Highly catalytic Prussian blue analogues and their application on the three-dimensional origami paper-based sweat sensors.

Prussian blue analogues (PBAs) are promising materials due to their rich active sites and straightforward synthesis. However, their limited conductivity and electron transfer inefficiency hinder practical applications. This study utilizes a simple one-pot synthesis approach to produce a tungsten-disulfide (WS2) and iron-cobalt Prussian blue analogue composite (WS2-PBA), enhancing conductivity and electron transfer rate performance. Through the inclusion of sodium citrate into the solution, the S-edge site concentration of WS2 increases. This augmentation introduces additional active sites and defects into the catalyst, enhancing its catalytic activity. The effectiveness of the WS2-PBA 3D-Origami paper device for lactate detection in sweat is also evaluated for biomedical applications. The device demonstrated a robust relationship between the lactate concentration and current intensity (R2 = 0.997), with a detection limit of 1.83 mM. Additionally, this platform has successfully detected lactate in clinical sweat, correlating with the high-performance liquid chromatography test results, suggesting promising prospects for clinical diagnosis. In the future, the excellent catalytic and Rct performance of the WS2-PBA will enable its use in biomedical applications.

Diagnosis of Mycobacterium tuberculosis using palladium-platinum bimetallic nanoparticles combined with paper-based analytical devices.

In this study, we demonstrate that palladium-platinum bimetallic nanoparticles (Pd@Pt NPs) as the nanozyme, combined with a multi-layer paper-based analytical device and DNA hybridization, can successfully detect Mycobacterium tuberculosis. This nanozyme has peroxidase-like properties, which can increase the oxidation rate of the substrate. Compared with horseradish peroxidase, which is widely used in traditional detection, the Michaelis constants of Pd@Pt NPs are fourteen and seventeen times lower than those for 3,3',5,5'-tetramethylbenzidine and H2O2, respectively. To verify the catalytic efficiency of Pd@Pt NPs, this study will execute molecular diagnosis of Mycobacterium tuberculosis. We chose the IS6110 fragment as the target DNA and divided the complementary sequences into the capture DNA and reporter DNA. They were modified on paper and Pd@Pt NPs, respectively, to detect Mycobacterium tuberculosis on a paper-based analytical device. With the above-mentioned method, we can detect target DNA within 15 minutes with a linear range between 0.75 and 10 nM, and a detection limit of 0.216 nM. These results demonstrate that the proposed platform (a DNA-nanozyme integrated paper-based analytical device, dnPAD) can provide sensitive and on-site infection prognosis in areas with insufficient medical resources.

Electrochemical biosensor for the evaluation of monoclonal antibodies targeting the N protein of SARS-CoV-2 virus.

The emergence of COVID-19 caused by the coronavirus SARS-CoV-2 has prompted a global pandemic that requires continuous research and monitoring. This study presents a design of an electrochemical biosensing platform suitable for the evaluation of monoclonal antibodies targeting the SARS-CoV-2 nucleocapsid (N) protein. Screen-printed carbon electrodes (SPCE) modified with gold nanostructures (AuNS) were applied to design a versatile and sensitive sensing platform. Electrochemical techniques, including electrochemical impedance spectroscopy (EIS) and square wave voltammetry (SWV), were used to investigate the interactions between immobilised recombinant N (rN) protein and several monoclonal antibodies (mAbs). The electrochemical characterisation of SPCE/AuNS/rN demonstrated a successful immobilisation of rN, enhancing the electron transfer kinetics. Affinity interactions between immobilised rN and four mAbs (mAb-4B3, mAb-4G6, mAb-12B2, and mAb-1G5) were explored. Although mAb-4B3 showed some non-linearity, the other monoclonal antibodies exhibited specific and well-defined interactions followed by the formation of an immune complex. The biosensing platform demonstrated high sensitivity in the linear range (LR) from 0.2 nM to 1 nM with limits of detection (LOD) ranging from 0.012 nM to 0.016 nM for mAb-4G6, mAb-12B2, and mAb-1G5 and limits of quantification (LOQ) values ranging from 0.035 nM to 0.139 nM, as determined by both EIS and SWV methods. These results highlight the system's potential for precise and selective detection of monoclonal antibodies specific to the rN. This electrochemical biosensing platform provides a promising route for the sensitive and accurate detection of monoclonal antibodies specific to the rN protein.

A Semi-Automatic Environmental Monitoring Device for Mercury and Cobalt Ion Detection.

A syringe-based, semi-automatic environmental monitoring device is developed for on-site detection of harmful heavy metal ions in water. This portable device consists of a spring-embedded syringe and a polydimethylsiloxane (PDMS) membrane-based flow regulator for semi-automatic fix-and-release fluidic valve actuation, and a paper-based analytical device (PAD) with two kinds of gold nanoclusters (AuNCs) for sensitive Hg2+ and Co2+ ion detection, respectively. The thickness of the elastic PDMS membrane can be adjusted to stabilize and modulate the flow rates generated by the pushing force provided by the spring attached to the plunger. Also, different spring constants can drastically alter the response time. People of all ages can extract the fix-volume sample solutions and then release them to automatically complete the detection process, ensuring high reliability and repeatability. The PAD comprises two layers of modified paper, and each layer is immobilized with bovine serum albumin-capped gold nanoclusters (R-AuNCs) and glutathione-capped gold clusters (G-AuNCs), respectively. The ligands functionalized on the surface of the AuNCs not only can fine-tune the optical properties of the nanoclusters but also enable specific and simultaneous detection of Hg2+ and Co2+ ions via metallophilic Au+ -Hg2+ interaction and the Co2+ -thiol complexation effect, respectively. The feasibility of the device for detecting heavy metal ions at low concentrations in various environmental water samples is demonstrated. The Hg2+ and Co2+ ions can be seen simultaneously within 20 min with detection limits as low as 1.76 nm and 0.27 µm, respectively, lower than those of the regulatory restrictions on water by the US Environmental Protection Agency and the European Union. we expect this sensitive, selective, portable, and easy-to-use device to be valid for on-site multiple heavy metal ion pollution screenings in resource-constrained settings.

A flash signal amplification approach for ultrasensitive and rapid detection of single nucleotide polymorphisms in tuberculosis.

In recent years, the demand for rapid, sensitive, and simple methods for diagnosing deoxyribonucleic acid (DNA) has grown due to the increase in the variation of infectious diseases. This work aimed to develop a flash signal amplification method coupled with electrochemical detection for polymerase chain reaction (PCR)-free tuberculosis (TB) molecular diagnosis. We exploited the slightly miscible properties of butanol and water to instantly concentrate a capture probe DNA, a single-stranded mismatch DNA, and gold nanoparticles (AuNPs) to a small volume to reduce the diffusion and reaction time in the solution. In addition, the electrochemical signal was enhanced once two strands of DNA were hybridized and bound to the surface of the gold nanoparticle at an ultra-high density. To eliminate non-specific adsorption and identify mismatched DNA, the self-assembled monolayers (SAMs) and Muts proteins were sequentially modified on the working electrode. This sensitive and specific approach can detect as low as attomolar levels of DNA targets (18 aM) and is successfully applied to detecting tuberculosis-associated single nucleotide polymorphisms (SNPs) in synovial fluid. More importantly, as this biosensing strategy can amplify the signal in only a few seconds, it possesses a great potential for point-of-care and molecular diagnosis applications.

Music Box-Inspired Semi-Automatic Hematocrit Validation Device.

A high hematocrit (HCT) level is strongly associated with the risk of cardiovascular disease. For early diagnosis of cardiovascular disease, it is vital to regularly measure the HCT, which is typically achieved by centrifuging a blood sample to measure the percentage of red blood cells. However, the centrifugal modalities are usually bulky, expensive, and require a stable electric input, which restrict the availability. This research develops a semi-automatic and portable centrifugal device for HCT measurement. This torque-actuated semi-automatic centrifuge, which we call the tFuge, is inspired by a music box, allowing different operators to generate the same rhythm. It is electricity-free and can be controlled based on a constant torque mechanism. Repeatable test results can be received from among different users regardless of their age, sex, and activity. With the assistance of the Boycott effect on the tFuge, we proved that the HCT level is in high linearity to the length of the sedimentation of the blood cells in a tube (R2 = 0.99, sample HCT range 10-60%). The tFuge takes less than 4 min and requires no more than 10 µL of blood that can be obtained by a less-invasive finger prick to complete the testing procedure. Calibrated gradient numbers are printed onto the rotation disc for instant HCT results that can be read by the naked eye. We expect this proposed point-of-care testing device possesses the potential to replace the microhematocrit centrifuge in the regions with limited resources.

Molecularly Imprinted Polymer-Based Electrochemical Sensors for the Diagnosis of Infectious Diseases.

The appearance of biological molecules, so-called biomarkers in body fluids at abnormal concentrations, is considered a good tool for detecting disease. Biomarkers are usually looked for in the most common body fluids, such as blood, nasopharyngeal fluids, urine, tears, sweat, etc. Even with significant advances in diagnostic technology, many patients with suspected infections receive empiric antimicrobial therapy rather than appropriate treatment, which is driven by rapid identification of the infectious agent, leading to increased antimicrobial resistance. To positively impact healthcare, new tests are needed that are pathogen-specific, easy to use, and produce results quickly. Molecularly imprinted polymer (MIP)-based biosensors can achieve these general goals and have enormous potential for disease detection. This article aimed to overview recent articles dedicated to electrochemical sensors modified with MIP to detect protein-based biomarkers of certain infectious diseases in human beings, particularly the biomarkers of infectious diseases, such as HIV-1, COVID-19, Dengue virus, and others. Some biomarkers, such as C-reactive protein (CRP) found in blood tests, are not specific for a particular disease but are used to identify any inflammation process in the body and are also under consideration in this review. Other biomarkers are specific to a particular disease, e.g., SARS-CoV-2-S spike glycoprotein. This article analyzes the development of electrochemical sensors using molecular imprinting technology and the used materials' influence. The research methods, the application of different electrodes, the influence of the polymers, and the established detection limits are reviewed and compared.

Therapeutic effects of kefir peptides on adjuvant-induced arthritis in rats through anti-inflammation and downregulation of matrix metalloproteinases.

AIMS: Rheumatoid arthritis (RA) is a chronic autoimmune disease. Its pathological features are synovial inflammation, bone erosion, and joint structural damages. Our previous studies have shown that kefir peptides (KPs) can reduce cardiovascular disease, osteoporosis and renal inflammation. In this study, we further evaluate the efficacy of KPs on adjuvant-induced arthritis (AIA) in a rat model. MAIN METHODS: After the 14th day of adjuvant induction, rats were subsequently orally administered KPs (83 or 166 mg/day/kg) or tofacitinib (6.2 mg/day/kg) for 14 days. On the 28th day, the rats were anesthetized with isoflurane for ultrasonic, in vivo imaging system (IVIS), and radiographic imaging and then sacrificed for ankle tissues collection and analysis. In vitro, IL-1ß-treated human synovial cells (SW982) were subjected to anti-arthritis mechanism study in the presence of KPs. KEY FINDINGS: The results of ultrasonography, radiograph, histology, the expression of matrix metalloproteinases (MMPs), inflammatory cytokines and RANKL/OPG ratio demonstrated decreasing severity of synovitis and bone erosion in the ankle joints after KPs treatment. Activation of the NF-κB and MAPK pathways was significantly reduced in KPs treated AIA group. Furthermore, KPs attenuated IL-1ß-induced inflammatory cytokine production and the expression of MMPs in a human synovial cell line SW982. These results demonstrated that KPs alleviated adjuvant-induced arthritis in rats by inhibiting IL-1ß-related inflammation and MMPs production. SIGNIFICANCE: We concluded that KPs can exhibit anti-inflammatory effects by reducing the levels of macrophage-related inflammatory cytokines and MMPs, thus alleviating bone erosion in the ankle joint and constituting a potential therapeutic strategy for rheumatoid arthritis.

Kefir peptides promote osteogenic differentiation to enhance bone fracture healing in rats.

AIMS: Fractures are the result of fragile bone structures after trauma caused by direct or indirect external impact or strong muscular contraction. Most fracture patients undergo surgical fixation to accelerate the healing process and restore the function of mutilated bone. Promoting the healing process remains an important issue for the treatment of bone fractures. Our previous studies demonstrated the remarkable bone-protective effects of kefir peptides (KPs) in ovariectomized rats and mice. In this study, we further evaluate the efficacy of KPs on fracture healing using a rat model of femoral fracture. MAIN METHODS: Fifteen 8-week-old male Sprague Dawley (SD) rats were divided into the sham, mock, and KPs groups, in which the mock and the KPs groups underwent femur-fracture surgery with nail fixation, while the sham group underwent a sham operation. The next day, rats were orally administered with daily 400 mg/kg of KPs (KPs group) or distilled water (sham and mock groups) for four weeks. X-ray imaging, histochemical staining and serum osteogenic markers were applied for fracture healing evaluation. In vitro, mouse bone marrow mesenchymal stem cells (BMMSCs) and MC3T3-E1 line were subjected to osteoblast differentiation in the presence of KPs and compared with no KPs treatment. KEY FINDINGS: The results demonstrated that KPs treatment improved the progression of the fracture healing process (p < 0.05) and significantly increased the expressions of Col1a1, Alp, Spp1, Vegfa and Cox2 mRNA in the femurs of the KPs-treated fractured rats compared to those of the mock-treated fracture rats. In vitro, KPs treatment promoted bone regeneration factor (Col1a1, Alp, M-csf and Phospho1) expression in MC3T3-E1-derived osteoblast cultures (on Day 3) and enhanced osteogenic differentiation and mineralization in BMMSC-derived osteoblast cultures (on Day 17 and Day 21). SIGNIFICANCE: This is the first study to show that KPs can help with fracture healing by promoting osteogenic differentiation, and it also suggests that KPs can be used as a nutritional supplement to accelerate fracture healing.

Palladium-platinum bimetallic nanomaterials and their application in Staphylococcus aureus detection on paper-based devices.

In this study, we demonstrate how palladium@platinum nanoparticles (Pd@Pt NPs) can be used as a nanozyme for the detection of Staphylococcus aureus (S. aureus) on a paper-based analytical device. Pt was doped with Pd to form bimetallic NPs that serve as a peroxidase mimetic, which can enhance the reaction area with a substrate (3, 3', 5, 5'-tetramethylbenzidine, TMB). After material characterization, we show the peroxidase-like activity of the Pd@Pt NPs featured a 13-times higher binding affinity value (Km) for TMB compared to horseradish peroxidase, which is currently widely used in immunoassays. By incorporating the Pd@Pt NPs in a paper-based immunoassay, we can detect protein A, the biomarker of S. aureus, within 30 min with a detection limit of 9.56 ng/mL. We have also successfully validated this nanozyme-immobilized paper-based analytical device (nPAD) for the detection of human immunoglobulin G to demonstrate the capability of the Pd@Pt NPs for different target analytes. This highly sensitive, rapid, and portable nPAD design has the potential for personalized medicine and point-of-care testing, which could expand on-site prognoses in resource-limited settings.

Determination of rSpike Protein by Specific Antibodies with Screen-Printed Carbon Electrode Modified by Electrodeposited Gold Nanostructures.

In this research, we assessed the applicability of electrochemical sensing techniques for detecting specific antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike proteins in the blood serum of patient samples following coronavirus disease 2019 (COVID-19). Herein, screen-printed carbon electrodes (SPCE) with electrodeposited gold nanostructures (AuNS) were modified with L-Cysteine for further covalent immobilization of recombinant SARS-CoV-2 spike proteins (rSpike). The affinity interactions of the rSpike protein with specific antibodies against this protein (anti-rSpike) were assessed using cyclic voltammetry (CV) and differential pulse voltammetry (DPV) methods. It was revealed that the SPCE electroactive surface area increased from 1.49 ± 0.02 cm2 to 1.82 ± 0.01 cm2 when AuNS were electrodeposited, and the value of the heterogeneous electron transfer rate constant (k0) changed from 6.30 × 10-5 to 14.56 × 10-5. The performance of the developed electrochemical immunosensor was evaluated by calculating the limit of detection and limit of quantification, giving values of 0.27 nM and 0.81 nM for CV and 0.14 nM and 0.42 nM for DPV. Furthermore, a specificity test was performed with a solution of antibodies against bovine serum albumin as the control aliquot, which was used to assess nonspecific binding, and this evaluation revealed that the developed rSpike-based sensor exhibits low nonspecific binding towards anti-rSpike antibodies.

Electrochemical Determination of Interaction between SARS-CoV-2 Spike Protein and Specific Antibodies.

The serologic diagnosis of coronavirus disease 2019 (COVID-19) and the evaluation of vaccination effectiveness are identified by the presence of antibodies specific to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this paper, we present the electrochemical-based biosensing technique for the detection of antibodies specific to the SARS-CoV-2 proteins. Recombinant SARS-CoV-2 spike proteins (rSpike) were immobilised on the surface of a gold electrode modified by a self-assembled monolayer (SAM). This modified electrode was used as a sensitive element for the detection of polyclonal mouse antibodies against the rSpike (anti-rSpike). Electrochemical impedance spectroscopy (EIS) was used to observe the formation of immunocomplexes while cyclic voltammetry (CV) was used for additional analysis of the surface modifications. It was revealed that the impedimetric method and the elaborate experimental conditions are appropriate for the further development of electrochemical biosensors for the serological diagnosis of COVID-19 and/or the confirmation of successful vaccination against SARS-CoV-2.

Distinct Age-Specific Effects on Olfactory Associative Learning in C57BL/6 Substrains.

C57BL/6 is the most widely used mouse strain in the laboratories. Two substrains of C57BL/6, C57BL/6J (B6J), and C57BL/6N (B6N) are well-known backgrounds for genetic modification and have been shown difference in quite a few tests, including open field test, rotarod test, and Morris water maze. However, difference between these two substrains in olfaction-dependent behaviors remains unknown. Here, we used olfactory two-alternative choice task, which is modified to have two training stages, to evaluate animals' ability in instrumental learning and olfactory association. In the first (rule learning) stage, the mice were trained to use the operant chamber to collect water rewards. An odor cue was provided in the procedure, with no indication about reward locations. In the following (discrimination learning) stage, two odor cues were provided, with each indicating a specific water port. The animals were rewarded upon correct port choices following cue deliveries. We found that during young adulthood (7-10 weeks old), proportionally more B6J than B6N mice were able to pass rule learning (58.3% vs. 29.2%) and ultimately acquire this task (54.2% vs. 25%), with the two substrains showing similar pass rates in discrimination learning (92.9% vs. 85.7%). Surprisingly, at a more mature age (17 weeks old), this substrain difference disappeared. Mature B6N mice had a significant improvement in pass percentages of rule learning and overall task, whereas similar improvement was not observed in the B6J counterparts. Instead, mature B6J mice had an improved speed in rule learning and overall task. We further examined behavioral patterns of 8-week-old B6J and B6N mice in the olfactory habituation or dishabituation test. We observed normal olfactory habituation from subjects of both substrains, with the B6J mice exhibiting stronger investigative responses to newly presented odorants. These results reveal for the first time that B6J and B6N mice are different in acquisition processes of a behavioral task that requires instrumental learning and olfactory association, and that maturation appears to employ different effects on these two substrains during these processes. Furthermore, young adult B6J and B6N mice might be similar in olfactory habituation but different in the olfactory aspects of novelty seeking.

Biosensors for the Determination of SARS-CoV-2 Virus and Diagnosis of COVID-19 Infection.

Monitoring and tracking infection is required in order to reduce the spread of the coronavirus disease 2019 (COVID-19), induced by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To achieve this goal, the development and deployment of quick, accurate, and sensitive diagnostic methods are necessary. The determination of the SARS-CoV-2 virus is performed by biosensing devices, which vary according to detection methods and the biomarkers which are inducing/providing an analytical signal. RNA hybridisation, antigen-antibody affinity interaction, and a variety of other biological reactions are commonly used to generate analytical signals that can be precisely detected using electrochemical, electrochemiluminescence, optical, and other methodologies and transducers. Electrochemical biosensors, in particular, correspond to the current trend of bioanalytical process acceleration and simplification. Immunosensors are based on the determination of antigen-antibody interaction, which on some occasions can be determined in a label-free mode with sufficient sensitivity.

Comparison of Different Dosages of Alteplase in Atrial Fibrillation-Related Acute Ischemic Stroke After Intravenous Thrombolysis: A Nationwide, Multicenter, Prospective Cohort Study in Taiwan.

Background Insufficient evidence is available for patients with acute ischemic stroke with atrial fibrillation (AF) to determine the efficacy and safety of different dosages of intravenous thrombolysis treatment. This study examined clinical outcomes in Chinese patients with stroke with and without AF after intravenous thrombolysis treatment with different intravenous thrombolysis doses. Methods and Results This multicenter, prospective cohort study recruited 2351 patients with acute ischemic stroke (1371 with AF and 980 without AF) treated with intravenous thrombolysis using alteplase. The Totaled Health Risks in Vascular Events score is a validated risk-scoring tool used for assessing patients with acute ischemic stroke with and without AF. We evaluated favorable functional outcome at day 90 and symptomatic intracranial hemorrhage within 24 to 36 hours and outcomes of the patients receiving different doses of alteplase. Compared with the non-AF group, the AF group exhibited a 2- to 3-fold increased risk of symptomatic intracranial hemorrhage according to the National Institute of Neurological Disorders and Stroke standard (relative risk [RR], 2.10 [95% CI, 1.35-3.26]). Favorable functional outcome at 90 days and symptomatic intracranial hemorrhage rates according to the European Cooperative Acute Stroke Study II and the Safe Implementation of Thrombolysis in Stroke-Monitoring Study standards did not significantly differ between the AF and non-AF groups. In addition, the low-dose alteplase subgroup exhibited an increased risk of symptomatic intracranial hemorrhage according to the National Institute of Neurological Disorders and Stroke standard (RR, 2.84 [95% CI, 1.63-4.96]). A validation study confirmed these findings after adjustment for scores determined using different stroke risk-scoring tools. Conclusions Different alteplase dosages did not affect functional status at 90 days in the AF and non-AF groups. Thus, the adoption of low-dose alteplase simply because of AF is not recommended.

Suppression of Dendritic Cell Maturation by Kefir Peptides Alleviates Collagen-Induced Arthritis in Mice.

Arthritis is a disorder that is characterized by joint inflammation and other symptoms. Rheumatoid arthritis (RA), an autoimmune disease, is one of the most common arthritis in worldwide. Inflammation of the synovium is the main factor that triggers bone erosion in the joints in RA, but the pathogenesis of RA is not clearly understood. Kefir grain-fermented products have been demonstrated to enhance immune function and exhibit immune-modulating bioactivities. This study aims to explore the role of kefir peptides (KPs) on the regulation of dendritic cell, which are found in RA synovial fluid, and the protection effects of KPs on mice with collagen-induced arthritis (CIA). Immature mouse bone marrow-derived dendritic cells (BMDCs) were treated with KPs (2.2 and 4.4 mg/ml) and then exposed to lipopolysaccharide (LPS) to study the immune regulation function of KPs in dendritic cells. Mice with CIA (n = 5 per group) were orally administrated KPs (3.75 and 7.5 mg/day/kg) for 21 days and therapeutic effect of KPs on mice with arthritis were assessed. In this study, we found that KPs could inhibit surface molecule expression, reduce inflammatory cytokine release, and repress NF-κB and MAPK signaling in LPS-stimulated mouse BMDCs. In addition, a high dose of KPs (7.5 mg/kg) significantly alleviated arthritis symptoms, decreased inflammatory cytokine expression, suppressed splenic DC maturation and decrease the percentage of Th1 and Th17 in the spleens on mice with CIA. Our findings demonstrated that KPs ameliorate CIA in mice through the mechanism of suppressing DC maturation and inflammatory cytokine releases.

KFP-1, a Novel Calcium-Binding Peptide Isolated from Kefir, Promotes Calcium Influx Through TRPV6 Channels.

INTRODUCTION: Kefir is an acidic and alcoholic fermented milk product with multiple health-promoting benefits. A previous study demonstrated that kefir enhanced calcium absorption in intestinal Caco-2 cells. In this study, kefir-fermented peptide-1 (KFP-1) is isolated from the kefir peptide fraction, and its function as a calcium-binding peptide is characterized. METHODS AND RESULTS: KFP-1 was identified as a 17-residue peptide with a sequence identical to that of κ-casein (residues 138-154) in milk protein. KFP-1 is demonstrated to promote calcium influx in Caco-2 and IEC-6 small intestinal cells in a concentration-dependent manner. TRPV6, but not L-type voltage-gated calcium channels, is associated with the calcium influx induced by KFP-1. An in vitro calcium binding assay indicates that the full-length KFP-1 peptide has a higher calcium-binding capacity than the two truncated KFP-1 peptides, KFP-1∆C5 and KFP-1C5. Alexa Fluor 594 labeling shows that KFP-1 is taken up by Caco-2 cells and interacts with calcium ions and TRPV6 protein. Moreover, KFP-1 is found moderately resistant to pepsin and pancreatin digestions and enhanced calcium uptake by intestinal enterocytes in vivo. CONCLUSION: These data suggest that KFP-1, a novel calcium-binding peptide, binds extracellular calcium ions and enters Caco-2 and IEC-6 cells, and promotes calcium uptake through TRPV6 calcium channels. The present study is of great importance for developing kefir-derived metal ion-binding peptides as functional nutraceutical additives.

Dietary supplementation of fermented lemon peel enhances lysozyme activity and susceptibility to Photobacterium damselae for orange-spotted grouper, Epinephelus coioides.

The waste recycling of lemon peel, as a functional feed additive in aquafeed was evaluated by estimating the effects of fermented lemon peel (FLP) supplementation in diet on growth performance, innate immune responses, and susceptibility to Photobacterium damselae of grouper, Epinephelus coioides. A basal diet was added FLP at 0%, 1%, 3%, and 5%. Four tested diets were each fed to juvenile grouper (initial weight: 15.89 ± 0.10 g, triplicate groups) in a recirculation rearing system for eight weeks. Fish fed diets with 0%-3% FLP exhibited higher (p < 0.05) final weight, weight gain, and feed efficiency than fish fed the 5% FLP-diet. After challenge test, fish fed the 3% FLP-diet appeared the lowest mortality, followed by fish fed the 1% FLP-diet, and lowest in fish fed 0% and 5% FLP-diets. Plasma lysozyme activities were higher in fish fed diets with FLP than in fish fed the FLP-free control diet before challenge test. After challenge, fish fed diets with 1% and 3% FLP showed highest lysozyme activities, followed by fish fed the diet with 5% FLP, and lowest in fish fed the control diet. Hepatic malondialdehyde content was higher in fish fed the control diet than in fish fed diets with 1%-3% FLP. Results found that diets supplemented with 1%-3% fermented lemon peel can enhance lysozyme activity and resistance to pathogen P. damselae of grouper.

Paper-based analytical devices for point-of-care blood tests.

Blood can be a window to health, and as a result, is the most intensively studied human biofluid. Blood tests can diagnose diseases, monitor therapeutic drugs, and provide information about the health of an individual. Rapid response blood tests are becoming increasingly essential, especially when subsequent treatment is required. Toward this need, paper-based devices have been excellent tools for performing blood tests due to their ability to conduct rapid and low-cost diagnostics and analyses in a non-laboratory environment. In this Perspective, we review recent advances in paper-based blood tests, particularly focusing on the specific techniques and assays applied. Additionally, we discuss the future of these paper-based devices, such as how the signal intensity can be enhanced and how the in situ synthesis of nanomaterials can be used to improve the sensitivity, functionality, and operational simplicity. With these advances, paper-based devices are becoming increasingly valuable tools for point-of-care blood tests in various practical scenarios.

Hand-Powered Microfluidics for Parallel Droplet Digital Loop-Mediated Isothermal Amplification Assays.

Droplet digital loop-mediated isothermal amplification (ddLAMP) is an important assay for pathogen detection due to its high accuracy, specificity, and ability to quantify nucleic acids. However, performing ddLAMP requires expensive instrumentation and the need for highly trained personnel with expertise in microfluidics. To make ddLAMP more accessible, a ddLAMP assay is developed, featuring significantly decreased operational difficulty and instrumentation requirements. The proposed assay consists of three simplified steps: (1) droplet generation step, in which a LAMP mixture can be emulsified just by manually pulling a syringe connected to a microfluidic device. In this step, for the first time, we verify that highly monodispersed droplets can be generated with unstable flow rates or pressures, allowing untrained personnel to operate the microfluidic device and perform ddLAMP assay; (2) heating step, in which the droplets are isothermally heated in a water bath, which can be found in most laboratories; and (3) result analysis step, in which the ddLAMP result can be determined using only a fluorescence microscopy and an open-source analyzing software. Throughout the process, no droplet microfluidic expertise or equipment is required. More importantly, the proposed system enables multiple samples to be processed simultaneously with a detection limit of 10 copies/µL. The test is simple and intuitive to operate in most laboratories for multi-sample detection, significantly enhancing the accessibility and detection throughput of the ddLAMP technique.