23, 95% CI 1.06-1.43, p =.008) and CV mortality (HRfor1ln unit increase: 1.26, 95% CI 1.03-1.54, p =.03). After multivariate adjustment, these associations were no longer significant (all-cause mortality, HRfor 1 ln unit increase: 1.16, 95% CI .99-1.36, p =.07; CV mortality, HRfor1ln unit increase: 1.18, 95% CI .95-1.46, p =.14). However, in fully adjusted interaction analyses, a doubling in the risk of this outcome in women was registered (Women, HRfor1ln unit increase: 1.88, 95% CI 1.27-2.78, p =.002; Men, HRfor1ln unit increase: 1.07, 95% CI .83-1.38, p =.61; p for effect modification: .02). PCSK9 levels are unrelated to all-cause mortality in haemodialysis patients but, like in studies of the general population, independently of other risk factors, entail a doubling in the risk of CV events in women in this population.

A paper-in-polymer-pond (PiPP) hybrid microfluidic microplate for multiplexed ultrasensitive detection of cancer biomarkers.

Conventional affinity-based colorimetric enzyme-linked immunosorbent assay (ELISA) is one of the most widely used methods for the detection of biomarkers. However, rapid point-of-care (POC) detection of multiple cancer biomarkers by conventional ELISA is limited by long incubation time, large reagent volume, and costly instrumentation along with low sensitivity due to the nature of colorimetric methods. Herein, we have developed a reusable and cost-effective paper-in-polymer-pond (PiPP) hybrid microfluidic microplate for ultrasensitive and high-throughput multiplexed detection of disease biomarkers within an hour without using specialized instruments. A piece of pre-patterned chromatography paper placed in the PMMA polymer pond facilitates rapid protein immobilization to avoid intricate surface modifications of polymer and can be changed with a fresh paper layer to reuse the device. Reagents can be simply delivered from the top PMMA layer to multiple microwells in the middle PMMA layer via flow-through microwells, thereby increasing the efficiency of washing and avoiding repeated manual pipetting or costly robots. Quantitative colorimetric analysis was achieved by calculating the brightness of images scanned by an office scanner or a smartphone camera. Sandwich-type immunoassay was performed in the PiPP hybrid device after the optimization of multiple assay conditions. Limits of detection of 0.32 ng mL-1 for carcinoembryonic antigen (CEA) and 0.20 ng mL-1 for prostate-specific antigen (PSA) were obtained, which were about 10-fold better than those of commercial ELISA kits. We envisage that this simple but versatile hybrid device can have broad applications in various bioassays in resource-limited settings.

Two birds with one stone: Gold “bridging” both nanozyme and immunosensor for sensitive double-response detection of T-2 toxin

Developing immunosensors with nanozymes as signal labels has made a great deal of progress and is no longer a novel concept. But even then, several research obstacles, e.g., suboptimal activity, uncertain antibody coupling, and redundant processes, still remain and must be tackled in this frontier. In this study, we constructed a colorimetric and fluorescent double-response enzyme-linked immunosorbent assay using CeO2@Au heterojunctions as signal labels (CeO2@Au-TFNLISA) for sensitive detection of T-2 toxin. Gold nanoparticles (AuNPs) acting as a bridge for CeO2 can connect and realize the bidirectional improvement of CeO2@Au heterojunctions: (1) enhancing the nanozyme activity significantly by multiple effects (charge transfer, AuNPs assistance, substrate affinity, and oxygen vacancy, etc.); (2) improving the affinity between antibodies and labels through electrostatic incorporation. Meanwhile, in this system, nanozyme catalysis generates the first signal, and fluorescence quenching forms the second signal via the inner-filter effect (IFE) to avoid the inaccuracy result brought on by the off-laboratory environment. The CeO2@Au-TFNLISA has increased the sensitivity by 42 and 18 times in colorimetric (TNLISA) and fluorescent modes (FNLISA) compared with traditional ELISA (0.00852 ng/mL for TNLISA, 0.02011 ng/mL for FNLISA, 0.36492 ng/mL for traditional ELISA), respectively, and saved half of the detection time (from 135 min to 50 min) due to the weakened non-specific adsorption. As a result, the CeO2@Au-TFNLISA method is expected to gain strong momentum to advance the rapid and precise detection of T-2 in food.

Framework-Directed Amino-Acid Insertions Generated over 55-Fold Affinity-Matured Antibody Fragments That Enabled Sensitive Luminescent Immunoassays of Cortisol.

We generated three single-chain Fv fragments (scFvs) specific to cortisol according to our original affinity-maturation strategy and verified their utility in developing immunoassays. These scFv mutants (m-scFvs) had insertion of one, four, or six amino acid(s) in the framework region 1 of the VH-domain and showed >55-fold higher affinity (Ka, 2.0 - 2.2 × 1010 M-1) than the unmodified scFv (wt-scFv). Each m-scFv was fused with NanoLuc luciferase (NLuc) for the use in enzyme-linked immunosorbent assays (ELISAs). In these ELISA, the m-scFv-NLuc fusions were competitively reacted with immobilized cortisol residues and cortisol standards, and then the bound NLuc activity was monitored luminometrically. The luminescent ELISAs generated dose-response curves with extremely low midpoints (approx. 3 pg/assay) and were >150-fold more sensitive than the colorimetric ELISAs using wt-scFv and >8000-fold more sensitive than the ELISA using the parental native antibody. The luminescent ELISAs showed acceptable cross-reactivity patterns with related steroids, and the determination of control sera afforded cortisol levels in the reference range with satisfactory parallelism.

Dopamine as a polymerizable reagent for enzyme-linked immunosorbent assay using horseradish peroxidase

We demonstrate that dopamine can be used as a reagent for colorimetric enzyme-linked immunosorbent assay (ELISA) using horseradish peroxidase (HRP). Dopamine was able to be polymerized in the presence of HRP and H2O2, and black polydopamine was obtained after the enzymatic reaction. Because of the black color, the absorbance was significantly changed in the whole range of the visible light region. Here, an indirect competitive ELISA based on the polymerization of dopamine was performed to detect a fluoroquinolone antibiotic, enrofloxacin. The antibiotic is commonly used in livestock farming. The anti-antibiotics antibody was produced from egg yolk from chicken hens. In the visible range, sufficient absorbance changes of ∼0.4∼0.5 and a low background level for the ELISA response were obtained, and the 50 % inhibitory concentration value at 450 nm was determined to be 26 ppb. The performance of the indirect competitive ELISA based on the polymerization of dopamine was compared to that based on the oxidation of catechol because dopamine has a catechol skeleton. By the complex of HRP and H2O2, catechol can be oxidized to o-benzoquinone having a maximum absorption wavelength of 420 nm. It was shown that the absorbance change in the case of polydopamine was about 2.5 times higher than that of catechol, where the background levels were similar. This confirms that the polymerization of dopamine significantly enhanced the photosignal.

Nanobody based immunoassay for alpha fetal protein detection using streptavidin-conjugated polymerized horseradish peroxidase for signal amplification.

The enzyme-linked immunosorbent assay (ELISA) offers several advantages, including simple operation, high throughput, and low cost, making it an ideal immunoassay method for efficient screening of disease-related biomarkers in clinical samples. However, the traditional colorimetric ELISA has relatively low sensitivity, which promotes the continuous emergence of various novel signal amplification technologies. In this work, we fused the AFP-specific nanobody (A1) with the streptavidin-binding peptide (SBP) to develop a fusion protein (A1-SBP) as biorecognition element in a colorimetric ELISA for detecting AFP. Besides, to further improve the sensitivity of the traditional colorimetric ELISA, the streptavidin-conjugated polymerized horseradish peroxidase (SA-PolyHRP) were selected as a detection probe for signal amplification. The proposed signal enhancement strategy demonstrated a limit of detection (LOD) of 0.597ng/mL for the SA-polyHRP-based ELISA, which is 7.67-fold lower than that of the traditional SA-HRP-based ELISA without additional steps. Furthermore, the proposed SA-polyHRP-based ELISA showed a good correlation with the detection of clinical samples using the Roche E601 chemiluminescence immunoassay analyzer. Therefore, the proposed signal enhancement strategy is an attractive approach for improving the sensitivity of immunoassay without requiring additional steps.

Colorimetric and photothermal immunosensor for sensitive detection of cancer biomarkers based on enzyme-mediated growth of gold nanostars on polydopamine

BackgroundDetecting cancer biomarker levels in body fluids is essential for medical diagnosis. Enzyme-linked immunosorbent assay (ELISA) has been broadly used to detect cancer biomarkers. However, colorimetric ELISA based solely on nanoparticles (NPs) are susceptible to environmental influences, which often results in the detection inaccuracy, being limited in clinical applications. In this regard, the dual-mode approach would add signal diversity to the detection, making the results more reliable. ResultsWe present colorimetric and photothermal immunosensor that enables direct reading of the color and temperature of the solution. A core-satellite nanoprobe constructed by polydopamine (PDA) as the core and gold seeds as satellites is rationally designed as the signal reporter. When ascorbic acid is present in the solution, PDA can cooperate with ascorbic acid to reduce chloroauric acid and mediate the growth of gold seeds on the PDA surface, inducing a redshift of the localized surface plasmon resonance peak of the nanosensor and the change in photothermal conversion efficiency. The method is further combined with the sandwiched immunoassay to construct an alkaline phosphatase based colorimetric and photothermal ELISA for the highly sensitive and accurate evaluation and detection of prostate-specific antigen (PSA). The linear range was from 0.05 to 100 ng mL−1 with a detection limit of 6.71 pg mL−1 for the colorimetric detection, while the linear range was from 0.5 to 90 ng mL−1 with a detection limit of 0.13 ng mL−1 in the photothermal analysis. The accurate detection of PSA levels in serum samples was well demonstrated with the dual-mode approach. SignificanceThe presented immunoassay allows straightforward, sensitive, and selective readout by color and temperature without advanced instrumentation. Particularly, the LOD was much lower than the threshold in clinical trials for PSA. Therefore, this method has a great prospect in the early diagnosis of cancer biomarkers based on a dual-mode multifunctional platform.

Unravelling blood-based epigenetic mechanisms: the impact of hsa-miR-146a and histone H3 acetylation in lead-induced inflammation among occupational workers.

Occupational and environmental exposure to lead (Pb) is a persistent health problem majorly in developing countries and has been implied to cause epigenetic alterations. Its effect on histone post-translational modifications is not explored in human population. MicroRNAs are epigenetic modulators reported to be differentially expressed under Pb exposure. The present study was targeted to find plausible association between the role of hsa-miR-146a and global histone (H3) acetylation in Pb-induced inflammation in occupationally exposed workers. A total of 100 occupationally exposed individuals working in different industries were recruited for the study and divided into 2 groups based on the median Pb levels [low Pb group (Pb < 5μg/dL) and High Pb group (Pb > 5μg/dL)]. The Pb levels were measured in whole blood using atomic absorption spectrometry to confirm Pb exposure. Histone H3 acetylation and serum interleukin-6 (IL-6) levels were measured using colorimetric methods and enzyme-linked immunosorbent assay (ELISA), respectively. MicroRNA-146a expression was quantified using TaqMan assay. The median BLL of the study population was 5μg/dL. BLL, IL-6, and Histone (H3) acetylation increased significantly with the duration of exposure. BLL level showed a significant positive correlation with IL-6 and histone H3 acetylation level. We also found that hsa-miR-146a exhibited significantly increased expression in the high Pb group compared to the low Pb group (Fold change: 2.56; P = 0.014). The linear regression model suggested that BLL has significantly predicted histone H3 acetylation, hsa-miR-146a, and IL-6 in the study subjects. The finding that hsa-miR146a was significantly upregulated in individuals with high BLL and had a significant negative correlation with serum IL-6 suggests that Pb-induced oxidative stress likely activates H3 acetylation, which then releases inflammatory cytokines like IL-6.

Deep learning-assisted ultra-accurate smartphone testing of paper-based colorimetric ELISA assays

Smartphone has long been considered as one excellent platform for disease screening and diagnosis, especially when combined with microfluidic paper-based analytical devices (μPADs) that feature low cost, ease of use, and pump-free operations. In this paper, we report a deep learning-assisted smartphone platform for ultra-accurate testing of paper-based microfluidic colorimetric enzyme-linked immunosorbent assay (c-ELISA). Different from existing smartphone-based μPAD platforms, whose sensing reliability is suffered from uncontrolled ambient lighting conditions, our platform is able to eliminate those random lighting influences for enhanced sensing accuracy. We first constructed a dataset that contains c-ELISA results (n=2048) of rabbit IgG as the model target on μPADs under eight controlled lighting conditions. Those images are then used to train four different mainstream deep learning algorithms. By training with these images, the deep learning algorithms can well eliminate the influences of lighting conditions. Among them, the GoogLeNet algorithm gives the highest accuracy (>97%) in quantitative rabbit IgG concentration classification/prediction, which also provides 4% higher area under curve (AUC) value than that of the traditional curve fitting results analysis method. In addition, we fully automate the whole sensing process and achieve the "image in, answer out" to maximize the convenience of the smartphone. A simple and user-friendly smartphone application has been developed that controls the whole process. This newly developed platform further enhances the sensing performance of μPADs for use by laypersons in low-resource areas and can be facilely adapted to the real disease protein biomarkers detection by c-ELISA on μPADs.

Comparability of biosimilar romiplostim with originator: Protein characterization, animal pharmacodynamics and pharmacokinetics

The results of preclinical studies of romiplostim analogue GP40141 are presented. The effect of a cell proliferation, phosphorylation of the TPO receptor and JAK2 phosphorylation were studied in the presence of romiplostim and in the presence of GP40141 in a cell line of mice (Mus musculus) lymphoblasts with stable expression of human TPO receptor 32D-hTPOR clone 63. Binding to the TPO receptor and to the neonatal Fc receptor (FcRn) was examined for both romiplostim and the developed analogue. In Sprague-Dawley rats, the dynamics of platelet count after the administration of romiplostim or GP40141 were determined. The pharmacokinetics of romiplostim and GP40141, as well as the dynamics of platelet count, were studied in cynomolgus monkeys. The serum concentrations of romiplostim were determined using a modified colorimetric enzyme-linked immunosorbent assay (ELISA). The data obtained allow us to assert the similarity of the biological action of Nplate® and GP40141.

Trace detection of bromadiolone and brodifacoum in biological and food samples: A label-free fluorescence immunoassay based on an in situ formation strategy

The widespread use of bromadiolone and brodifacoum poses a threat to public health and initiates poisoning incidents. Conventional colorimetric enzyme-linked immunosorbent assays (ELISAs) suffer from poor sensitivity, making it difficult to meet the trace detection of analytes. In this work, we constructed a sensitive and label-free fluorescence immunoassay based on the in situ formation strategy, for the quantification of bromadiolone and brodifacoum in biological and food samples. Alkaline phosphatase, an essential enzyme in ELISA, catalyzed the substrate sodium ascorbyl phosphate to L-ascorbic acid, which reacted with ο-phenylenediamine and triggered an in situ fluorescence signal with excitation/emission wavelengths of 350/430 nm. To reduce the reaction time, Fe3+ was utilized for the in situ immunoassay system. The fluorescence signal system was introduced to conventional ELISA without any label procedures, thus resulting in higher sensitivity with a 3.8-fold improvement in the half-maximal inhibitory concentration for bromadiolone, compared with colorimetric ELISA. The calculated limits of detection for bromadiolone and brodifacoum were 0.45–0.76 and 0.34–0.58 ng/mL (ng/g) in the human serum and corn samples, respectively, with recovery values ranging from 82.0 % to 93.1 %, and a coefficient of variation lower than 13.4 %. Due to its advantages of high sensitivity, cost-effectiveness, and easy substrate fabrication, this immunoassay may offer promise for applications in poisoning diagnosis and food analysis of bromadiolone and brodifacoum.

Metabotropic Effect of Probiotic Supplementation and High-Intensity Interval Training in Menopause-Induced Metabolic Syndrome in Rats.

This study aimed to investigate the interactive effect of supplementation with a native potential probiotic lactobacillus and 8-week high-intensity interval training (HIIT) on insulin resistance and dyslipidemia in a menopause-induced metabolic syndrome. A total of 40 ovariectomized (OVX) Wistar rats were divided into five groups: control (OVX + Vehicle), exercise (EXE) (OVX + Exe), probiotic (Prob) (OVX + Prob), exercise and probiotic (OVX + Exe + Prob), and sham surgery. After the end of the treatment interventions, body weight, body mass index (BMI), waist circumference (WC), visceral fat, and serum concentrations of glucose, insulin, lipid profile, and adiponectin were measured using colorimetric analysis and enzyme-linked immunosorbent assay, respectively. Data revealed a significant decrease in weight, waist circumference , visceral fat, BMI, and levels of glucose, insulin, homeostasis model assessment of insulin resistance, triacylglyceride, total cholesterol, and low-density lipoprotein (LDL), but an increase in high-density lipoprotein and adiponectin levels (P = 0.001), in OVX + Exe + Prob compared with the OVX + Vehicle group. The present study indicates that native probiotic lactobacillus combined with HIIT effectively reduces body weight, visceral fat, and levels of LDL, glucose, and insulin and increases adiponectin level, although exercise contributes more to fat reduction and probiotics to insulin resistance.

Astragaloside IV ameliorates spinal cord injury through controlling ferroptosis in H2O2-damaged PC12 cells in vitro.

Spinal cord injury (SCI) is associated with significant paralysis and high fatality. Recent research has revealed that ferroptosis participates in the pathogenesis of SCI. Astragaloside IV (AS-IV), the main active ingredient of the plant Astragalus membranaceus, has been reported to promote motor function recovery in rats with SCI. This study explored the effects of AS-IV in H2O2-treated PC12 pheochromocytoma cells. The optimal concentration and duration of AS-IV treatment in PC12 cells was assessed using the cell counting kit 8 (CCK-8) assay. Subsequently, the SCI cell model was established in PC12 cells using H2O2. The effects of AS-IV, FIN56, and transcription factor EB (TFEB) small interfering (si)RNA on cell viability and apoptosis in the SCI model were determined using the CCK-8 assay and flow cytometry, respectively. Caspase‑3 and lactate dehydrogenase (LDH) levels were measured by colorimetric assay and enzyme-linked immunosorbent assay (ELISA), respectively. Cellular reactive oxygen species (ROS) were detected by flow cytometry combined with dichloro-dihydro-fluorescein diacetate (DCFH-DA) assay. The cellular ultrastructure was analyzed by transmission electron microscopy (TEM). The ferroptosis pathway-related proteins were confirmed using Western blot analysis. TFEB expression was confirmed by Western blot and immunofluorescence. The optimal concentration and duration of AS-IV treatment in PC12 cells was determined to be 1.0 µM and 48 h, respectively. AS-IV markedly accelerated proliferation, suppressed apoptosis, and reduced ROS and LDH accumulation. Furthermore, AS-IV enhanced TFEB expression in H2O2-damaged PC12 cells. The effects of AS-IV on SCI were inhibited by si-TFEB, and this inhibition was further reinforced by the addition of FIN56. The results of this investigation using the SCI cell model suggested that AS-IV alleviated SCI by promoting TFEB expression and subsequently mediating ferroptosis. This may represent a potential clinical treatment for SCI.

Pd@Pt Nanodendrites as Peroxidase Nanomimics for Enhanced Colorimetric ELISA of Cytokines with Femtomolar Sensitivity.

Colorimetric enzyme-linked immunosorbent assay (ELISA) has been widely applied as the gold-standard method for cytokine detection over decades. However, it has become a critical challenge to further improve the detection sensitivity of ELISA as limited by the catalytic activity of enzymes. Herein, we report an enhanced colorimetric ELISA for ultrasensitive detection of interleukin-6 (IL-6, as a model cytokine for demonstration) using core@shell nanodendrites ( NDs) as peroxidase nanomimics (named " ND ELISA"), pushing the sensitivity up to femtomolar level. Specifically, the NDs are rationally engineered by depositing Pt atoms on Pd nanocubes (NCs) to generate rough dendrite-like Pt skins on the Pd surfaces via Volmer-Weber growth mode. They can be produced on a large scale with highly uniform size, shape, composition, and structure. They exhibit significantly enhanced peroxidase-like catalytic activity with catalytic constants () more than 2000-fold higher than those of horseradish peroxidase (HRP, an enzyme commonly used in ELISA). Using NDs as the signal labels, the ND ELISA presents strong colorimetric signals for the quantitative determination of IL-6 with a wide dynamic range of 0.05-100 pg mL-1 and an ultralow detection limit of 0.044 pg mL-1 (1.7 fM). This detection limit is 21-fold lower than that of conventional HRP-based ELISA. The reproducibility and specificity of the ND ELISA are excellent. More significantly, the ND ELISA was validated for analyzing IL-6 in human serum samples with high accuracy and reliability through recovery tests. Our results demonstrate that the colorimetric ND ELISA is a promising biosensing tool for ultrasensitive determination of cytokines and thus is expected to be applied in a variety of clinical diagnoses and fundamental biomedical studies.

The evaluation of serum lipid profile and apolipoprotein C-1 in the Iranian patients of Oral Squamous Cell Carcinoma.

The key role of apolipoprotein C-1 (ApoC-1) is reported in breast, pancreas and lung cancer. However, no information is available on potential difference of ApoC-1 between OSCC patients and healthy individuals. This work aimed to examine the serum ApoC-1 level as well as lipid profile values between OSCC patients and healthy control groups. In this study, 44 blood samples from 22 OSCC patients and 22 healthy individuals were collected to determine the values of lipid profile and ApoC-1 concentration using colorimetric method and Enzyme-Linked Immunosorbent Assay (ELISA), respectively. A significant decrease in serum lipid profile and ApoC-1 concentration was observed between OSCC and healthy control groups (P = 0.001). Our results confirm the previous findings on the significant differences of lipid profile between OSCC and controls, also show the lower serum level of ApoC-1 in OSCC as compared to the controls. Future studies would further elaborate the association of ApoC-1 with OSCC.

An Electromechanical Lab-on-a-Chip Platform for Colorimetric Detection of Serum Creatinine.

Chronic kidney disease (CKD) is a high-cost disease that affects approximately one in ten people globally, progresses rapidly, results in kidney failure or dialysis, and triggers other diseases. Although clinically used serum creatinine tests are used to evaluate kidney functions, these tests are not suitable for frequent and regular control at-home settings that obstruct the regular monitoring of kidney functions, improving CKD management with early intervention. This study introduced a new electromechanical lab-on-a-chip platform for point-of-care detection of serum creatinine levels using colorimetric enzyme-linked immunosorbent assay (ELISA). The platform was composed of a chip containing microreservoirs, a stirring bar coated with creatinine-specific antibodies, and a phone to detect color generated via ELISA protocols to evaluate creatinine levels. An electromechanical system was used to move the stirring bar to different microreservoirs and stir it inside them to capture and detect serum creatinine in the sample. The presented platform allowed automated analysis of creatinine in ∼50 min down to ∼1 and ∼2 mg/dL in phosphate-buffered saline (PBS) and fetal bovine serum (FBS), respectively. Phone camera measurements in hue, saturation, value (HSV) space showed sensitive analysis compared to a benchtop spectrophotometer that could allow low-cost analysis at point-of-care.

Colorimetric ELISA based on urease catalysis curcumin as a ratiometric indicator for the sensitive determination of aflatoxin B1 in grain products

There is an urgent need to measure aflatoxin B1 (AFB1) in food to prevent contaminated food consumption. In this work, a novel colorimetric enzyme-linked immunosorbent assay (ELISA) was developed for the detection of AFB1 using curcumin as a colorimetric indicator. An indirect competitive enzyme-label immunoassay was developed using urease and rabbit anti-mouse immunoglobulin G labeled with gold nanoparticles as the signal-transduction tag. Urease catalyzed the hydrolysis of urea to produce ammonia, which increased the pH of the solution. The phenolic hydroxyl group of curcumin ionized into phenolic oxygen anions under alkaline conditions, which strengthened the synergistic effect of electron supply and absorption in curcumin. As a result, the color of curcumin changed from yellow to reddish-brown, producing a visible color change. Under optimal conditions, AFB1 could be qualitatively determined with the naked eye, and quantitatively assessed by measuring the ratio of absorbance at wavelengths of 550 and 428 nm. The change in the ratio of absorbance Δ550/Δ428 decreased linearly in a range of 0.01–5 ng mL−1, and the limit of detection was 67 pg mL−1. Therefore, the selectivity and reliability of this proposed method were well validated. This method was also successfully used for the quantitation of AFB1 in spiked rice flour and wheat flour samples. This approach may broaden the application field of colorimetric ELISA for aflatoxin, providing a promising platform for the rapid screening of aflatoxin in food.