Low Sample Volume Research Articles

Ultrafast Magic Angle Spinning Solid-State NMR Spectroscopy: Advances in Methodology and Applications.

Solid-state NMR spectroscopy is one of the most commonly used techniques to study the atomic-resolution structure and dynamics of various chemical, biological, material, and pharmaceutical systems spanning multiple forms, including crystalline, liquid crystalline, fibrous, and amorphous states. Despite the unique advantages of solid-state NMR spectroscopy, its poor spectral resolution and sensitivity have severely limited the scope of this technique. Fortunately, the recent developments in probe technology that mechanically rotate the sample fast (100 kHz and above) to obtain "solution-like" NMR spectra of solids with higher resolution and sensitivity have opened numerous avenues for the development of novel NMR techniques and their applications to study a plethora of solids including globular and membrane-associated proteins, self-assembled protein aggregates such as amyloid fibers, RNA, viral assemblies, polymorphic pharmaceuticals, metal-organic framework, bone materials, and inorganic materials. While the ultrafast-MAS continues to be developed, the minute sample quantity and radio frequency requirements, shorter recycle delays enabling fast data acquisition, the feasibility of employing proton detection, enhancement in proton spectral resolution and polarization transfer efficiency, and high sensitivity per unit sample are some of the remarkable benefits of the ultrafast-MAS technology as demonstrated by the reported studies in the literature. Although the very low sample volume and very high RF power could be limitations for some of the systems, the advantages have spurred solid-state NMR investigation into increasingly complex biological and material systems. As ultrafast-MAS NMR techniques are increasingly used in multidisciplinary research areas, further development of instrumentation, probes, and advanced methods are pursued in parallel to overcome the limitations and challenges for widespread applications. This review article is focused on providing timely comprehensive coverage of the major developments on instrumentation, theory, techniques, applications, limitations, and future scope of ultrafast-MAS technology.

Towards portable rapid TB biosensor: Detecting Mycobacterium tuberculosis in raw sputum samples using functionalized screen printed electrodes

Due to lack of robust, sensitive and low cost detection strategies, Tuberculosis (TB) remains a significant global health issue. WHO reports 1.5 million deaths per year, ∼80 % cases occur in low- to middle-income countries, where resource limitations complicate the diagnosis. Robust detection of TB infection is important to contain the spread and treat disease. We developed a label-free DNA biosensor based on commercially available screen printed electrodes (SPEs) (DropSens and Zensors) that can detect TB robustly, sensitively, and specifically via DNA hybridization with its IS6110 gene marker, in purified DNA and raw sputum samples. The fabricated biosensor was morphologically characterized by scanning electron microscopy coupled with energy dispersive X-ray spectroscopy. Cyclic voltammetry and Differential Pulse Voltammetry was used for electrochemical analysis of the modified electrode. The fabricated biosensor demonstrated satisfactory selectivity for Mycobacterium tuberculosis (MTB) against Salmonella typhimurium and Escherichia coli and was able to detect MTB; the limit of detection (LOD) of 1.90 nM with R2 = 0.993, when analyzed over a range of concentrations of DNA (0.5–10 nM). It is being exploited to detect target MTB from clinical samples, without DNA purification. The approach is robust, sensitive, and specific, requires low sample volume and can be extended towards portable point of care diagnosis of TB.

A paper chromatographic-based electrochemical analytical device for the separation and simultaneous detection of carbofuran and carbaryl pesticides

We developed an electrochemical paper-based device (ePAD) for the online separation and simultaneous determination of structurally similar carbofuran (CBF) and carbaryl (CBR). Our device combined paper chromatography and an electrochemical sensor as a single system for increasing the selectivity to simultaneous detection of the two pesticides. During the simultaneous detection of CBF and CBR, the separation of the two carbamate compounds depends on the differences between the movements of the substances on the chromatographic paper in the moving phase flow. The isolated pesticides were electrochemically measured using an amperometric method. Under optimal conditions, which include the effect of the mobile phase, separation channel, and the applied potential, the proposed device provides a linear range for the simultaneous determination of CBF and CBR from 0.1 to 2.0 and 0.5–7.5 mg L−1, with the limits of detection at 0.06 and 0.40 mg L−1, respectively. Furthermore, we successfully used the developed device to determine CBF and CBR in water, cucumber, and cabbage samples. The obtained results correspond to results obtained from a standard method. This developed ePAD is low-cost and uses a low sample volume (2 µL). Moreover, it is disposable, portable, and a promising tool for the on-site quantification of pesticides in actual samples.

Quantitative analysis of minor and trace elements in blood serum of dairy cattle (Bos taurus) by WDXRF


 
 
 
 Assessment of heavy metal content in biological fluids is essential to determine human and animal health as a result of exposure to contaminated environments, food, and water. WDXRF is a non-destructive method that involves no chemical treatments. It is possible to perform fast chemical analysis without producing hazardous waste and uses a small sample volume (± 50 mL), which is useful to veterinary usage. Therefore, the aim of this work was to implement an alternative method for minor and trace elements in blood serum samples of dairy cattle (Bos taurus) using WDXRF. It was implemented for the determination of Al, Cr, Co, Fe, Cu, As, and Hg, and the method was evaluated by comparing WDXRF and ICP-OES results of a set of 14 blood serum samples of dairy cattle using principal component analysis (PCA). Results of WDXRF and ICP-OES were statistically equivalent to all analyzed elements, except for Fe and Al, which were still comparable. WDXRF proved to be a suitable alternative methodology for heavy metal detection in animal blood serum samples due to the low sample volume. The results of this study enable a new and fast method for veterinary internal medicine, which requires a technique that quickly detects animals with a high concentration of toxic elements to assure the correct protocols to prevent the animal's death.
 
 
 

A Dual-Scale CSRRs-Based Sensor for Dielectric Characterization of Solid Materials

A compact planar sensor is presented in this letter, which could measure the dielectric constants of solid materials at microwave and sub-THz frequencies, simultaneously. The lower band resonator is based on a complementary split ring resonator (CSRR), fed by a coplanar waveguide line on the board operating at 4.76 GHz, and the upper band sensor is a 3 × 3 CSRR array, excited by a pair of WR-10 open-ended waveguide probes operating at 112 GHz. The upper band CSRR array is nested in the lower band CSRR resonator, but their sensing functions are independent of each other. A coincident sensing area is obtained that needs a testing sample with small volume. Dielectric films composed of polydimethylsiloxane and barium-titanate with different volume fractions are used to verify the performance of the sensor in both frequency scales. The advantages of the proposed design, including compact size, high sensitivity, low sample volume, and dual-scale operation, make it a competitive solution for detecting solid materials with similar dielectric properties in a single frequency band, for application such as the food industry, material quality control, and biomedical.

An Ultra-High-Performance Liquid Chromatography-Tandem Mass Spectrometry Method for Simultaneous Determination of 4 β-Lactam Antibiotics, Tazobactam, and Linezolid in Human Plasma Samples.

Optimization of antimicrobial therapy is a challenge in critically ill patients who develop extreme interindividual and intraindividual pharmacokinetic variability. Therapeutic drug monitoring is a valuable tool for maximizing the effect of a drug and minimizing its adverse and unwanted effects. The aim of the current work was to develop and validate an ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method to determine multiple antibiotics in clinical plasma samples from critically ill patients; low sample volume and rapid processing of samples were considered the main criteria. A separation method based on an online combination of UHPLC-MS/MS was developed for the simultaneous determination of 4 β-lactam antibiotics (cefepime, meropenem, cefotaxime, and piperacillin), tazobactam, and linezolid in human plasma samples. The volume of plasma sample used for analysis was 20 µL. The developed method was validated according to Food and Drug Administration guidelines. The chromatographic run time was 8 minutes. Calibration curves were linear for concentration ranges of 0.1-100 mcg/mL (r 2 > 0.99) for tazobactam, meropenem, cefotaxime, linezolid, and piperacillin and 1-100 mcg/mL (r 2 > 0.99) for cefepime. The intraday and interday accuracy of the method ranged from 92.4% to 110.7% and 93.6% to 113.3%, respectively. The intraday and interday precision values were ≤17.3% and ≤17.4%, respectively. No interfering and carryover analytes were observed. The developed UHPLC-MS/MS method is an appropriate and practical tool for therapeutic drug monitoring of the selected antibiotics. Owing to its rapidity, requirement of low sample volume, and high selectivity, sensitivity, and reliability, it can be effectively implemented in routine clinical laboratory tests for critically ill patients.

A high-throughput LC-MS/MS method for simultaneous determination of isoniazid, ethambutol and pyrazinamide in human plasma.

Tuberculosis (TB) remains a challenging global infectious disease, mainly affecting the lungs. First-line anti-TB drugs play a crucial role in slowing down the rapid spread of TB. In addition, the patient might benefit from therapeutic drug monitoring since it has become an accepted clinical tool for optimizing TB treatment. A simple and sensitive liquid chromatography/tandem mass spectrometry method was developed to monitor the plasma level of isoniazid, ethambutol and pyrazinamide in plasma samples. A one-step extraction procedure using an Ostro™ plate was applied, and extracts were analyzed by gradient elution followed by detection on a mass spectrometer by multiple reaction monitoring mode. The analytes were separated within 4.2min and over the concentration range of 0.2-10μg/ml for isoniazid and ethambutol and 1-65 μg/ml for pyrazinamide. The method was successfully validated according to the European Medicine Agency guideline for the selectivity, linearity and lower limit of detection, precision and accuracy, matrix effect, extraction recovery, carryover, dilution integrity and stability, and applied for quantification of analytes in clinical samples from TB patients. The presented method allows sensitive and reproducible determination of selected anti-TB drugs with advantages such as low sample volume requirement, short run time of analysis, one-step sample preparation procedure with capabilities for phospholipids removal, and a low quantification limit as well as a high degree of selectivity.

In-line sample trap columns with diatomite for large-volume injection in CZE-IM-MS.

The analysis of low-abundant compounds with capillary zone electrophoresis-drift-tube ion mobility spectrometry-mass spectrometry (CZE-DTIMS-MS) is compromised due to the low injectable sample volumes in CZE and low duty cycle in DTIMS. Fritless packed in-line trap columns, using solid-phase extraction sorbent particles, have been used to increase injection volumes in CZE, but these columns are difficult to prepare and exhibit rapidly increasing back pressures. To provide smooth and complete filling of trap columns as well as to ensure higher and sustained flow rates though the columns, blends of cation and anion exchange particles with diatomite were used. The application of diatomite blends ensured the use of trap columns for at least 100 injections, with maximum injection volumes over 10µl, which corresponds to an enrichment factor of more than 1000 over conventional injections in CZE-MS, enabling the detection of low nM concentrations of N-glycans with CZE-IMS-MS.

New challenges in sample preparation: Miniaturized stir bar sorptive dispersive microextraction as a high-throughput and feasible approach for low-availability sample preparation

The miniaturization of stir bar sorptive dispersive microextraction (mSBSDME) for the analysis of low-availability samples is presented. This new methodology is based on the principles of stir bar sorptive dispersive microextraction, but the amount of sorbent and, most importantly, the amount of sample are considerably reduced to a tiny amount and a few microliters, respectively. Thus, affordable 400-μL flat-base glass inserts and minute bar-shape neodymium magnets (3 mm length x 2 mm diameter) were used as extraction devices hold by a specifically designed multiextraction assembly, which comprises a high-rate stirring plate and a 3D-printed support to treat 15 samples simultaneously. This new approach allows a fast, affordable, portable, and high-throughput analysis of low-volume samples, expanding the potential of the technique. The same extraction device is used along the different stages, thus avoiding transfers, which reduces sample handling. Besides, the reduction in the sample, sorbent and organic solvent amounts allows a considerable decrease of the waste generation, and thus pursues a green sample preparation for bioanalysis. As a proof-of-concept of this new methodology, cortisone and cortisol were determined in human saliva using a composite material made of a reversed phase polymer (Strata™-X-RP) and CoFe2O4 magnetic nanoparticles. Liquid chromatography coupled to tandem mass spectrometry was used to measure both analytes obtaining good analytical features in terms of linearity (R2 > 0.997), method limits of detection and quantification (22.6 and 75.5 ng L−1 for cortisone, and 19.3 and 64.3 ng L−1 for cortisol, respectively), repeatability (RSD ≤11%) and relative recoveries (78–134%).

Statistical comparisons of 0.08 g/100 ml fortified blood alcohol samples from 6-ml and 10-ml gray-top tubes.

This experiment supplements the study, "Statistical comparisons of blood alcohol samples from 6-ml and 10-ml grey-top tubes". The initial study analyzed fortified samples for blood alcohol concentration (BAC) using two sizes of gray-top tubes: a 10-ml tube containing a nominal 1% sodium fluoride (NaF), a preservative, and a 6-ml tube containing 0.25% NaF, using the variables of time, storage temperature, fill volume, and concentration. From that study, paired t-tests determined no difference in BAC from data sets of the two tube sizes, however, Analysis of Variance, or ANOVA, testing determined the two tube sizes yielded different results at 0.08 g/100 ml. To investigate this potential disparity, this study fortified new samples at 0.08 g/100 and focused on two variables: fill volume and storage duration, while increasing the sample population (to n=8). For this study, the two tube types yielded equivalent concentrations under the majority of conditions studied. Differences between the two tube types were found using paired t-test for the high-volume samples on Days 7 and 30; ANOVA yielded the same results but also determined one additional statistical difference for the Day 30 low-volume samples. However, the differences observed between tube sizes fall within standard deviation ranges established for the analytical method precision profile, indicating statistical differences are not analytically significant. Further studies are needed to investigate the comparability of the tubes under real-world conditions, under which oxygen and/or additives are not added during sample preparation steps or by usage of blood bank products.

Automated Proteomics Sample Preparation of Phosphatidylserine-Positive Extracellular Vesicles from Human Body Fluids.

Extracellular vesicles (EVs) are ubiquitously secreted by almost every cell type and are present in all body fluids. Blood-derived EVs can be used as a promising source for biomarker monitoring in disease. EV proteomics is currently being analyzed in clinical specimens. However, their EV proteomics preparation methods are limited in throughput for human subjects. Here, we introduced a novel automated EV isolation and sample preparation method using a magnetic particle processing robot for automated 96-well processing of magnetic particles for EV proteomics analysis that can be started with a low volume of multiple clinical samples. The automation of EV purification reduced the coefficient of variation of protein quantification from 3.5 to 2.2% compared with manual purification, enabling the quantification of 1120 proteins in 1 h of MS analysis. This automated proteomics EV sample preparation is attractive for processing large cohort samples for biomarker development, validation, and routine testing.

A Microfluidic Platform Revealing Interactions between Leukocytes and Cancer Cells on Topographic Micropatterns.

Immunoassay for detailed analysis of immune-cancer intercellular interactions can achieve more promising diagnosis and treatment strategies for cancers including nasopharyngeal cancer (NPC). In this study, we report a microfluidic live-cell immunoassay integrated with a microtopographic environment to meet the rising demand for monitoring intercellular interactions in different tumor microenvironments. The developed assay allows: (1) coculture of immune cells and cancer cells on tunable (flat or micrograting) substrates, (2) simultaneous detection of different cytokines in a wide working range of 5-5000 pg/mL, and (3) investigation of migration behaviors of mono- and co-cultured cells on flat/grating platforms for revealing the topography-induced intercellular and cytokine responses. Cytokine monitoring was achieved on-chip by implementing a sensitive and selective microbead-based sandwich assay with an antibody on microbeads, target cytokines, and the matching fluorescent-conjugated detection antibody in an array of active peristaltic mixer-assisted cytokine detection microchambers. Moreover, this immunoassay requires a low sample volume down to 0.5 μL and short assay time (30 min) for on-chip cytokine quantifications. We validated the biocompatibility of the co-culture strategy between immune cells and NPC cells and compared the different immunological states of undifferentiated THP-1 monocytic cells or PMA-differentiated THP-1 macrophages co-culturing with NP460 and NPC43 on topographical and planar substrates, respectively. Hence, the integrated microfluidic platform provides an efficient, broad-range and precise on-chip cytokine detection approach, eliminates the manual sampling procedures and allows on-chip continuous cytokine monitoring without perturbing intercellular microenvironments on different topographical ECM substrates, which has the potential of providing clinical significance in early immune diagnosis, personalized immunotherapy, and precision medicine.

Hydrophilic Interaction Liquid Chromatography–Tandem Mass Spectrometry of 5-Aminolevulinic Acid for Low Concentration and Small Sample Volumes

Hydrophilic Interaction Liquid Chromatography–Tandem Mass Spectrometry of 5-Aminolevulinic Acid for Low Concentration and Small Sample Volumes

A miniaturized all-in-one optical nose based on 3D printing technology and patterned paper substrate for discrimination of volatile organic compounds

Herein, an integrated design of a paper-based colorimetric sensor array, which includes both sensor array and sampling vessel is described. The paper substrate was patterned for providing hydrophobic barriers to deposit sensing elements, while a hydrophilic regions were embedded on the paper for sample solution. In this way, the paper substrate works as a sensing device and sampling vessel, simultaneously. A miniaturized cubic reaction chamber sizing (30 × 30 × 0.02) mm, equipped with a small aperture for sample injection was fabricated using 3D printing technology to hold the paper substrate. The required sample volume was lowered down dramatically (13.0 μL) due to the all-in-one sampling and sensing device. This miniaturized sensor array concept was employed to detect and discriminate volatile organic compounds (VOCs) relating to different solvent families that are known as cancer biomarkers. A colorimetric sensor array was fabricated by spotting 12 indicators belonging to three families of solvatochromic dyes, pH indicators and metallochromic dyes. This simple proposed device was able to discriminate the studied solvents into three chemical families including polar protic, dipolar aprotic and nonpolar with 93% accuracy. In addition, this sensor showed potential for quantitative and semi-quantitative analysis of volatile compounds in their mixtures. In total, the designed device is portable, rapid, and cost-effective and requires low volumes of samples that can be considered a good candidate for various VOC analysis with limited volume.

Analysis of microsamples of biological fluids via inductively coupled plasma-tandem mass spectrometry using a micro-flow injection system coupled to a high-efficiency sample introduction setup

In many fields, the sample volume available is the limiting factor for ultra-trace elemental analysis, and thus, the development of novel methods that can extract more information from lower sample amounts is a topic that deserves attention. In this work, the performance of a micro-flow injection system coupled to either (1) a traditional or (2) a high-efficiency sample introduction system has been evaluated for the analysis of liquid microsamples using inductively coupled plasma-mass spectrometry (ICP-MS). The figures of merit with each sample introduction system have been studied in a wide range of sample volumes and sample uptake rates. The use of a high-efficiency low-volume sample introduction system provided a > 4-fold enhanced sensitivity as compared to the traditional one, while the former setup was demonstrated being capable of accurately delivering volumes as low as 5 or 10 μL to the ICP. Even for such low sample amounts, good linearity (>0.9998) and repeatability (RSD down to 0.2%) were experimentally demonstrated, while matrix effects were lower with the high-efficiency sample introduction system. The use of an internal standard adequately corrected for changes in sample introduction conditions, allowing quantitative results to be obtained by using a single set of calibration data obtained at fixed operating conditions (sample volume and sample uptake rate), with a bias <15% (typical acceptance criterion for clinical QC analysis). The set-up characterized was successfully applied to the analysis of microsamples of biological fluids (down to 0.4 μL of sample volume). Tandem ICP-MS (ICP-MS/MS) was used to overcome spectral overlap (chemical resolution with a mixture of NH3/He) affecting the determination of six clinically relevant metals (Co, Cr, Mn, Ni, Ti and V) at ultra-trace concentration levels. No significant differences were found between the results obtained in this work and the reference values, even when using different sample introduction conditions for the measurement of calibration standards and samples.

Photoluminescent Histidine-Stabilized Gold Nanoclusters as Efficient Sensors for Fast and Easy Visual Detection of Fe Ions in Water Using Paper-Based Portable Platform.

Herein is presented a novel and efficient portable paper-based sensing platform using paper-incorporated histidine stabilized gold nanoclusters (His-AuNCs), for the sensitive and selective detection of Fe ions from low-volume real water samples based on photoluminescence (PL) quenching. Highly photoluminescent colloidal His-AuNCs are obtained via a novel microwave-assisted method. The His-AuNCs-based sensor reveals a limit of detection (LOD) as low as 0.2 μM and a good selectivity towards Fe ions, in solution. Further, the fabricated portable sensing device based on paper impregnated with His-AuNCs proves to be suitable for the easy detection of hazardous Fe levels from real water samples, under UV light exposure, through evaluating the level of PL quenching on paper. Photographic images are thereafter captured with a smartphone camera and the average blue intensity ratio (I/I0) of the His-AuNCs-paper spots is plotted against [Fe2+] revealing a LOD of 3.2 μM. Moreover, selectivity and competitivity assays performed on paper-based sensor prove that the proposed platform presents high selectivity and accuracy for the detection of Fe ions from water samples. To validate the platform, sensing assays are performed on real water samples from local sources, spiked with 35 μM Fe ions (i.e., Fe2+). The obtained recoveries prove the high sensitivity and accuracy of the proposed His-AuNCs-paper-based sensor pointing towards its applicability as an easy-to-use, fast, quantitative and qualitative sensor suitable for on-site detection of toxic levels of Fe ions in low-volume real water samples.

Inkjet Electrodes for Developing Wearable Sensors for the Detection of Peptides and Neurotransmitters in Sweat Using Flexible Materials

In recent years, there has been an increased interest in the research and development of 2D materials for use and application in different fields, such as new technologies, medicine, health care, flexible biosensors, and wearable devices. Our goal is based on developing a flexible wearable biosensor for the skin to detect in real-time neurotransmitters and neuropeptides in human sweat. The development of flexible biosensors for detecting molecules in sweat has been of great interest. However, the problem is that most research has focused on a limited number of molecules such as lactate, chloride and potassium ions, glucose, and pH, and there is a great need to develop flexible biosensors that can monitor real-time molecules that are not yet studied in sweat. One of our molecules of great interest is neuropeptide Y (NPY). NPY has an essential role in the energy balance and is related to different diseases and conditions such as diabetes, obesity, depression, anxiety, and sleep problems and has recently been found to be associated with cardiovascular problems.Our biosensor consists of flexible, low-cost materials that do not require much manufacturing time. The proposed flexible sensor system consists of paper-based microfluidics that serve as our system's passive flow method and silver inkjet electrodes printed on the PET surface. Microfluidic systems can be characterized as active or passive, depending on the force applied to the sample or flow. We can perform minimal flow or particles in a minimum amount of liquid or a sample flow with paper-based microfluidics systems. Among the passive microfluidic systems, paper-based microfluidics, also known as micro-pads, are among the most promising methods because they are easy to manufacture, inexpensive, and have low sample volume. One of the most popular techniques to develop electrochemical wearable biosensors is the fabrication of microelectrode arrays through inkjet printing. To fabricate our electrode arrays, we first created the design in AutoCAD and then printed it using silver conductive ink and a commercial inkjet printer. Since sweat is a complex fluid, it is necessary to modify the working electrode's surface to give the electrode's selectivity towards the molecule to be analyzed or detected. To achieve the detection of NPY, we first add carboxyl groups to the surface of the working electrode, which will serve as an anchor for the specific aptamer for NPY. We use different buffer solutions like artificial cerebrospinal fluid, phosphate buffer solutions, and artificial sweat to perform electrochemical characterization on our silver inkjet electrodes through cyclic voltammetry and electrochemical impedance spectroscopy (EIS). We decreased the oxidation of the surface of our electrodes by changing the buffer and modifying the surface for greater selectivity towards NPY. Using 2D materials, we managed to manufacture and electrochemically characterize our flexible biosensor system prototypes to detect neuropeptides and neurotransmitters in sweat, applying the use of paper-based microfluidics and silver inkjet electrodes printed on PET and paper surfaces.

On the Move-Sensitive Fluorescent Aptassay on Board Catalytic Micromotors for the Determination of Interleukin-6 in Ultra-Low Serum Volumes for Neonatal Sepsis Diagnostics.

A graphene oxide/nickel/platinum nanoparticle micromotor (MM)-based fluorescent aptassay is proposed to determine interleukin-6 (IL-6) in serum samples from low-birth-weight infants (gestational age of less than 32 weeks and birthweight below 1000 g) with sepsis suspicion. In this kind of patients, IL-6 has demonstrated good sensitivity and specificity for the diagnosis of sepsis, both for early and late onset sepsis. The approach was based on the adsorption of the aptamer for IL-6 tagged with 6-FAM as a fluorescent label (AptIL-6, λem = 520 nm) on the graphene oxide external layer (MMGO-AptIL-6) inducing fluorescence quenching (OFF state) and a subsequent on-the-move affinity recognition of IL-6 from AptIL-6 (IL-6-AptIL-6 complex) recovering the fluorescence (ON state). An aptamer against IL-6 was selected and developed by the systematic evolution of ligands by exponential enrichment technology. This approach displayed a suitable linear range of 0.07-1000 pg mL-1 (r = 0.995) covering the cut-off and clinical practice levels, allowing direct determination without any dilution and simplifying the analysis as well as exhibiting an excellent sensitivity (LOD = 0.02 pg mL-1) in ultralow volumes of diagnostic clinical samples (2 μL). A high agreement between IL-6 levels obtained from our MM-based approach and the method used by the Hospital was obtained (relative error < 3%). The MM-based aptassay is competitive in comparison with that of the Hospital, in terms of a significant reduction of the sample volume (15 times less) and enhanced sensitivity, employing similar analysis times. These results position MM technology with enough potential to achieve high sensitivities in low sample volumes, opening new avenues in diagnosis based on low sample volumes.

Nanostructured Silicon Enabled HR-MS for the Label-Free Detection of Biomarkers in Colorectal Cancer Plasma Small Extracellular Vesicles

Despite improvements in treatment options for advanced colorectal cancer (CRC), survival outcomes are still best for patients with non-metastasised disease. Diagnostic tools to identify blood-based biomarkers and assist in CRC subtype classification could afford a means to track CRC progression and treatment response. Cancer cell-derived small extracellular vesicles (EVs) circulating in blood carry an elevated cargo of lipids and proteins that could be used as a signature of tumour suppressor/promoting events or stages leading up to and including metastasis. Here, we used pre-characterised biobanked plasma samples from surgical units, typically with a low volume (~100 µL), to generate and discover signatures of CRC-derived EVs. We employed nanostructured porous silicon (pSi) surface assisted-laser desorption/ionisation (SALDI) coupled with high-resolution mass spectrometry (HR-MS), to allow sensitive detection of low abundant analytes in plasma EVs. When applied to CRC samples, SALDI-HR-MS enabled the detection of the peptide mass fingerprint of cancer suppressor proteins, including serine/threonine phosphatases and activating-transcription factor 3. SALDI-HR-MS also allowed the detection of a spectrum of glycerophospholipids and sphingolipid signatures in metastatic CRC. We observed that lithium chloride enhanced detection sensitivity to elucidate the structure of low abundant lipids in plasma EVs. pSi SALDI can be used as an effective system for label-free and high throughput analysis of low-volume patient samples, allowing rapid and sensitive analysis for CRC classification.

Experimental infection of bovine spongi-form encephalopathy in guinea pigs: clinical symptoms, pathohistology and immunohistochemistry

Prion diseases are infectious neurodegenerative diseases affecting humans and animals that are invariably fatal and have no currently available prevention or treatment methods. These diseases are also known as transmissible spongiform encephalopathies (TSE), characterized by similar neurological disorders, vacuolization of the neuroparenchyma, accumulation of the pathogenic isoform of the prion protein in it, the absence of an immunological response to the pathogen, and the inevitable death. The most famous of them is the classic bovine spongiform encephalopathy, because. has unprecedented social and economic consequences. C-BSE of cattle is also dangerous for humans, causing one of the forms of Creutzfeldt-Jakob disease, leading to a gradual progression of irreversible C-BSE lesions, leading to the death of a sick person. Laboratory animal models make a huge contribution to the study of the biological properties of prions and the diseases they cause. Thus, mouse models of prion infections have played a major role in deciphering fundamental questions of prion biology and assessing the potential transmissibility of neurodegenerative diseases. However, despite the economic acceptability and other positive properties of this model, it has a number of disadvantages. It produces low-volume tissue samples, and it is very difficult to obtain the required volume of serum and cerebrospinal fluid for many studies. Therefore, the search for and study of the disease in new models of laboratory animals sensitive to bovine C-BSE is of great scientific importance in the study of prion diseases. This work shows that during experimental intracerebral infection of guinea pigs with the causative agent of classical bovine spongiform encephalopathy, they are 100% sensitive to the pathogen. The clinical picture of the disease, developing severe pathomorphological changes in all parts of the brain with vacuolization of neurons and neropil and massive deposition of the prion protein PrPSc are described. Guinea pigs can be a highly sensitive object for laboratory study of the causative agent of bovine spongiform encephalopathy.