Non-invasive iron deficiency diagnosis: a saliva-based approach using capillary flow microfluidics.

Iron deficiency anemia (IDA) is a condition characterized by lower-than-average iron (Fe) levels in the body, affecting a substantial number of young children and pregnant women globally. Existing diagnostic methods for IDA rely on invasive analysis of stored Fe in ferritin from blood samples, posing challenges, especially for toddlers and young children. To address this issue, saliva has been proposed as a non-invasive sample matrix for IDA diagnosis. However, conventional Fe analysis techniques often necessitate complex and costly instrumentation. This study presents the first non-invasive, saliva-based preliminary screening test for IDA using a nitrocellulose lateral flow system. In this study, we introduce a novel approach using the ferroin reaction with bathophenanthroline (Bphen) and ferrous (Fe2+) ions to quantify Fe levels in saliva. Our methodology involves a capillary flow-driven microfluidic device integrated into a lateral flow system utilizing nitrocellulose membranes. Here, we present the first instance of saliva on a nitrocellulose substrate to detect salivary Fe levels. The optimized system yielded a linear response over the 1-200 ppm range in buffer solution, with a limit of detection (LoD) of 5.6 ppm. Furthermore, the system demonstrated a linear response in pooled saliva samples across the 1-1000 ppm range, with a LoD of 55.1 ppm. These results underscore the potential of our capillary flow-driven microfluidic device as a viable non-invasive diagnostic tool for IDA, particularly in remote and resource-limited settings.

Office paper and laser printing: a versatile and affordable approach for fabricating paper-based analytical devices with multimodal detection capabilities.

Multiple protocols have been reported to fabricate paper-based analytical devices (PADs). However, some of these techniques must be revised because of the instrumentation required. This paper describes a versatile and globally affordable method to fabricate PADs using office paper as a substrate and a laser printing technique to define hydrophobic barriers on paper surfaces. To demonstrate the feasibility of the alternatives proposed in this study, the fabrication of devices for three types of detection commonly associated with using PADs was demonstrated: colorimetric detection, electrochemical detection, and mass spectrometry associated with a paper-spray ionization (PSI-MS) technique. Besides that, an evaluation of the type of paper used and chemical modifications required on the substrate surface are also presented in this report. Overall, the developed protocol was suitable for using office paper as a substrate, and the laser printing technique as an efficient fabrication method when using this substrate is accessible at a resource-limited point-of-need. Target analytes were used as a proof of concept for these detection techniques. Colorimetric detection was carried out for acetaminophen, iron, nitrate, and nitrite with limits of detection of 0.04 µg, 4.5 mg mL-1, 2.7 µmol L-1, and 6.8 µmol L-1, respectively. A limit of detection of 0.048 fg mL-1 was obtained for the electrochemical analysis of prostate-specific antigen. Colorimetric and electrochemical devices revealed satisfactory performance when office paper with a grammage of 90 g m-2 was employed. Methyldopa analysis was also carried out using PSI-MS, which showed a good response in the same paper weight and behavior compared to chromatographic paper.

Direct immunoassay on a polyester microwell plate for colorimetric detection of the spike protein in swab and saliva samples.

This study presents the development of a polyester microplate for detecting the S-protein of the SARS-CoV-2 virus in saliva and nasopharyngeal swab samples using direct enzyme-linked immunosorbent assay (ELISA) technology. The polyester microplate was designed to contain 96 zones with a 3 mm diameter each, and a volume of 2-3 µL. The experimental conditions including reagent concentration and reaction time were optimized. The microplate image was digitized and analyzed using graphical software. The linear range obtained between protein S concentrations and pixel intensity was 0-10 µg mL-1, with a correlation coefficient of 0.99 and a limit of detection of 0.44 µg mL-1. The developed methodology showed satisfactory intraplate and interplate repeatability with RSD values lower than 7.8%. The results achieved through immunoassay performed on polyester microplates were consistent with those of the RT-PCR method and showed a sensitivity of 100% and 90% and specificity of 85.71% and 100% for saliva and nasopharyngeal samples, respectively. The proposed direct immunoassay on polyester microplates emerges as an alternative to conventional immunoassays performed on commercial polystyrene plates, given the low cost of the device, low consumption of samples and reagents, lower waste generation, and shorter analysis time. Moreover, the immunoassay has shown great potential for diagnosing COVID-19 with precision and accuracy.

Voltammetric Determination of 3-Methylmorphine Using Glassy Carbon Electrode Modified with rGO and Bismuth Film.

This work reports the development and application of a simple, rapid and low-cost voltammetric method for the determination of 3-methylmorphine at nanomolar levels in clinical and environmental samples. The proposed method involves the combined application of a glassy carbon electrode modified with reduced graphene oxide, chitosan and bismuth film (Bi-rGO-CTS/GCE) via square-wave voltammetry using 0.04 mol L-1 Britton-Robinson buffer solution (pH 4.0). The application of the technique yielded low limit of detection of 24 × 10-9 mol L-1 and linear concentration range of 2.5 × 10-7 to 8.2 × 10-6 mol L-1. The Bi-rGO-CTS/GCE sensor was successfully applied for the detection of 3-methylmorphine in the presence of other compounds, including paracetamol and caffeine. The results obtained also showed that the application of the sensor for 3-methylmorphine detection did not experience any significant interference in the presence of silicon dioxide, povidone, cellulose, magnesium stearate, urea, ascorbic acid, humic acid and croscarmellose. The applicability of the Bi-rGO-CTS/GCE sensor for the detection of 3-methylmorphine was evaluated using synthetic urine, serum, and river water samples through addition and recovery tests, and the results obtained were found to be similar to those obtained for the high-performance liquid chromatography method (HPLC)-used as a reference method. The findings of this study show that the proposed voltammetric method is a simple, fast and highly efficient alternative technique for the detection of 3-methylmorphine in both biological and environmental samples.

Prostate Cancer Diagnosis in the Clinic Using an 8-Protein Biomarker Panel.

The inability to distinguish aggressive from indolent prostate cancer is a longstanding clinical problem. Prostate specific antigen (PSA) tests and digital rectal exams cannot differentiate these forms. Because only ∼10% of diagnosed prostate cancer cases are aggressive, existing practice often results in overtreatment including unnecessary surgeries that degrade patients' quality of life. Here, we describe a fast microfluidic immunoarray optimized to determine 8-proteins simultaneously in 5 µL of blood serum for prostate cancer diagnostics. Using polymeric horseradish peroxidase (poly-HRP, 400 HRPs) labels to provide large signal amplification and limits of detection in the sub-fg mL-1 range, a protocol was devised for the optimization of the fast, accurate assays of 100-fold diluted serum samples. Analysis of 130 prostate cancer patient serum samples revealed that some members of the protein panel can distinguish aggressive from indolent cancers. Logistic regression was used to identify a subset of the panel, combining biomarker proteins ETS-related gene protein (ERG), insulin-like growth factor-1 (IGF-1), pigment epithelial-derived factor (PEDF), and serum monocyte differentiation antigen (CD-14) to predict whether a given patient should be referred for biopsy, which gave a much better predictive accuracy than PSA alone. This represents the first prostate cancer blood test that can predict which patients will have a high biopsy Gleason score, a standard pathology score used to grade tumors.

Use of data processing for rapid detection of the prostate-specific antigen biomarker using immunomagnetic sandwich-type sensors.

Diagnosis of cancer using electroanalytical methods can be achieved at low cost and in rapid assays, but this may require the combination with data treatment for determining biomarkers in real samples. In this paper, we report an immunomagnetic nanoparticle-based microfluidic sensor (INµ-SPCE) for the amperometric detection of the prostate-specific antigen (PSA) biomarker, the data of which were treated with information visualization methods. The INµ-SPCE consists of eight working electrodes, reference and counter electrodes. On the working electrodes, magnetic nanoparticles with secondary antibodies with the enzyme horseradish peroxidase were immobilized for the indirect detection of PSA in a sandwich-type procedure. Under optimal conditions, the immunosensor could operate within a wide range from 12.5 to 1111 fg·L-1, with a low detection limit of 0.062 fg·L-1. Multidimensional projections combined with feature selection allowed for the distinction of cell lysates with different levels of PSA, in agreement with results from the traditional enzyme-linked immunosorbent assay. The approaches for immunoassays and data processing are generic, and therefore the strategies described here may provide a simple platform for clinical diagnosis of cancers and other types of diseases.

Ultrasensitive immunoassay for detection of Citrus tristeza virus in citrus sample using disposable microfluidic electrochemical device.

Tristeza is a disease that affects citrus crops in general, caused by the Citrus tristeza virus (CTV). It is considered an economically important virus diseases in citrus, which is present in the main citrus producing regions all around the world. Early detection of CTV is crucial to avoid any epidemics and substantial economic losses for the citrus growers. Consequently, the development of rapid, accurate, and sensitive methods capable of detecting the virus in the early stages of the disease is highly desired. Based on that, a low-cost and rapid magneto-immunoassay methodology to detect the capsid protein from CTV (CP-CTV) was proposed. For this, magnetic beads were decorated with antibodies anti-CP-CTV and horseradish peroxidase enzyme (HRP) and applied for the capture and separation of CP-CTV from the sample solutions. The magnetically captured biomarker was detected using a simple disposable microfluidic electrochemical device (DµFED) constructed by rapid prototyping technique and composed by an array of immunosensors. In DµFED, the electrodes were modified with monoclonal antibody anti-CP-CTV and the detection was carried out using amperometry, based on the hydroquinone/H2O2 catalytic redox reaction due to the presence of HRP label in an immune-sandwich structure. The proposed immunoassay presented excellent linearity with a wide linear range of concentration of 1.95-10.0 × 103 fg mL-1 and ultralow detection limit of 0.3 fg mL-1. The disposable device was successfully applied for the detection of Citrus tristeza virus in healthy and infected plant samples, where it showed good agreements with the comparative method of enzyme-linked immunosorbent assay (ELISA). The developed immunoassay methodology showed a sensitive and selective way in the detection of CTV. Hence, it can be considered as a promising analytical alternative for rapid and low-cost diagnosis of Tristeza disease in citrus.

Novel enzyme-free immunomagnetic microfluidic device based on Co0.25Zn0.75Fe2O4 for cancer biomarker detection.

Early diagnosis of cancer by biomarker detection has been widely studied since it can lead to an increase in patient survival rates. Magnetic nanoparticles (MNPs) play an important role in this field acting as a valuable tool in the biomarker immunocapture and detection. In this work, Co0.25Zn0.75Fe2O4 (CoZnFeONPs) nanoparticles were synthesized and applied as enzyme mimics of peroxidase-like catalysis in a disposable enzyme-free microfluidic immunoarray device (µID). The catalytic activity of CoZnFeONPs was evaluated by hydrogen peroxide detection using cyclic voltammetry and the apparent Michaelis-Menten constant was estimated by Lineweaver-Burk equation showing good Km values. In µID, the immunosensors were assembled with monoclonal antibody against CYFRA 21-1 covalently immobilized on graphene oxide previously deposited on the screen-printed carbon-based electrodes. Under optimized conditions, the method presented a good linear response for CYFRA 21-1 in the range of 3.9-1000 fg mL-1 achieving an ultralow limit of detection (LOD) of 0.19 fg mL-1. For comparison, Fe3O4 nanoparticles (FeONPs) was also synthetized and presented results slight inferior to that obtained with CoZnFeONPs. The methods developed using both MNPs exhibited countless advantages when compared with the immunosensors developed for CYFRA-21-1, previously reported in the literature. The methods were successful applied for the detection of CYFRA 21-1 in real serum samples of healthy and prostate cancer patients and showed good correlation with results obtained with the enzyme-linked immunosorbent assay (ELISA). The CoZnFeONPs associated with the disposable microfluidic immunoarray device provides a simple and effective method for biomarker detection that could satisfy the need for a low-cost and rapid test for early diagnosis of cancer.

Disposable inkjet-printed electrochemical platform for detection of clinically relevant HER-2 breast cancer biomarker.

Rapidly fabricated, disposable sensor platforms hold tremendous promise for point-of-care detection. Here, we present an inexpensive (< $0.25) fully inkjet printed electrochemical sensor with integrated counter, reference, and working electrodes that is easily scalable for commercial fabrication. The electrochemical sensor platform featured an inkjet printed gold working 8-electrode array (WEA) and counter electrode (CE), along with an inkjet -printed silver electrode that was chlorinated with bleach to produce a Ag/AgCl quasi-reference electrode (RE). As proof of concept, the electrochemical sensor was successfully applied for detection of clinically relevant breast cancer biomarker Human Epidermal Growth Factor Receptor 2 (HER-2). Capture antibodies were bound to a chemically modified surface on the WEA and placed into a microfluidic device. A full sandwich immunoassay was constructed following a simultaneous injection of target protein, biotinylated antibody, and polymerized horseradish peroxide labels into the microfluidic device housing the WEA. With an ultra fast assay time, of only 15mins a clinically relevant limit of detection of 12pgmL-1 was achieved. Excellent reproducibility and sensitivity were observed through recovery assays preformed in human serum with recoveries ranging from 76% to 103%. These easily fabricated and scalable electrochemical sensor platforms can be readily adapted for multiplex detection following this rapid assay protocol for cancer diagnostics.