Medical Relevance, State-of-the-Art and Perspectives of "Sweet Metacode" in Liquid Biopsy Approaches.

This review briefly introduces readers to an area where glycomics meets modern oncodiagnostics with a focus on the analysis of sialic acid (Neu5Ac)-terminated structures. We present the biochemical perspective of aberrant sialylation during tumourigenesis and its significance, as well as an analytical perspective on the detection of these structures using different approaches for diagnostic and therapeutic purposes. We also provide a comparison to other established liquid biopsy approaches, and we mathematically define an early-stage cancer based on the overall prognosis and effect of these approaches on the patient's quality of life. Finally, some barriers including regulations and quality of clinical validations data are discussed, and a perspective and major challenges in this area are summarised.

Glycoprofiling of proteins as prostate cancer biomarkers: A multinational population study.

The glycoprofiling of two proteins, the free form of the prostate-specific antigen (fPSA) and zinc-α-2-glycoprotein (ZA2G), was assessed to determine their suitability as prostate cancer (PCa) biomarkers. The glycoprofiling of proteins was performed by analysing changes in the glycan composition on fPSA and ZA2G using lectins (proteins that recognise glycans, i.e. complex carbohydrates). The specific glycoprofiling of the proteins was performed using magnetic beads (MBs) modified with horseradish peroxidase (HRP) and antibodies that selectively enriched fPSA or ZA2G from human serum samples. Subsequently, the antibody-captured glycoproteins were incubated on lectin-coated ELISA plates. In addition, a novel glycoprotein standard (GPS) was used to normalise the assay. The glycoprofiling of fPSA and ZA2G was performed in human serum samples obtained from men undergoing a prostate biopsy after an elevated serum PSA, and prostate cancer patients with or without prior therapy. The results are presented in the form of an ROC (Receiver Operating Curve). A DCA (Decision Curve Analysis) to evaluate the clinical performance and net benefit of fPSA glycan-based biomarkers was also performed. While the glycoprofiling of ZA2G showed little promise as a potential PCa biomarker, the glycoprofiling of fPSA would appear to have significant clinical potential. Hence, the GIA (Glycobiopsy ImmunoAssay) test integrates the glycoprofiling of fPSA (i.e. two glycan forms of fPSA). The GIA test could be used for early diagnoses of PCa (AUC = 0.83; n = 559 samples) with a potential for use in therapy-monitoring (AUC = 0.90; n = 176 samples). Moreover, the analysis of a subset of serum samples (n = 215) revealed that the GIA test (AUC = 0.81) outperformed the PHI (Prostate Health Index) test (AUC = 0.69) in discriminating between men with prostate cancer and those with benign serum PSA elevation.

Negative Charge-Carrying Glycans Attached to Exosomes as Novel Liquid Biopsy Marker.

Prostate cancer (PCa) is the second most common cancer. In this paper, the isolation and properties of exosomes as potential novel liquid biopsy markers for early PCa liquid biopsy diagnosis are investigated using two prostate human cell lines, i.e., benign (control) cell line RWPE1 and carcinoma cell line 22Rv1. Exosomes produced by both cell lines are characterised by various methods including nanoparticle-tracking analysis, dynamic light scattering, scanning electron microscopy and atomic force microscopy. In addition, surface plasmon resonance (SPR) is used to study three different receptors on the exosomal surface (CD63, CD81 and prostate-specific membrane antigen-PMSA), implementing monoclonal antibodies and identifying the type of glycans present on the surface of exosomes using lectins (glycan-recognising proteins). Electrochemical analysis is used to understand the interfacial properties of exosomes. The results indicate that cancerous exosomes are smaller, are produced at higher concentrations, and exhibit more nega tive zeta potential than the control exosomes. The SPR experiments confirm that negatively charged α-2,3- and α-2,6-sialic acid-containing glycans are found in greater abundance on carcinoma exosomes, whereas bisecting and branched glycans are more abundant in the control exosomes. The SPR results also show that a sandwich antibody/exosomes/lectins configuration could be constructed for effective glycoprofiling of exosomes as a novel liquid biopsy marker.

MXene-based electrochemical devices applied for healthcare applications.

The initial part of the review provides an extensive overview about MXenes as novel and exciting 2D nanomaterials describing their basic physico-chemical features, methods of their synthesis, and possible interfacial modifications and techniques, which could be applied to the characterization of MXenes. Unique physico-chemical parameters of MXenes make them attractive for many practical applications, which are shortly discussed. Use of MXenes for healthcare applications is a hot scientific discipline which is discussed in detail. The article focuses on determination of low molecular weight analytes (metabolites), high molecular weight analytes (DNA/RNA and proteins), or even cells, exosomes, and viruses detected using electrochemical sensors and biosensors. Separate chapters are provided to show the potential of MXene-based devices for determination of cancer biomarkers and as wearable sensors and biosensors for monitoring of a wide range of human activities.

Glycosylation in extracellular vesicles: Isolation, characterization, composition, analysis and clinical applications.

This review provides a comprehensive overview of our understanding of the role that glycans play in the formation, loading and release of extracellular vesicles (EVs). The capture of EVs (typically with a size of 100-200 nm) is described, including approaches based on glycan recognition with glycan-based analysis offering highly sensitive detection of EVs. Furthermore, detailed information is provided about the use of EV glycans and glycan processing enzymes as potential biomarkers, therapeutic targets or tools applied for regenerative medicine. The review also provides a short introduction into advanced methods for the characterization of EVs, new insights into the biomolecular corona covering EVs and bioanalytical tools available for glycan analysis.

Amperometric Miniaturised Portable Enzymatic Nanobiosensor for the Ultrasensitive Analysis of a Prostate Cancer Biomarker.

Screen-printing technology is a game changer in many fields including electrochemical biosensing. Two-dimensional nanomaterial MXene Ti3C2Tx was integrated as a nanoplatform to immobilise enzyme sarcosine oxidase (SOx) onto the interface of screen-printed carbon electrodes (SPCEs). A miniaturised, portable, and cost-effective nanobiosensor was constructed using chitosan as a biocompatible glue for the ultrasensitive detection of prostate cancer biomarker sarcosine. The fabricated device was characterised with energy-dispersive X-ray spectroscopy (EDX), electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). Sarcosine was detected indirectly via the amperometric detection of H2O2 formed during enzymatic reaction. The nanobiosensor could detect sarcosine down to 7.0 nM with a maximal peak current output at 4.10 ± 0.35 × 10-5 A using only 100 µL of a sample per measurement. The assay run in 100 µL of an electrolyte showed the first linear calibration curve in a concentration window of up to 5 µM with a slope of 2.86 µA·µM-1, and the second linear calibration curve in the range of 5-50 µM with a slope of 0.32 ± 0.01 µA·µM-1 (R2 = 0.992). The device provided a high recovery index of 92.5% when measuring an analyte spiked into artificial urine, and could be used for detection of sarcosine in urine for at least a period of 5 weeks after the preparation.

How to choose proper magnetic particles for bioaffinity interactions? The case for immobilised glyconanoconjugate.

In this study, an assay for detection of the cancer biomarker Thomsen-nouvelle (Tn) antigen on the ELISA plates format was designed and developed. The effects of size and the interfacial density of the negative charge of magnetic beads (MBs) on the specific sensitivity of the bioaffinity interaction were studied. In particular, glyconanoconjugate, i.e. glycan Tn antigen conjugated to bovine serum albumin (BSA) was covalently immobilised on MBs for the bioaffinity detection of anti-Tn antibodies as cancer biomarkers. Six different MBs were used in the study, i.e. carboxy-modified MBs of 250 nm, 500 nm, 1000 nm and 2800 nm and epoxy-modified MBs of 2800 nm and 4500 nm. In order to evaluate which MBs are the best suited for detection of the analyte anti-Tn antibodies, sensitivities of detection (slopes from calibration curves) were calculated. Next, specific sensitivities were calculated for each type of MBs as a ratio of sensitivity of detection to the mass of MBs. From zeta potential ζ for each type of MBs, the interfacial charge density on MBs was calculated, expressed as the density of zeta potential ζd (ratio of zeta potential to surface area of MBs, i.e. ζd = Î¶/A). Then, we evaluated the effect of size and ζd on the specific sensitivity of detection of anti-Tn antibodies in order to understand the immobilisation process on nanoscale. We also identified an optimal value of ζd on MBs; this was essential to achieve highly sensitive detection of the analyte, which made it possible to attain limit of detection (LOD) of (0.31 ± 0.01) ng mL-1 or (2.10 ± 0.04) pM for analyte detection. In addition, the optimal assay configuration was highly selective and enabled reliable detection of the analyte in human serum with a recovery index in the range of 102-104%.

MXene as a novel cartridge for N-glycan enrichment.

In this study, we applied MXene as column cartridge for N-glycan enrichment from human samples with a focus on the analysis of sialic acid linkages using a derivatisation protocol followed by glycan analysis via Matrix Assisted Laser Desorption Ionisation-Time Of Flight Mass Spectrometry (MALDI-TOF-MS). The MXene-based cartridge enriches a higher number of glycans (i.e., sialylated and bisecting N-glycans) when compared to the commercial HILIC columns. We demonstrate the strong potential of MXene as a stationary phase in MS glycomic analysis.

Redox features of hexaammineruthenium(III) on MXene modified interface: Three options for affinity biosensing.

In this article we describe construction of a bioreceptive interface for detection of a breast cancer biomarker carbohydrate antigen CA15-3. The conductive interface was patterned by a 2D nanomaterial MXene, to which a mixed layer containing sulfobetaine and carboxybetaine was electrochemically grafted through a diazonium moiety. Such a modified interface was then applied for covalent immobilisation of anti-CA15-3 antibody as a bioreceptive probe for detection of a breast cancer biomarker. Two different strategies were applied for final construction of an immunosensor i.e. an interface finally blocked by bovine serum albumin or an immunosensor without such modification. Finally, electrochemical reading was accomplished using a soluble redox probe Ru(NH3)63+ ion for detection of CA15-3 in a clinically relevant range up to 50 U mL-1. The results indicate that immunosensor based on non-blocked interface can be applied for biosensing using two modes of action: 1. differential pulse voltammetry (a plot of a peak current vs. analyte concentration) and 2. an electrochemical impedance spectroscopy (a plot of a charge transfer resistance vs. analyte concentration). The electrode blocked by bovine serum albumin (BSA) can be used by additional 3. mode of action: through detection of changes in the potential (a plot Epvs. c). Additionally, we reveal and explain that Ru(NH3)63+ is redox probe, which can be applied as interfacial molecular nanoscale ruler to distinguish negatively charged protein molecules present in the close proximity (≤ 6 nm) of the electrode (in our case adsorbed BSA molecules) from the negatively charged protein molecules at a larger distance (>12 nm) from the electrode (i.e. CA15-3 analyte).

Facile fabrication of screen-printed MoS2 electrodes for electrochemical sensing of dopamine.

Molybdenum disulfide (MoS2) screen-printed working electrodes were developed for dopamine (DA) electrochemical sensing. MoS2 working electrodes were prepared from high viscosity screen-printable inks containing various concentrations and sizes of MoS2 particles and ethylcellulose binder. Rheological properties of MoS2 inks and their suitability for screen-printing were analyzed by viscosity curve, screen-printing simulation and oscillatory modulus. MoS2 inks were screen-printed onto conductive FTO (Fluorine-doped Tin Oxide) substrates. Optical microscopy and scanning electron microscopy with energy-dispersive X-ray spectroscopy (SEM/EDX) analysis were used to characterize the homogeneity, topography and thickness of the screen-printed MoS2 electrodes. The electrochemical performance was assessed through differential pulse voltammetry. Results showed an extensive linear detection of dopamine from 1 µM to 300 µM (R2 = 0.996, sensitivity of 5.00 × 10-8 A µM-1), with the best limit of detection being 246 nM. This work demonstrated the possibility of simple, low-cost and rapid preparation of high viscosity MoS2 ink and their use to produce screen-printed FTO/MoS2 electrodes for dopamine detection.

Exosomes from prostate cancer cell lines: Isolation optimisation and characterisation.

Exosomes are considered to be a rich source of biomarkers, hence in this article we examine the best procedure for their isolation. We examine several isolation procedures, exosome storage conditions and other conditions affecting exosome production by prostate cell lines. We selected four different commercially available kits based on different principles to achieve exosome isolation, the best being magnetic-based. In addition, we found storage at - 20 °C to be good for storing isolated exosomes and that exosomes were produced from the cancerous prostate cell line 22Rv1 in much greater amounts than the non-cancerous prostate cell line RWPE1. We also found differences in the response of both cell lines in the production of exosomes as a result of stress, i.e. exposure to hydrogen peroxide and starvation. The effect of Triton X-100 on exosome lysis was examined using two different surfactant concentrations by analysis of the exosome count and change in the exosome size. The final part of the article details the advantages of the use of a 2D biochip prepared in-house over a commercially available 3D biochip for monitoring the interaction of exosomes via its surface receptors (CD63) with an immobilised ligand (anti-CD63 antibodies) using surface plasmon resonance. The final experiment shows the potential of lectin fluorescent microarrays for the analysis of glycans present in lysed exosomes.

CCNE1 amplification is synthetic lethal with PKMYT1 kinase inhibition.

Amplification of the CCNE1 locus on chromosome 19q12 is prevalent in multiple tumour types, particularly in high-grade serous ovarian cancer, uterine tumours and gastro-oesophageal cancers, where high cyclin E levels are associated with genome instability, whole-genome doubling and resistance to cytotoxic and targeted therapies1-4. To uncover therapeutic targets for tumours with CCNE1 amplification, we undertook genome-scale CRISPR-Cas9-based synthetic lethality screens in cellular models of CCNE1 amplification. Here we report that increasing CCNE1 dosage engenders a vulnerability to the inhibition of the PKMYT1 kinase, a negative regulator of CDK1. To inhibit PKMYT1, we developed RP-6306, an orally bioavailable and selective inhibitor that shows single-agent activity and durable tumour regressions when combined with gemcitabine in models of CCNE1 amplification. RP-6306 treatment causes unscheduled activation of CDK1 selectively in CCNE1-overexpressing cells, promoting early mitosis in cells undergoing DNA synthesis. CCNE1 overexpression disrupts CDK1 homeostasis at least in part through an early activation of the MMB-FOXM1 mitotic transcriptional program. We conclude that PKMYT1 inhibition is a promising therapeutic strategy for CCNE1-amplified cancers.

Glycan signatures for the identification of cisplatin-resistant testicular cancer cell lines: Specific glycoprofiling of human chorionic gonadotropin (hCG).

BACKGROUND: Testicular cancer (TC) is the most frequent type of cancer among young men aged between 15 and 34 years. TC is treated using cisplatin, but 3%-5% of TC patients fail to respond to cisplatin, with a very bad to fatal prognosis. Accordingly, it is most important to quickly and readily identify those TC patients who are resistant to cisplatin treatment. METHODS: This study seeks to investigate changes in the glycosylation associated with cisplatin resistance to TC cell lines. RESULTS: A specific glycoprofiling of human chorionic gonadotropin (hCG) was analysed in three TC cell lines and one cell line of female origin. A typical calibration curve for hCG glycoprofiling showed a dynamic range up to 50 ng/ml, with a limit of detection of 0.3 ng/ml and assay reproducibility represented by relative standard deviation of 3.0%. Changes in the glycan signatures on hCG were analysed in cisplatin-sensitive cell lines and in their cisplatin-resistant sub-lines using an enzyme-linked lectin assay (ELLA) protocol. An immobilised antibody was applied to a selective capture of hCG from a cytoplasmic fraction of cell lysates with final incubation using a lectin from a panel of 17 lectins. CONCLUSION: The results suggest that one particular lectin Dolichos biflorus agglutinin (DBA) can selectively discriminate sensitive TC cell lines from resistant TC cell lines. Moreover, there are additional lectins which can provide useful information about the strength of cisplatin resistance.

Amplified suspension magnetic bead-based assay for sensitive detection of anti-glycan antibodies as potential cancer biomarkers.

The development of a novel SUspension Magnetic-Bead-based Assay (SUMBA) for the detection of antibodies against aberrant glycans (AGA) as potential cancer biomarkers is presented here. The SUMBA method was extensively optimised by choosing proper commercially available AGA able to specifically, and with high affinity, recognise aberrant glycans, which were attached to the protein backbone working as a molecular scaffold (a glycoconjugate). The whole SUMBA was optimised using several analytical techniques such as Surface Plasmon Resonance and Energy Dispersive X-ray Spectroscopy. Additionally, the SUMBA method was extensively optimised for signal enhancement. With all steps optimised, we were able to detect AGA ultrasensitively with a limit of detection of 0.45 pM. Moreover, AGA could be detected in serum samples with a recovery index in the range of 98%-104%.

Tandem Osmotic Engine Based on Hydrogel Particles with Antipolyelectrolyte and Polyelectrolyte Effect Fuelled by Both Salinity Gradient Modes.

In this study, we propose a new approach to attain energy by salinity gradient engines with pistons based on hydrogels possessing polyelectrolyte and antipolyelectrolyte effects in a tandem arrangement, providing energy in each salinity gradient mode in a repeatable manner. The swelling of hydrogel with a polyelectrolyte effect and shrinking of hydrogel particles possessing an antipolyelectrolyte effect in desalinated water, and subsequent shrinking of hydrogel with polyelectrolyte and swelling of hydrogel antipolyelectrolyte effect in saline water, generate power in both increasing and decreasing salinity modes. To investigate the energy recovery, we scrutinized osmotic engine assemblies by a setup arrangement of pistons with hydrogel particles, with polyelectrolyte and antipolyelectrolyte effects, in tandem. The energy recovery from the tandem engine setup (calculated based on dry form for each polyelectrolyte polyacrylate-based hydrogel-SPA) and antipolyelectrolyte-sulfobetaine-based gel with methacrylate polymeric backbone-SBE) up to 581 J kg-1 and a mean power of 0.16 W kg-1 was obtained by the tandem setup of SPA and SBE hydrogel containing 3% crosslinking density and particle size of 500 microns with an external load of 3.0 kPa. Exchange of sulfobetaine with methacrylamide (SBAm), the main polymer backbone, revealed a positive increase in energy recovery of 670 J kg-1 with a mean power of 0.19 W kg-1 for the tandem system operating under the same parameters (SPA@SBAm). The energy recovery can be controlled, modulated and tuned by selecting both hydrogels with antipolyelectrolyte and polyelectrolyte effects and their performing parameters. This proof of concept provides blue energy harvesting by contributing both polyelectrolyte and antipolyelectrolyte effects in a single tandem setup; together with easy accessibility (diaper-based materials (SPA)) and known antibiofouling, these properties offer a robust alternative for energy harvesting.

Identification of Whole-Serum Glycobiomarkers for Colorectal Carcinoma Using Reverse-Phase Lectin Microarray.

Colorectal cancer (CRC) is one of the most common types of cancer among men and women worldwide. Efforts are currently underway to find novel and more cancer-specific biomarkers that could be detected in a non-invasive way. The analysis of aberrant glycosylation of serum glycoproteins is a way to discover novel diagnostic and prognostic CRC biomarkers. The present study investigated a whole-serum glycome with a panel of 16 different lectins in search for age-independent and CRC-specific glycomarkers using receiver operating characteristic (ROC) curve analyses and glycan heat matrices. Glycosylation changes present in the whole serum were identified, which could lead to the discovery of novel biomarkers for CRC diagnostics. In particular, the change in the bisecting glycans (recognized by Phaseolus vulgaris erythroagglutinin) had the highest discrimination potential for CRC diagnostics in combination with human L selectin providing area under the ROC curve (AUC) of 0.989 (95% CI 0.950-1.000), specificity of 1.000, sensitivity of 0.900, and accuracy of 0.960. We also implemented novel tools for identification of lectins with strong discrimination power.

Breast cancer glycan biomarkers: their link to tumour cell metabolism and their perspectives in clinical practice.

INTRODUCTION: Breast cancer (BCa) is the most common cancer type diagnosed in women and 5th most common cause of deaths among all cancer deaths despite the fact that screening program is at place. This is why novel diagnostics approaches are needed in order to decrease number of BCa cases and disease mortality. AREAS COVERED: In this review paper, we aim to cover some basic aspects regarding cellular metabolism and signalling in BCa behind altered glycosylation. We also discuss novel exciting discoveries regarding glycan-based analysis, which can provide useful information for better understanding of the disease. The final part deals with clinical usefulness of glycan-based biomarkers and the clinical performance of such biomarkers is compared to already approved BCa biomarkers and diagnostic tools based on imaging. EXPERT OPINION: Recent discoveries suggest that glycan-based biomarkers offer high accuracy for possible BCa diagnostics in blood, but also for better monitoring and management of BCa patients. The review article was written using Web of Science search engine to include articles published between 2019 and 2021.

Exchange Counterion in Polycationic Hydrogels: Tunability of Hydrophobicity, Water State, and Floating Capability for a Floating pH Device.

Smart gel materials are capable of controlling and switching swelling, water state, and wettability properties triggered by external stimuli. In this study, we fabricated a series of polyelectrolyte hydrogels bearing a 3-trimethylammoniumpropyl pendant to a methacrylamide-based backbone and examined the switchability with hydrophobic-like counteranions. The exchange between the initial chloride and camphor sulfate (CaS), dodecyl sulfate (DS), and perfluorooctanoate (PFO) counterions was investigated. The kinetics of the exchange showed that the fast exchange (within 4 h) of PFO allowed for a favorable coordination for ion pairing, resulting in a decrease in hydration. The reversibility of the exchange to the Cl- ion was only enabled for the CaS ion due to its bulkiness, while the PFO and DS hydrogels were unable to exchange, even by using tetrabutylammonium chloride, which is a structurally similar reagent, due to aggregation or the coagulates in the collapsed state of the linear tails of the counterions. The hydrogels exhibited a modulable water state and water swelling. Moreover, the hydrogels containing DS and PFO, as counterions, showed surface hydrophobic (contact angle 90°) and high hydrophobic (110°) behavior, respectively. The Raman spectrometry fluorescence with a pyrene probe indicated an increase in strong hydrogen-bonded water molecules, water confinement, and hydrophobic domains in the PFO hydrogel. Moreover, the PFO-modified hydrogel demonstrated a free-floating ability on the water surface, with a strong water repellency, showing that it has the potential to be applied in a floating pH detection device to distinguish between volatile and nonvolatile bases in a controlled manner.

Nicotinamide-based supergelator self-assembling via asymmetric hydrogen bonding NH⋯OC and H⋯Br- pattern for reusable, moldable and self-healable nontoxic fuel gels.

HYPOTHESIS: Development of highly efficient low-molecular weight gelators (LMWGs) for safe energy storage materials is of great demand. Energy storage materials as fuel gels are often achieved by construction of hybrid organic frameworks capable of multiple noncovalent interactions in self-assembly, which allow tuning required properties at the molecular level by altering individual building blocks of the LMWG. However, LMWGs have limited rechargeable capability due to their chemical instability. EXPERIMENTS: We designed, synthesized and characterized a novel, bio-inspired chiral gemini amphiphile derivative 1 containing N-hexadecyl aliphatic tails from quaternized nicotinamide-based segment and bromide anion showing supergelation ability in water, alcohols, aprotic polar and aromatic solvents, with critical gel concentrations as low as 0.1 and 0.035 wt% in isopropanol and water, respectively. FINDINGS: Nanostructural architecture of the network depended on the solvent used and showed variations in size and shape of 1D nanofibers. Supergelation is attributed to a unique asymmetric NH⋯OC, H⋯Br- hydrogen bonding pattern between H-2 hydrogens from nicotinamide-based segment, amide functional groups from chiral trans-cyclohexane-1,2-diamide-based segment and bromide ions, supporting the intermolecular amide-amide interactions appearing across one strand of the self-assembly. Gels formed from 1 exhibit high stiffness, self-healing, moldable and colorable properties. In addition, isopropanol gels of 1 are attractive as reusable, shape-persistent non-toxic fuels maintaining the chemical structure with gelation efficiency for at least five consecutive burning cycles.