Diagnosis of viral infectious diseases through sensitive detection of human serum antibodies using a modified label-free electrochemical biosensor.

Rapid virus identification is crucial for preventing outbreaks. The COVID-19 pandemic has highlighted the critical nature of rapid virus detection. Here, we designed a label-free electrochemical biosensor modified with gold nanoparticles (AuNPs) to detect IgG antibodies from human serum, enabling rapid point-of-care diagnostics. AuNPs were synthesized and characterized. A multivariate optimization was carried out to determine the optimal condition for functionalizing AuNPs with anti-IgG. Subsequently, using a glassy carbon electrode (GCE), a modified AuNPs/GCE electrochemical biosensor was developed for IgG detection. The results indicated that AuNPs displayed a spherical morphology with a size distribution of 19.54 nm. Additionally, the zeta potential was recorded at -7.84 mV. Central composite design (CCD) analysis determined the optimal conditions for functionalizing AuNPs to be an anti-IgG concentration of 320 µg mL-1, a temperature of 25 °C, and pH of 7.4. The characterization study confirmed the successful synthesis and functionalization of AuNPs. Through electrochemical impedance spectroscopy measurement, the biosensor demonstrated a limit of detection (LOD) of 0.2 ng mL-1 and limit of quantification (LOQ) of 0.8 ng mL-1. Furthermore, tests in real samples showed the interaction between IgG antibodies in serum samples and AuNPs/GCE, confirming the biosensor's ability to detect and quantify IgG in clinical samples.

Understanding the relationship between viral infections and trace elements from a metallomics perspective: implications for COVID-19.

Recently, the World Health Organization (WHO) declared a pandemic situation due to a new viral infection (COVID-19) caused by a novel virus (Sars-CoV-2). COVID-19 is today the leading cause of death from viral infections in the world. It is known that many elements play important roles in viral infections, both in virus survival, and in the activation of the host's immune system, which depends on the presence of micronutrients to maintain the integrity of its functions. In this sense, the metallome can be an important object of study for understanding viral infections. Therefore, this work presents an overview of the role of trace elements in the immune system and the state of the art in metallomics, highlighting the challenges found in studies focusing on viral infections.

A feasible strategy based on high ultrasound frequency and mass spectrometry for discriminating individuals diagnosed with bipolar disorder and schizophrenia through ionomic profile.

RATIONALE: A viable and accurate method based on high-power ultrasound-assisted microextraction and inductively coupled plasma mass spectrometry was developed to determine metals in human serum from patients with bipolar disorder and schizophrenia. METHODS: A simple and rapid sample preparation method using a cup-horn sonoreactor was developed. The acid concentration of HNO3 (10, 20, and 40% v/v) and HCl (1, 5, 15, and 30% v/v) of the extraction solution, the sonication time (1, 3, 6, and 10 min), and the sonication amplitude (20, 40, 60, and 80%) were evaluated. Cd, Cu, Fe, Li, Pb, and Zn were determined in serum samples from patients with bipolar disorder and schizophrenia, and from healthy controls. Quantitative metal recoveries using the proposed method were compared under the same conditions using an ultrasonic bath, magnetic stirring, and microwave-assisted digestion. RESULTS: Optimum extraction conditions were obtained using HNO3 (40% v/v) + HCl (30% v/v) as the extraction solution with 3 min sonication time and 60% sonication amplitude. Significant differences were observed among the methods compared. On application of the sample preparation method based on high-power ultrasound-assisted microextraction coupled with inductively coupled plasma mass spectrometry, Pb and Cd in all the studied samples were below the limit of detection of our method. Compared with healthy controls, the concentration of Cu, Li, Fe, and Zn was found to be significantly higher for the bipolar disorder group, while these metals and Li were found at a lower level for the group diagnosed with schizophrenia. CONCLUSIONS: Principal component analysis showed a significant separation for the groups studied based on their ionomic profiles after the application of high-power ultrasound-assisted microextraction as a sample preparation strategy.

Metallomics-based platforms for comparing the human blood serum profiles between bipolar disorder and schizophrenia patients.

RATIONALE: An evaluation of bipolar disorder (BD) and schizophrenia (SCZ) was carried out, from a metallomics point of view, using native conditions, attempting to preserve the interaction between metals and biomolecules. METHOD: For this task, blood serum samples from healthy individuals and patients were compared. In addition, the profiles of metal ions and metalloids involved in the pathologies were quantified, and a comparison was carried out of the protein profile in serum samples of healthy individuals and diseased patients. RESULTS: After optimization and accuracy evaluation of the method, different concentrations of Li, Mg, Mn and Zn were observed in the samples of BD patients and high levels of copper for SCZ patients, indicating an imbalance in the homeostasis of important micronutrients. The treatment, especially with lithium, may be related to competition between metallic ions. BD-related metallobiomolecules were detected, preserving the binding between metal ions and biomolecules, with four fractions detected in the ultraviolet range (280 nm). Four fractions were collected by high-performance liquid chromatography/inductively coupled plasma mass spectrometry (HPLC/ICP-MS) and the proteins were identified by liquid chromatography/tandem mass spectrometry (LC/MS/MS). The Ig lambda chain V-IV region Hil, immunoglobulin heavy constant gama 1 (IGHG1) and beta-2-glycoprotein 1 (or ApoH) was identified in SCZ samples, suggesting its relationship with mood disorders. Surprisingly, Protein IGKV2D-28 was identified only in BD samples, opening up new possibilities for studies regarding the role of this protein in BD. CONCLUSIONS: This approach brings new perspectives to the comprehension of mood disorders, highlighting the importance of metallomics science in disease development. This strategy showed an innovative potential for evaluating mood disorders at the proteomic level, making it possible to identify proteins related to mood disorders and BD.

Optimization of a Methodology for Quantification and Removal of Zinc Gives Insights Into the Effect of This Metal on the Stability and Function of the Zinc-Binding Co-chaperone Ydj1.

Ydj1, a class B J-protein (Hsp40) in yeast, has two zinc finger domains in each monomer and belongs to an important co-chaperone family that plays crucial roles in cells, such as recognizing and binding partially folded proteins and assisting the Hsp70 chaperone family in protein folding. Yeast cells with ydj1 deletion were less efficient at coping with zinc stress than wild-type cells, and site-directed mutagenesis studies that impair or delete the zinc finger region have confirmed the importance of this region to the function of Ydj1; however, little is known about whether the presence of zinc is critical for the function of the protein. To gain insights into the effect of zinc on the structure and function of Ydj1 without having to modify its primary structure, a method was developed and optimized to quantify and remove the zinc from the protein. Recombinant Ydj1 was produced and purified, and its zinc content was determined by ICP-MS. The result showed that two zinc atoms were bound per monomer of protein, a good indicator that all sites were saturated. To optimize the removal of the bound zinc, variations on chelating agent (EDTA, EGTA, 1,10-phenanthroline), chelator concentration, reaction time, pH, and temperature were tested. These procedures had no effect on the overall secondary structure of the protein, since no significant changes in the circular dichroism spectrum were observed. The most significant removal (91 ± 2%, n = 3) of zinc was achieved using 1,10-phenanthroline (1 × 10-3 mol L-1) at 37°C with a pH 8.5 for 24 h. Zinc removal affected the stability of the protein, as observed by a thermal-induced unfolding assay showing that the temperature at the middle of the transition (Tm) decreased from 63 ± 1°C to 60 ± 1°C after Zn extraction. In addition, the effect on the ability of Ydj1 to protect a model protein (luciferase) against aggregation was completely abolished after the Zn removal procedure. The main conclusion is that zinc plays an important role in the stability and activity of Ydj1. Additionally, the results highlight the medical importance of chaperones, as altered zinc homeostasis is implicated in many diseases, such as neurodegenerative disorders.

Sample Preparation Focusing on Plant Omics.

Because of strong impact of omics in many fields, and the complexity of the samples when focusing on areas such as genomics, (metallo)proteomics, metabolomics, among others, it is easy to rationalize the great importance that sample preparation has for achieving reliable results, mainly considering plant science. Then, this chapter points out applications of the sample preparation focusing on such areas, and a diversity of strategies, techniques, and procedures is highlighted and commented.

Quantifying proteins at microgram levels integrating gel electrophoresis and smartphone technology.

A combination between gel electrophoresis and smartphone technology is applied for quantifying proteins in a serum sample. Electrophoresis not only allows one to separate different proteins but also to build-up a calibration curve for those proteins. To expand its applicability, a smartphone allows one to capture a gel image, through its camera. Additionally, the treatment of the data extracted through each protein band is performed using a freely available program (PhotoMetrix), which is downloaded to one's own smartphone. Through this approach, the quantification of proteins such as albumin, immunoglobulin, and carbonic anhydrase is performed in a serum sample by acquiring images using 32 × 32 pixels for each image in the region of the protein bands. An LOQ from 1.9 to 2.4 µg and r2 > 0.980 may be obtained. Comparing results through the analyses of the gels using Image Master 2D Platinum 6.0 software reflects good agreement (95% confidence level) between the results. SIGNIFICANCE STATEMENT: In many biological studies involving structural analysis or biophysical and biochemical characterization after purification process, or even in assays of protein:protein interaction, the estimation of protein quantity and protein purity is a fundamental step. However, even although many methodologies are proposed in the literature for quantifying proteins, they present some limitations, once are frequently expensive and require solution sample. In addition, they usually do not quantify the specific active concentration, but the total amount. Thus, it is necessary to develop an easy, fast and low-cost method that allows to quantify and to evaluate protein purity of reactional system, for example, a protein:protein interaction. In this way, we presented a simple strategy based on the integration of Smartphone technology and gel electrophoresis, where SDS-PAGE provides multiple information regarding the quality of the protein, such as the presence of degradation products, as well as if there was interaction reaction between proteins. Then, the smartphone detects the proteins in the SDS-PAGE gel, allowing to evaluate purity degree and the quantity, at microgram level, of the protein. We believe that the combination of these features may help to increase the productivity and accuracy of gel electrophoresis analysis, and the results obtained through a smartphone, easily achieved in our pockets.

Chemical Speciation and Metallomics.

Chemical speciation approaches is an inherent part of metallomics, once metals/metalloids and organic structures need to be currently evaluated for attaining metallomics studies. Then, this chapter focuses on the applications of the chemical speciation applied to the human health risk, food and human diet, drugs, forensic, nanoscience, and geological metallomics, also pointing out the advances in such area. Some aspects regarding sample preparation is commented along this chapter, and some strategies for maintaining the integrity of the metallomics information are also emphasized.

Simplifying the human serum proteome for discriminating patients with bipolar disorder of other psychiatry conditions.

PURPOSE: An exploratory analysis using proteomic strategies in blood serum of patients with bipolar disorder (BD), and with other psychiatric conditions such as Schizophrenia (SCZ), can provide a better understanding of this disorder, as well as their discrimination based on their proteomic profile. METHODS: The proteomic profile of blood serum samples obtained from patients with BD using lithium or other drugs (N=14), healthy controls, including non-family (HCNF; N=3) and family (HCF; N=9), patients with schizophrenia (SCZ; N=23), and patients using lithium for other psychiatric conditions (OD; N=4) were compared. Four methods for simplifying the serum samples proteome were evaluated for both removing the most abundant proteins and for enriching those of lower-abundance: protein depletion with acetonitrile (ACN), dithiothreitol (DTT), sequential depletion using DTT and ACN, and protein equalization using commercial ProteoMiner® kit (PM). For proteomic evaluation, 2-D DIGE and nanoLC-MS/MS analysis were employed. RESULTS: PM method was the best strategy for removing proteins of high abundance. Through 2-D DIGE gel image comparison, 37 protein spots were found differentially abundant (p<0.05, Student's t-test), which exhibited ≥2.0-fold change of the average value of normalized spot intensities in the serum of SCZ, BD and OD patients compared to subject controls (HCF and HCNF). From these spots detected, 13 different proteins were identified: ApoA1, ApoE, ApoC3, ApoA4, Samp, SerpinA1, TTR, IgK, Alb, VTN, TR, C4A and C4B. CONCLUSIONS: Proteomic analysis allowed the discrimination of patients with BD from patients with other mental disorders, such as SCZ. The findings in this exploratory study may also contribute for better understanding the pathophysiology of these disorders and finding potential serum biomarkers for these conditions.

Evaluation of a novel metal-organic framework as an adsorbent for the extraction of multiclass pesticides from coconut palm (Cocos nucifera L.): An analytical approach using matrix solid-phase dispersion and liquid chromatography.

We report the synthesis, characterization, and application of [Zn(1,4-benzenedicarboxylate)(H2 O)2 ]n , Zn(1,4-benzenedicarboxylate)0.99 (NH2 -1,4-benzenedicarboxylate)0.01 (H2 O)2 ]n , [Zn(1,4-benzenedicarboxylate)0.95 (NH2 -1,4-benzenedicarboxylate)0.05 (H2 O)2 ]n , and [Zn(1,4-benzenedicarboxylate)0.9 (NH2 -1,4-benzenedicarboxylate)0.1 (H2 O)2 ]n as sorbents for the extraction of multiclass pesticides from coconut palm. Liquid chromatography with ultraviolet diode array detection was used as the analysis technique, and the experiments were performed at one fortification level (0.1 µg/g). The recoveries were 47-67, 51-70, 58-72, and 64-76% for [Zn(1,4-benzenedicarboxylate)(H2 O)2 ]n , Zn(1,4-benzenedicarboxylate)0.99 (NH2 -1,4-benzenedicarboxylate)0.01 (H2 O)2 ]n , [Zn(1,4-benzenedicarboxylate)0.95 (NH2 -1,4-benzenedicarboxylate)0.05 (H2 O)2 ]n , and [Zn(1,4-benzenelate)0.95 (NH2 -1,4-benzenedicarboxylate)0.05 (H2 O)2 ]n , and [Zn(1,4-benzenedicarboxylate)0.9 (NH2 -1,4-benzenedicarboxylate)0.1 (H2 O)2 ]n , respectively, with relative standard deviation ranging from 1 to 7% (n = 3). Detection and quantification limits were 0.01-0.05 and 0.05-0.2 µg/g, respectively, for the different pesticides studied. The method developed was linear over the range tested (0.01-10.0 µg/g) with r2  > 0.9991. A direct comparison of [Zn(1,4-benzenedicarboxylate)0.9 (NH2 -1,4-benzenedicarboxylate)0.1 (H2 O)2 ]n with the commercially available neutral alumina showed that [Zn(1,4-benzenedicarboxylate)0.9 (NH2 -1,4-benzenedicarboxylate)0.1 (H2 O)2 ]n was a similar extracting phase for the pesticides investigated.

Depleting high-abundant and enriching low-abundant proteins in human serum: An evaluation of sample preparation methods using magnetic nanoparticle, chemical depletion and immunoaffinity techniques.

The efficiency of three different depletion methods to remove the most abundant proteins, enriching those human serum proteins with low abundance is checked to make more efficient the search and discovery of biomarkers. These methods utilize magnetic nanoparticles (MNPs), chemical reagents (sequential application of dithiothreitol and acetonitrile, DTT/ACN), and commercial apparatus based on immunoaffinity (ProteoMiner, PM). The comparison between methods shows significant removal of abundant protein, remaining in the supernatant at concentrations of 4.6±0.2, 3.6±0.1, and 3.3±0.2µgµL-1 (n=3) for MNPs, DTT/ACN and PM respectively, from a total protein content of 54µgµL-1. Using GeLC-MS/MS analysis, MNPs depletion shows good efficiency in removing high molecular weight proteins (>80kDa). Due to the synergic effect between the reagents DTT and ACN, DTT/ACN-based depletion offers good performance in the depletion of thiol-rich proteins, such as albumin and transferrin (DTT action), as well as of high molecular weight proteins (ACN action). Furthermore, PM equalization confirms its efficiency in concentrating low-abundant proteins, decreasing the dynamic range of protein levels in human serum. Direct comparison between the treatments reveals 72 proteins identified when using MNP depletion (43 of them exclusively by this method), but only 20 proteins using DTT/ACN (seven exclusively by this method). Additionally, after PM treatment 30 proteins were identified, seven exclusively by this method. Thus, MNPs and DTT/ACN depletion can be simple, quick, cheap, and robust alternatives for immunochemistry-based protein depletion, providing a potential strategy in the search for disease biomarkers.

Bipolar disorder: recent advances and future trends in bioanalytical developments for biomarker discovery.

In this manuscript we briefly describe bipolar disorder (a depressive and manic mental disease), its classification, its effects on the patient, which sometimes include suicidal tendencies, and the drugs used for treatment. We also address the status quo with regard to diagnosis of bipolar disorder and recent advances in bioanalytical approaches for biomarker discovery. These approaches focus on blood samples (serum and plasma) and proteins as the main biomarker targets, and use various strategies for protein depletion. Strategies include use of commercially available kits or other homemade strategies and use of classical proteomics methods for protein identification based on "bottom-up" or "top-down" approaches, which used SELDI, ESI, or MALDI as sources for mass spectrometry, and up-to-date mass analyzers, for example Orbitrap. We also discuss some future objectives for treatment of this disorder and possible directions for the correct diagnosis of this still-unclear mental illness.