Falls During Inpatient Rehabilitation After Spinal Cord Injury: Characterization, Clock-Hour Visualization, and Time to Event Predictors.

OBJECTIVES: To (1) determine fall characteristics (eg, cause, location, witnesses) of inpatients with spinal cord injury (SCI) and whether they were different for ambulatory persons vs wheelchair users; (2) visualize the total number of daily falls per clock-hour for different inpatients' features (eg, cause of injury, age); (3) compare clinical and demographic characteristics of inpatients who experienced a first fall event vs inpatients who did not experience such event; and (4) identify first fall event predictors. DESIGN: Retrospective observational cohort study. SETTING: Institution for inpatient neurologic rehabilitation. PARTICIPANTS: Persons with SCI (N=1294) admitted to a rehabilitation facility between 2005 and 2022. INTERVENTIONS: Not applicable. MAIN OUTCOME MEASURES: Functional independence measure (FIM), Hospital Anxiety and Depression Scale (HADS), American Spinal Injury Association Impairment Scale (AIS), and Spinal Cord Independence Measure (SCIM) at admission. Kaplan-Meier survival curves and Cox proportional hazards models were used. RESULTS: A total of 502 fall events were experienced by 369 ambulatory inpatients (19.8%) and wheelchair users (80.2%) in 63.9% of cases being alone, with cause, situation, and location significantly different in both groups. Clock-hour visualizations revealed an absolute peak at 12 AM (complete or incomplete injuries, with paraplegia or tetraplegia) but a relative peak at 9 AM mainly including incomplete patients with paraplegia. Of the (n=1294) included patients, 16.8% experienced at least 1 fall. Fallen patients reported higher levels of HADS depression, lower total SCIM, and longer time since injury to admission, with no differences in age, sex, educational level, FIM (quasi-significant), and AIS grade. Multivariable Cox proportional hazards identified time since injury to admission and AIS grade D as significant predictors of first fall event. CONCLUSIONS: Falls identification, characterization, and clock-hour visualization can support decisions for mitigation strategies specifically addressed to inpatients with SCI. Fall predictors were identified as a first step for future research.

Risk for Recurrent Venous Thromboembolism in Patients With Subsegmental Pulmonary Embolism Managed Without Anticoagulation : A Multicenter Prospective Cohort Study.

BACKGROUND: The incidence of pulmonary embolism has been increasing, but its case-fatality rate is decreasing, suggesting a lesser severity of illness. The clinical importance of patients with pulmonary embolism isolated to the subsegmental vessels is unknown. OBJECTIVE: To determine the rate of recurrent venous thromboembolism in patients with subsegmental pulmonary embolism managed without anticoagulation. DESIGN: Multicenter prospective cohort study. (ClinicalTrials.gov: NCT01455818). SETTING: Eighteen sites between February 2011 and February 2021. PATIENTS: Patients with isolated subsegmental pulmonary embolism. INTERVENTION: At diagnosis, patients underwent bilateral lower-extremity venous ultrasonography, which was repeated 1 week later if results were negative. Patients without deep venous thrombosis did not receive anticoagulant therapy. MEASUREMENTS: The primary outcome was recurrent venous thromboembolism during the 90-day follow-up period. RESULTS: Recruitment was stopped prematurely because the predefined stopping rule was met after 292 of a projected 300 patients were enrolled. Of the 266 patients included in the primary analysis, the primary outcome occurred in 8 patients, for a cumulative incidence of 3.1% (95% CI, 1.6% to 6.1%) over the 90-day follow-up. The incidence of recurrent venous thromboembolism was 2.1% (CI, 0.8% to 5.5%) and 5.7% (CI, 2.2% to 14.4%) over the 90-day follow-up in patients with single and multiple isolated subsegmental pulmonary embolism, respectively. No patients had a fatal recurrent pulmonary embolism. LIMITATION: The study was restricted to patients with low-risk subsegmental pulmonary embolism. CONCLUSION: Overall, patients with subsegmental pulmonary embolism who did not have proximal deep venous thrombosis had a higher-than-expected rate of recurrent venous thromboembolism. PRIMARY FUNDING SOURCE: Heart and Stroke Foundation of Canada and French Ministry of Health Programme Hospitalier de Recherche Clinique.

Biosensing Tacrolimus in Human Whole Blood by Using a Drug Receptor Fused to the Emerald Green Fluorescent Protein.

Tacrolimus (FK506) is an immunosuppressant drug (ISD) used to prevent organ rejection after transplantation that exhibits a narrow therapeutic window and is subject to wide inter- and intra-individual pharmacokinetic fluctuations requiring careful monitoring. The immunosuppressive capacity of FK506 arises from the formation of a complex with immunophilin FKBP1A. This paper describes the use of FKBP1A as an alternative to common antibodies for biosensing purposes. Bioassays use recombinant FKBP1A fused to the emerald green fluorescent protein (FKBP1A-EmGFP). Samples containing the immunosuppressant are incubated with the recombinant protein, and free FKBP1A-EmGFP is captured by magnetic beads functionalized with FK506 to generate a fluorescence signal. Recombinant receptor-drug interaction is evaluated by using a quartz crystal microbalance and nuclear magnetic resonance. The limit of detection (3 ng mL-1) and dynamic range thus obtained (5-70 ng mL-1) fulfill therapeutic requirements. The assay is selective for other ISD usually coadministered with FK506 and allows the drug to be determined in human whole blood samples from organ transplant patients with results comparing favorably with those of an external laboratory.

Recombinant antibodies and their use for food immunoanalysis.

Antibodies are widely employed as biorecognition elements for the detection of a plethora of compounds including food and environmental contaminants, biomarkers, or illicit drugs. They are also applied in therapeutics for the treatment of several disorders. Recent recommendations from the EU on animal protection and the replacement of animal-derived antibodies by non-animal-derived ones have raised a great controversy in the scientific community. The application of recombinant antibodies is expected to achieve a high growth rate in the years to come thanks to their versatility and beneficial characteristics in comparison to monoclonal and polyclonal antibodies, such as stability in harsh conditions, small size, relatively low production costs, and batch-to-batch reproducibility. This review describes the characteristics, advantages, and disadvantages of recombinant antibodies including antigen-binding fragments (Fab), single-chain fragment variable (scFv), and single-domain antibodies (VHH) and their application in food analysis with especial emphasis on the analysis of biotoxins, antibiotics, pesticides, and foodborne pathogens. Although the wide application of recombinant antibodies has been hampered by a number of challenges, this review demonstrates their potential for the sensitive, selective, and rapid detection of food contaminants.

Molecular super-gluing: a straightforward tool for antibody labelling and its application to mycotoxin biosensing.

Mycotoxins are low molecular weight toxic compounds, which can cause severe health problems in animals and humans. Immunoassays allow rapid, simple and cost-effective screening of mycotoxins. Sandwich assays with a direct readout provide great improvement in terms of selectivity and sensitivity, compared to the widely used competitive assay formats, for the analysis of low molecular weight molecules. In this work, we report a non-competitive fluorescence anti-immune complex (IC) immunoassay, based on the specific recognition of HT-2 toxin with a pair of recombinant antibody fragments, namely antigen-binding fragment (Fab) (anti-HT-2 (10) Fab) and single-chain variable fragment (scFv) (anti-IC HT-2 (10) scFv). The SpyTag and SpyCatcher glue proteins were applied for the first time as a bioconjugation tool for the analysis of mycotoxins. To this aim, a SpyTag-mScarlet-I (fluorescent protein) and scFv-SpyCatcher fusion proteins were constructed, produced and fused in situ during the assay by spontaneous Tag-Catcher binding. The assay showed an excellent sensitivity with an EC50 of 4.8 ± 0.4 ng mL-1 and a dynamic range from 1.7 ± 0.3 to 13 ± 2 ng mL-1, an inter-day reproducibility of 8.5% and a high selectivity towards HT-2 toxin without cross-reactivity with other Fusarium toxins. The bioassay was applied to the analysis of the toxin in an oat reference material and in oat samples, with a LOD of 0.6 µg kg-1, and the results were validated by analysing a certificate reference material and by HPLC-MS/MS.

Thoracic imaging tests for the diagnosis of COVID-19.

BACKGROUND: Our March 2021 edition of this review showed thoracic imaging computed tomography (CT) to be sensitive and moderately specific in diagnosing COVID-19 pneumonia. This new edition is an update of the review. OBJECTIVES: Our objectives were to evaluate the diagnostic accuracy of thoracic imaging in people with suspected COVID-19; assess the rate of positive imaging in people who had an initial reverse transcriptase polymerase chain reaction (RT-PCR) negative result and a positive RT-PCR result on follow-up; and evaluate the accuracy of thoracic imaging for screening COVID-19 in asymptomatic individuals. The secondary objective was to assess threshold effects of index test positivity on accuracy. SEARCH METHODS: We searched the COVID-19 Living Evidence Database from the University of Bern, the Cochrane COVID-19 Study Register, The Stephen B. Thacker CDC Library, and repositories of COVID-19 publications through to 17 February 2021. We did not apply any language restrictions. SELECTION CRITERIA: We included diagnostic accuracy studies of all designs, except for case-control, that recruited participants of any age group suspected to have COVID-19. Studies had to assess chest CT, chest X-ray, or ultrasound of the lungs for the diagnosis of COVID-19, use a reference standard that included RT-PCR, and report estimates of test accuracy or provide data from which we could compute estimates. We excluded studies that used imaging as part of the reference standard and studies that excluded participants with normal index test results. DATA COLLECTION AND ANALYSIS: The review authors independently and in duplicate screened articles, extracted data and assessed risk of bias and applicability concerns using QUADAS-2. We presented sensitivity and specificity per study on paired forest plots, and summarized pooled estimates in tables. We used a bivariate meta-analysis model where appropriate. MAIN RESULTS: We included 98 studies in this review. Of these, 94 were included for evaluating the diagnostic accuracy of thoracic imaging in the evaluation of people with suspected COVID-19. Eight studies were included for assessing the rate of positive imaging in individuals with initial RT-PCR negative results and positive RT-PCR results on follow-up, and 10 studies were included for evaluating the accuracy of thoracic imaging for imagining asymptomatic individuals. For all 98 included studies, risk of bias was high or unclear in 52 (53%) studies with respect to participant selection, in 64 (65%) studies with respect to reference standard, in 46 (47%) studies with respect to index test, and in 48 (49%) studies with respect to flow and timing. Concerns about the applicability of the evidence to: participants were high or unclear in eight (8%) studies; index test were high or unclear in seven (7%) studies; and reference standard were high or unclear in seven (7%) studies. Imaging in people with suspected COVID-19 We included 94 studies. Eighty-seven studies evaluated one imaging modality, and seven studies evaluated two imaging modalities. All studies used RT-PCR alone or in combination with other criteria (for example, clinical signs and symptoms, positive contacts) as the reference standard for the diagnosis of COVID-19. For chest CT (69 studies, 28285 participants, 14,342 (51%) cases), sensitivities ranged from 45% to 100%, and specificities from 10% to 99%. The pooled sensitivity of chest CT was 86.9% (95% confidence interval (CI) 83.6 to 89.6), and pooled specificity was 78.3% (95% CI 73.7 to 82.3). Definition for index test positivity was a source of heterogeneity for sensitivity, but not specificity. Reference standard was not a source of heterogeneity. For chest X-ray (17 studies, 8529 participants, 5303 (62%) cases), the sensitivity ranged from 44% to 94% and specificity from 24 to 93%. The pooled sensitivity of chest X-ray was 73.1% (95% CI 64. to -80.5), and pooled specificity was 73.3% (95% CI 61.9 to 82.2). Definition for index test positivity was not found to be a source of heterogeneity. Definition for index test positivity and reference standard were not found to be sources of heterogeneity. For ultrasound of the lungs (15 studies, 2410 participants, 1158 (48%) cases), the sensitivity ranged from 73% to 94% and the specificity ranged from 21% to 98%. The pooled sensitivity of ultrasound was 88.9% (95% CI 84.9 to 92.0), and the pooled specificity was 72.2% (95% CI 58.8 to 82.5). Definition for index test positivity and reference standard were not found to be sources of heterogeneity. Indirect comparisons of modalities evaluated across all 94 studies indicated that chest CT and ultrasound gave higher sensitivity estimates than X-ray (P = 0.0003 and P = 0.001, respectively). Chest CT and ultrasound gave similar sensitivities (P=0.42). All modalities had similar specificities (CT versus X-ray P = 0.36; CT versus ultrasound P = 0.32; X-ray versus ultrasound P = 0.89). Imaging in PCR-negative people who subsequently became positive For rate of positive imaging in individuals with initial RT-PCR negative results, we included 8 studies (7 CT, 1 ultrasound) with a total of 198 participants suspected of having COVID-19, all of whom had a final diagnosis of COVID-19. Most studies (7/8) evaluated CT. Of 177 participants with initially negative RT-PCR who had positive RT-PCR results on follow-up testing, 75.8% (95% CI 45.3 to 92.2) had positive CT findings. Imaging in asymptomatic PCR-positive people For imaging asymptomatic individuals, we included 10 studies (7 CT, 1 X-ray, 2 ultrasound) with a total of 3548 asymptomatic participants, of whom 364 (10%) had a final diagnosis of COVID-19. For chest CT (7 studies, 3134 participants, 315 (10%) cases), the pooled sensitivity was 55.7% (95% CI 35.4 to 74.3) and the pooled specificity was 91.1% (95% CI 82.6 to 95.7). AUTHORS' CONCLUSIONS: Chest CT and ultrasound of the lungs are sensitive and moderately specific in diagnosing COVID-19. Chest X-ray is moderately sensitive and moderately specific in diagnosing COVID-19. Thus, chest CT and ultrasound may have more utility for ruling out COVID-19 than for differentiating SARS-CoV-2 infection from other causes of respiratory illness. The uncertainty resulting from high or unclear risk of bias and the heterogeneity of included studies limit our ability to confidently draw conclusions based on our results.

Limited Chest Ultrasound to Replace CXR in Diagnosis of Pneumothorax Post Image-Guided Transthoracic Interventions.

PURPOSE: To assess the diagnostic accuracy of limited chest ultrasound in detecting pneumothorax following percutaneous transthoracic needle interventions using chest X-ray (CXR) as the reference standard. METHODS: With IRB approval, after providing consent, asymptomatic patients after percutaneous transthoracic needle interventions were enrolled to undergo limited chest ultrasound in addition to CXR. A chest Radiologist blinded to the patient's prior imaging performed a bedside ultrasound, scanning only the first 3 anterior intercostal spaces. Pneumothorax diagnosed on CXR was categorized as small or large and on ultrasound as grades 1, 2, or 3 when detected in 1, 2, or 3 intercostal spaces, respectively. RESULTS: 38 patients underwent 36 biopsies (34 lungs, 1 pleura, and 1 mediastinum) and 2 coil localizations. CXR showed pneumothorax in 13 patients. Ultrasound was positive in 10 patients, with 9 true-positives, 1 false-positive, 4 false-negatives, and 24 true-negatives. The false positive results were due to apical subpleural bullae. The false-negative results occurred in 2 small apical and 2 focal pneumothoraces at the needle entry sites. Four pneumothoraces were categorized as large on CXR, all of which were categorized as grade 3 on ultrasound. Sensitivity and specificity of US for detection of pneumothorax of any size were 69.23% (95%CI 38.6%, 90.1%) and 96.0% (95%CI 79.6%, 99.9%), and for detection of large pneumothorax were 100% (95%CI 39.8%, 100%) and 100% (95%CI 89.7%, 100%). CONCLUSIONS: Results of this prospective study is promising. Limited chest ultrasound could potentially replace CXR in the management of postpercutaneous transthoracic needle intervention patients.

Recombinant Peptide Mimetic NanoLuc Tracer for Sensitive Immunodetection of Mycophenolic Acid.

Mycophenolic acid (MPA) is an immunosuppressant drug commonly used to prevent organ rejection in transplanted patients. MPA monitoring is of great interest due to its small therapeutic window. In this work, a phage-displayed peptide library was used to select cyclic peptides that bind to the MPA-specific recombinant antibody fragment (Fab) and mimic the behavior of MPA. After biopanning, several phage-displayed peptides were isolated and tested to confirm their epitope-mimicking nature in phage-based competitive immunoassays. After identifying the best MPA mimetic (ACEGLYAHWC with a disulfide constrained loop), several immunoassay approaches were tested, and a recombinant fusion protein containing the peptide sequence with a bioluminescent enzyme, NanoLuc, was developed. The recombinant fusion enabled its direct use as the tracer in competitive immunoassays without the need for secondary antibodies or further labeling. A bioluminescent sensor, using streptavidin-coupled magnetic beads for the immobilization of the biotinylated Fab antibody, enabled the detection of MPA with a detection limit of 0.26 ng mL-1 and an IC50 of 2.9 ± 0.5 ng mL-1. The biosensor showed good selectivity toward MPA and was applied to the analysis of the immunosuppressive drug in clinical samples, of both healthy and MPA-treated patients, followed by validation by liquid chromatography coupled to diode array detection.

Biosensing based on upconversion nanoparticles for food quality and safety applications.

Food safety and quality regulations inevitably call for sensitive and accurate analytical methods to detect harmful contaminants in food and to ensure safe food for the consumer. Both novel and well-established biorecognition elements, together with different transduction schemes, enable the simple and rapid analysis of various food contaminants. Upconversion nanoparticles (UCNPs) are inorganic nanocrystals that convert near-infrared light into shorter wavelength emission. This unique photophysical feature, along with narrow emission bandwidths and large anti-Stokes shift, render UCNPs excellent optical labels for biosensing because they can be detected without optical background interferences from the sample matrix. In this review, we show how this exciting technique has evolved into biosensing platforms for food quality and safety monitoring and highlight recent applications in the field.

Bioluminescent detection of zearalenone using recombinant peptidomimetic Gaussia luciferase fusion protein.

The development of a bioluminescent immunosensor is reported for the determination of zearalenone (ZEA) based on a peptide mimetic identified by phage display. The peptide mimetic GW, with a peptide sequence GWWGPYGEIELL, was used to create recombinant fusion proteins with the bioluminescent Gaussia luciferase (GLuc) that were directly used as tracers for toxin detection in a competitive immunoassay without the need for secondary antibodies or further labeling. The bioluminescent sensor, based on protein G-coupled magnetic beads for antibody immobilization, enabled determination of ZEA with a detection limit of 4.2 ng mL-1 (corresponding to 420 µg kg-1 in food samples) and an IC50 value of 11.0 ng mL-1. The sensor performance was evaluated in spiked maize and wheat samples, with recoveries ranging from 87 to 106% (RSD < 20%, n = 3). Finally, the developed method was applied to the analysis of a naturally contaminated reference matrix material and good agreement with the reported concentrations was obtained.Graphical abstract.

[Historical overview of acute infectious diarrhea in Mexico and future preventive strategies]. / Panorama histórico de la enfermedad diarreica aguda en México y el futuro de su prevención.

OBJECTIVE: To analyze the epidemiological aspects of AID through Mexican history and the potential strategies to pre- vent AID in Mexican population. MATERIALS AND METHODS: A systematic review was performed exploring the key words, diarrhea, morbidity, mortality, Mexico, health promotion for the last 20 years (1978-2018). RESULTS: Over 8 600 articles were obtained; all of them were evaluated to consider those follow the aim of the present work. CONCLUSIONS: The result of the performed systematic review denoted the influence of AID in Mexican public health policy; the adopted actions diminished the AID's associated risks and allowed future strategies to prevent it; those actions must include hygienic and dietetic measures, pharmaceutical innovations and technological tools applied to health policies.

OBJETIVO: Revisar los aspectos epidemiológicos de la enfermedad diarreica aguda (EDA) a través de la historia de México y analizar las estrategias que potencialmente podrán prevenir su aparición en la población mexicana. MATERIAL Y MÉTODOS: Se realizó una búsqueda sistematizada utilizando los siguientes descriptores de las ciencias de la salud: diarrea, morbilidad, mortalidad, México y promoción de la salud de los últimos 20 años (1878-2018). RESULTADOS: Se obtuvieron más de 8 600 artículos que fueron evaluados en función de los objetivos de la presente publicación. CONCLUSIONES: Como resultado de una revisión sistemática se observó que, gracias a las estrategias implementadas a lo largo del tiempo, se ha logrado graduar los matices de riesgo de la EDA; ello permite ahora plantear estrategias que guiarán a la prevención de ese padecimiento, de la mano de políticas que incluyan aspectos higiénico-dietéticos, innovaciones farmacéuticas y aplicaciones tecnológicas en medidas sanitarias.

Tag-Specific Affinity Purification of Recombinant Proteins by Using Molecularly Imprinted Polymers.

Epitope tagging is widely used to fuse a known epitope to proteins for which no affinity receptor is available by using recombinant DNA technology. One example is FLAG epitope (DYKDDDDK), which provides better purity and recoveries than the favorite poly histidine tag. However, purification requires using anti-FLAG antibody resins, the high cost and nonreusability of which restrict widespread use. One cost-effective solution is provided by the use of bioinspired anti-FLAG molecularly imprinted polymers (MIPs). This work describes the development of MIPs, based on the epitope approach, synthesized from the tetrapeptide DYKD as template that affords purification of FLAG-derived recombinant proteins. Polymer was optimized by using a combinatorial approach to select the functional monomer(s) and cross-linker(s), resulting in the best specific affinity toward FLAG and the peptide DYKD. The imprinted resin obtained was used to purify mCherry proteins tagged with either FLAG or DYKD epitopes from crude cell lysates. Both mCherry variants were highly efficiently purified ( R ≥ 95%, RSD ≤ 15%, n = 3) and impurities were removed. Unlike existing antibody-based resins, the proposed tag-imprinting strategy provides a general method for meeting the growing demand for efficient, inexpensive, and versatile materials for tagged proteins purification.

The 2018 Nobel Prize in Chemistry: phage display of peptides and antibodies.

One-half of the 2018 Nobel Prize in Chemistry was awarded jointly to George P. Smith and Sir Gregory P. Winter "for the phage display of peptides and antibodies". This feature article summarizes significant achievements leading to the development of phage display of peptides and antibodies, where a bacteriophage is genetically modified to display peptides and proteins, with the primary aim of producing new biopharmaceuticals. These significant achievements are proven to be useful for the development of phage-based bioassays and biosensors.

Development and comparison of mimotope-based immunoassays for the analysis of fumonisin B1.

Mycotoxins can be found as natural contaminants in many foods and feeds, and owing to their toxic effects, it is essential to detect them before they enter the food chain. An interesting approach for the analysis of mycotoxins by competitive immunoassays is the use of epitope-mimicking peptides, or mimotopes, which can replace the toxin conjugates traditionally used in such assays. Mimotopes can be selected from phage-displayed peptide libraries even without any prior knowledge of the antibody-antigen interaction, and after identifying the target specific clones, individual clones can be efficiently amplified in bacteria and used directly in the immunoassay. Following such approach, we have previously selected and identified a dodecapeptide which functions as a mimotope for the mycotoxin fumonisin B1. In this work, we present the development and comparison of various immunoassays based on this mimotope, named A2, which has been used in the phage-displayed format in which it was selected, but also as a fluorescent recombinant fusion protein or as a synthetic peptide. The highest sensitivity was obtained with a magnetic bead-based assay using the synthetic peptide and enzymatic detection which provided a detection limit of 0.029 ng mL-1. Analysis of the binding kinetics by surface plasmon resonance (SPR) further reinforced the suitability of the synthetic peptide for the competitive immunoassays, as this mimotope showed a slightly lower affinity for the target antibody in comparison with the recombinant fusion protein. Graphical abstract.

Sensitive Rapid Fluorescence Polarization Immunoassay for Free Mycophenolic Acid Determination in Human Serum and Plasma.

In this Article, we describe a fluorescence polarization immunoassay (FPIA) using a new label-near-infrared fluorescent dye. The developed FPIA method was optimized for the rapid analysis of free mycophenolic acid (MPA) in plasma of transplanted patients. The approach is based on the fluorescence competitive assay between the target immunosuppressant and a novel emissive near-infrared fluorescent dye-tagged MPA and MPA-AO for the binding sites of the anti-MPA antibody. The fluorescent analogue of MPA exhibits emission at 654 nm upon excitation at 629 nm (λexcmax) and shows a good photochemical stability and a significant emission quantum yield (0.16) in phosphate buffer media. Free mycophenolic acid was isolated from blood or plasma samples using ultrafiltration prior to analysis. The sample was incubated for 20 min with 5 µg/mL of anti-MPA antibody and 1 nM of MPA-AO before the measurements. The developed FPIA displays a limit of detection of 0.8 ng/mL (10% binding inhibition) and a dynamic range of 1.7-39 ng/mL (20%-80% binding inhibition) in a PBST buffer, fitting the therapeutic requirements. The immunoassay selectivity was evaluated by measuring the cross-reactivity to other immunosuppressive drugs administered in combination with MPA (cyclosporin A and tacrolimus), as well as for the metabolite MPA glucuronide. The assay has been successfully applied to the analysis of free MPA in the blood of a heart-transplanted patient after oral administration of both mycophenolate mofetil (MMF) and tacrolimus, and the results have been compared with those obtained by rapid-resolution liquid chromatography with diode array detection (RRLC-DAD).

Highly Fluorescent Magnetic Nanobeads with a Remarkable Stokes Shift as Labels for Enhanced Detection in Immunoassays.

Fluorescence immunoassays are popular for achieving high sensitivity, but they display limitations in biological samples due to strong absorption of light, background fluorescence from matrix components, or light scattering by the biomacromolecules. A powerful strategy to overcome these problems is introduced here by using fluorescent magnetic nanobeads doped with two boron-dipyrromethane dyes displaying intense emission in the visible and near-infrared regions, respectively. Careful matching of the emission and absorption features of the dopants leads to a virtual Stokes shift larger than 150 nm achieved by an intraparticle Förster resonance energy transfer (FRET) process between the donor and the acceptor dyes. Additionally, the magnetic properties of the fluorescent beads allow preconcentration of the sample. To illustrate the usefulness of this approach to increase the sensitivity of fluorescence immunoassays, the novel nanoparticles are employed as labels for quantification of the widely used Tacrolimus (FK506) immunosuppressive drug. The FRET-based competitive inhibition immunoassay yields a limit of detection (LOD) of 0.08 ng mL-1 , with a dynamic range (DR) of 0.15-2.0 ng mL-1 , compared to a LOD of 2.7 ng mL-1 and a DR between 4.1 and 130 ng mL-1 for the immunoassay carried out with direct excitation of the acceptor dye.

Bioinspired recognition elements for mycotoxin sensors.

Mycotoxins are low molecular weight molecules produced as secondary metabolites by filamentous fungi that can be found as natural contaminants in many foods and feeds. These toxins have been shown to have adverse effects on both human and animal health, and are the cause of significant economic losses worldwide. Sensors for mycotoxin analysis have traditionally applied elements of biological origin for the selective recognition purposes. However, since the 1970s there has been an exponential growth in the use of genetically engineered or synthetic biomimetic recognition elements that allow some of the limitations associated with the use of natural receptors for the analyses of these toxins to be circumvented. This review provides an overview of recent advances in the application of bioinspired recognition elements, including recombinant antibodies, peptides, aptamers, and molecularly imprinted polymers, to the development of sensors for mycotoxins based on different transduction elements. Graphical abstract Novel analytical methods based on bioinspired recognition elements, such as recombinant antibodies, peptides, aptamers, and molecularly imprinted polymers, can improve the detection of mycotoxins and provide better tools than their natural counterparts to ensure food safety.

Optical Biosensors for Label-Free Detection of Small Molecules.

Label-free optical biosensors are an intriguing option for the analyses of many analytes, as they offer several advantages such as high sensitivity, direct and real-time measurement in addition to multiplexing capabilities. However, development of label-free optical biosensors for small molecules can be challenging as most of them are not naturally chromogenic or fluorescent, and in some cases, the sensor response is related to the size of the analyte. To overcome some of the limitations associated with the analysis of biologically, pharmacologically, or environmentally relevant compounds of low molecular weight, recent advances in the field have improved the detection of these analytes using outstanding methodology, instrumentation, recognition elements, or immobilization strategies. In this review, we aim to introduce some of the latest developments in the field of label-free optical biosensors with the focus on applications with novel innovations to overcome the challenges related to small molecule detection. Optical label-free methods with different transduction schemes, including evanescent wave and optical fiber sensors, surface plasmon resonance, surface-enhanced Raman spectroscopy, and interferometry, using various biorecognition elements, such as antibodies, aptamers, enzymes, and bioinspired molecularly imprinted polymers, are reviewed.

Biosensing Based on Nanoparticles for Food Allergens Detection.

Food allergy is one of the major health threats for sensitized individuals all over the world and, over the years, the food industry has made significant efforts and investments to offer safe foods for allergic consumers. The analysis of the concentration of food allergen residues in processing equipment, in raw materials or in the final product, provides analytical information that can be used for risk assessment as well as to ensure that food-allergic consumers get accurate and useful information to make their food choices and purchasing decisions. The development of biosensors based on nanomaterials for applications in food analysis is a challenging area of growing interest in the last years. Research in this field requires the combined efforts of experts in very different areas including food chemistry, biotechnology or materials science. However, the outcome of such collaboration can be of significant impact on the food industry as well as for consumer’s safety. These nanobiosensing devices allow the rapid, selective, sensitive, cost-effective and, in some cases, in-field, online and real-time detection of a wide range of compounds, even in complex matrices. Moreover, they can also enable the design of novel allergen detection strategies. Herein we review the main advances in the use of nanoparticles for the development of biosensors and bioassays for allergen detection, in food samples, over the past few years. Research in this area is still in its infancy in comparison, for instance, to the application of nanobiosensors for clinical analysis. However, it will be of interest for the development of new technologies that reduce the gap between laboratory research and industrial applications.

Microarray-Based Immunoassay with Synthetic Mimotopes for the Detection of Fumonisin B1.

Mimotopes, or epitope mimics, can be applied to competitive immunoassays, for the detection of low molecular weight natural toxicants, as an alternative to toxin-conjugates. In this work, we report the development of a microarray-based immunoassay for the detection of fumonisin B1 using a novel mimotope selected by phage display technology. Fumonisin-specific antibody was used to isolate mimotopes from a 12-mer peptide library in successive selection rounds. Enrichment of antibody binding phages was observed after three panning rounds, and sequence analysis of randomly selected monoclonal phages revealed two conserved peptide sequences. Clone A2, with peptide sequence VTPNDDTFDPFR, showed the best response in enzyme-linked immunosorbent assay (ELISA) in terms of sensitivity and reproducibility and was selected for microarray development. A biotinylated synthetic derivative of this mimotope was immobilized onto epoxy-glass slides, and fumonisin B1 was detected in a competitive binding inhibition assay using the antifumonisin antibody and a labeled secondary antibody. The array showed an IC50 value of 37.1 ± 2.4 ng mL-1 (n = 9), a detection limit of 11.1 ng mL-1, and a dynamic range from 17.3 to 79.6 ng mL-1. Good specificity toward fumonisin B1 and its structural analog, fumonisin B2, was observed, together with negligible cross-reactivity for other mycotoxins produced by the same fungi species. The mimotope microarray was applied to the analysis of fumonisin B1 in spiked maize and wheat samples. The method enabled quantification of the mycotoxin at the levels set by European legislation and holds promise for future adaptation to include other mycotoxins for multiplex detection.