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.

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.

Analysis of serum glycome by lectin microarrays for prostate cancer patients - a search for aberrant glycoforms.

This is the first work focused on glycoprofiling of whole N- and O- glycome using lectins in an array format applied for analysis of serum samples from healthy individuals, benign prostate hyperplasia (BPH) patients, and prostate cancer (PCa) patients. Lectin microarray was prepared using traditional lectins with the incorporation of 2 recombinant bacterial lectins and 3 human lectins (17 lectins in total). Clinical validation of glycans as biomarkers was done in two studies: discrimination of healthy individuals with BPH patients vs. PCa patients (C vs. PCa) and discrimination of healthy individuals vs. BPH and PCa patients (H vs. PCond). Single lectins (17 lectins) and a combination of two lectins (136 binary lectin combinations) were applied in the clinical validation of glycan biomarkers providing 153 AUC values from ROC curves for both studies (C vs. PCa and H vs. PCond). Potential N- and O-glycans as biomarkers were identified and possible carriers of these glycans are shortly discussed.

Magnetization of active inclusion bodies: comparison with centrifugation in repetitive biotransformations.

BACKGROUND: Physiological aggregation of a recombinant enzyme into enzymatically active inclusion bodies could be an excellent strategy to obtain immobilized enzymes for industrial biotransformation processes. However, it is not convenient to recycle "gelatinous masses" of protein inclusion bodies from one reaction cycle to another, as high centrifugation forces are needed in large volumes. The magnetization of inclusion bodies is a smart solution for large-scale applications, enabling an easier separation process using a magnetic field. RESULTS: Magnetically modified inclusion bodies of UDP-glucose pyrophosphorylase were recycled 50 times, in comparison, inclusion bodies of the same enzyme were inactivated during ten reaction cycles if they were recycled by centrifugation. Inclusion bodies of sialic acid aldolase also showed good performance and operational stability after the magnetization procedure. CONCLUSIONS: It is demonstrated here that inclusion bodies can be easily magnetically modified by magnetic iron oxide particles prepared by microwave-assisted synthesis from ferrous sulphate. The magnetic particles stabilize the repetitive use of the inclusion bodies .

Electrochemical performance of Ti3C2Tx MXene in aqueous media: towards ultrasensitive H2O2 sensing.

An extensive characterization of pristine and oxidized Ti3C2Tx (T: =O, -OH, -F) MXene showed that exposure of MXene to an anodic potential in the aqueous solution oxidizes the nanomaterial forming TiO2 layer or TiO2 domains with subsequent TiO2 dissolution by F- ions, making the resulting nanomaterial less electrochemically active compared to the pristine Ti3C2Tx. The Ti3C2Tx could be thus applied for electrochemical reactions in a cathodic potential window i.e. for ultrasensitive detection of H2O2 down to nM level with a response time of approx. 10 s. The manuscript also shows electrochemical behavior of Ti3C2Tx modified electrode towards oxidation of NADH and towards oxygen reduction reactions.

Mixed Zwitterion-Based Self-Assembled Monolayer Interface for Impedimetric Glycomic Analyses of Human IgG Samples in an Array Format.

An impedimetric lectin biosensor for the detection of changes in the glycan structure of antibodies isolated from human serum is here correlated with the progression of rheumatoid arthritis (RA). The biosensor was built up from a mixed self-assembled monolayer (SAM) on gold consisting of two different thiolated zwitterionic derivatives, carboxybetaine and sulfobetaine, to resist nonspecific interactions. The carboxyl-terminated one was applied also for the covalent immobilization of lectin Ricinus communis agglutinin I (RCA-I). The process of building a bioreceptive layer was optimized and characterized using a diverse range of techniques. Impedimetric assays were integrated on a chip consisting of eight gold working electrodes, which is an important step toward the achievement of a moderate level of multiplexing for the analysis of human serum samples. At the end, the results obtained by the impedimetric analysis of immunoglobulins G (IgGs) isolated from serum samples were compared with those of two other standard bioanalytical methods employing lectins, that is, lectin microarrays (MAs) and enzyme-linked lectin binding assays (ELLBAs). The impedimetric results agreed very well with the DAS28 index (RA disease activity score 28), suggesting that impedimetric assays could be used for the development of a new diagnostic procedure sensitive to glycosylation changes in human IgGs and thus RA progression.

Carboxybetaine Modified Interface for Electrochemical Glycoprofiling of Antibodies Isolated from Human Serum.

Impedimetric lectin biosensors capable of recognizing two different carbohydrates (galactose and sialic acid) in glycans attached to antibodies isolated from human serum were prepared. The first step entailed the modification of a gold surface by a self-assembled monolayer (SAM) deposited from a solution containing a carboxybetaine-terminated thiol applied to the subsequent covalent immobilization of lectins and to resist nonspecific protein adsorption. In the next step, Sambucus nigra agglutinin (SNA) or Ricinus communis agglutinin (RCA) was covalently attached to the SAM, and the whole process of building a bioreceptive layer was optimized and characterized using a diverse range of techniques including electrochemical impedance spectroscopy, cyclic voltammetry, quartz crystal microbalance, contact angle measurements, zeta-potential assays, X-ray photoelectron spectroscopy, and atomic force microscopy. In addition, the application of the SNA-based lectin biosensor in the glycoprofiling of antibodies isolated from the human sera of healthy individuals and of patients suffering from rheumatoid arthritis (RA) was successfully validated using an SNA-based lectin microarray. The results showed that the SNA lectin, in particular, is capable of discriminating between the antibodies isolated from healthy individuals and those from RA patients based on changes in the amount of sialic acid present in the antibodies. In addition, the results obtained by the application of RCA and SNA biosensors indicate that the abundance of galactose and sialic acid in antibodies isolated from healthy individuals is age-related.

Ultrasensitive impedimetric lectin biosensors with efficient antifouling properties applied in glycoprofiling of human serum samples.

Ultrasensitive impedimetric lectin biosensors recognizing different glycan entities on serum glycoproteins were constructed. Lectins were immobilized on a novel mixed self-assembled monolayer containing 11-mercaptoundecanoic acid for covalent immobilization of lectins and betaine terminated thiol to resist nonspecific interactions. Construction of biosensors based on Concanavalin A (Con A), Sambucus nigra agglutinin type I (SNA), and Ricinus communis agglutinin (RCA) on polycrystalline gold electrodes was optimized and characterized with a battery of tools including electrochemical impedance spectroscopy, various electrochemical techniques, quartz crystal microbalance (QCM), Fourier transform infrared (FT-IR) spectroscopy, atomic force microscopy (AFM), and X-ray photoelectron spectroscopy (XPS) and compared with a protein/lectin microarray. The lectin biosensors were able to detect glycoproteins from 1 fM (Con A), 10 fM (Ricinus communis agglutinin (RCA), or 100 fM (SNA) with a linear range spanning 6 (SNA), 7 (RCA), or 8 (Con A) orders of magnitude. Furthermore, a detection limit for the Con A biosensor down to 1 aM was achieved in a sandwich configuration. A nonspecific binding of proteins for the Con A biosensor was only 6.1% (probed with an oxidized invertase) of the signal toward its analyte invertase and a negligible nonspecific interaction of the Con A biosensor was observed in diluted human sera (1000×), as well. The performance of the lectin biosensors was finally tested by glycoprofiling of human serum samples from healthy individuals and those having rheumatoid arthritis, which resulted in a distinct glycan pattern between these two groups.

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%.

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%.

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.

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.

Encapsulation of recombinant E. coli expressing cyclopentanone monooxygenase in polyelectrolyte complex capsules for Baeyer-Villiger biooxidation of 8-oxabicyclo[3.2.1]oct-6-en-3-one.

Recombinant Escherichia coli cells, over-expressing cyclopentanone monooxygenase activity, were immobilized in polyelectrolyte complex capsules, made of sodium alginate, cellulose sulfate, poly(methylene-co-guanidine), CaCl(2) and NaCl. More than 90% of the cell viability was preserved during the encapsulation process. Moreover, the initial enzyme activity was fully maintained within encapsulated cells while it halved in free cells. Both encapsulated and free cells reached the end point of the Baeyer-Villiger biooxidation of 8-oxabicyclo[3.2.1]oct-6-en-3-one to 4,9-dioxabicyclo[4.2.1]non-7-en-3-one at the same time (48 h). Similarly, the enantiomeric excess above 94% was identical for encapsulated and free cells.

Coencapsulation of oxygen carriers and glucose oxidase in polyelectrolyte complex capsules for the enhancement of D-gluconic acid and delta-gluconolactone production.

A novel encapsulated oxidative biocatalyst comprising glucose oxidase (GOD) coencapsulated with oxygen carriers within polyelectrolyte complex capsules was developed for the production of D-gluconic acid and delta-gluconolactone. The capsules containing immobilized GOD were produced by polyelectrolyte complexation with sodium alginate (SA) and cellulose sulfate (CS) as polyanions, poly(methylene-co-guanidine) (PMCG) as the polycation, CaCl(2) as the gelling agent and NaCl as the antigelling agent (GOD-SA-CS/PMCG capsules). Poly(dimethylsiloxane) (PDMS) and an emulsion of n-dodecane (DOD) or perfluorodecaline (PFD) with PDMS were used as the oxygen carriers and MnO(2) was used as a hydrogen peroxide decomposition catalyst. Water-soluble PDMS was found to act as both an oxygen carrier and an emulsifier of water-insoluble DOD and PFD. Stable microcapsules could be produced with concentrations of up to 4% (w/w) of PDMS, 10% (w/w) of DOD and PFD, and 25% (w/w) of MnO(2) in the polyanion solution of SA and CS. Roughly a two-fold increase in the GOD activity from 21.0+/-1.1 to 38.4+/-2.0 U*g(-1) and product space-time yields (STY) from 44.3+/-2.0 to 83.4+/-3.4 g*H*day(-1) could be achieved utilizing coencapsulated oxygen carriers compared to GOD encapsulated in the absence of oxygen carriers. This enhanced production does not significantly depend on the selected oxygen carrier under the conditions used in this study.

Ultrasensitive Ti3C2TX MXene/Chitosan Nanocomposite-Based Amperometric Biosensor for Detection of Potential Prostate Cancer Marker in Urine Samples.

Two-dimensional layered nanomaterial Ti3C2TX (a member of the MXene family) was used to immobilise enzyme sarcosine oxidase to fabricate a nanostructured biosensor. The device was applied for detection of sarcosine, a potential prostate cancer biomarker, in urine for the first time. The morphology and structures of MXene have been characterised by atomic force microscopy (AFM) and scanning electron microscopy (SEM). Electrochemical measurements, SEM and AFM analysis revealed that MXene interfaced with chitosan is an excellent support for enzyme immobilisation to fabricate a sensitive biosensor exhibiting a low detection limit of 18 nM and a linear range up to 7.8 µM. The proposed biosensing method also provides a short response time of 2 s and high recovery index of 102.6% for detection of sarcosine spiked into urine sample in a clinically relevant range.

Synthesis and characterization of Au nanoshells with a magnetic core and betaine derivatives.

The article describes preparation, characterization and further modification of hybrid magnetic particles (Au nanoshells with a magnetic core (MPs@silica@Au)) by zwitterionic molecules bearing diazonium functional groups. Such hybrid magnetic particles modified by zwitterionic molecules exhibit the following features: •Responsiveness towards external magnetic field applicable for various enrichment strategies due to magnetic core;•Golden outer layer exhibiting free surface plasmons could be used for grafting of zwitterionic molecules via diazonium functionality;•Zwitterionic interface on such particles provides resistivity towards non-specific protein binding; and at the same time such interface was applied for immobilization of antibodies against prostate specific antigen (PSA) applied for selective enrichment of PSA from serum samples with subsequent electrochemical assays. The approach presented here using hybrid magnetic particles can be easily applied for immobilization of antibodies using a highly robust surface patterning protocols i.e. by formation of a self-assembled monolayer with delivery of functional groups on the outer surface of magnetic particles. Hybrid magnetic particles with immobilized antibodies are applied for highly efficient and quick separation of protein of interest i.e. PSA from complex sample. Finally, hybrid magnetic particles with "fished-out" protein molecules could be incubated with lectins to form a sandwich configuration for glycoprofiling of PSA.

Advanced impedimetric biosensor configuration and assay protocol for glycoprofiling of a prostate oncomarker using Au nanoshells with a magnetic core.

In this paper several advances were implemented for glycoprofiling of prostate specific antigen (PSA), what can be applied for better prostate cancer (PCa) diagnostics in the future: 1) application of Au nanoshells with a magnetic core (MP@silica@Au); 2) use of surface plasmons of Au nanoshells with a magnetic core for spontaneous immobilization of zwitterionic molecules via diazonium salt grafting; 3) a double anti-fouling strategy with integration of zwitterionic molecules on Au surface and on MP@silica@Au particles was implemented to resist non-specific protein binding; 4) application of anti-PSA antibody modified Au nanoshells with a magnetic core for enrichment of PSA from a complex matrix of a human serum; 5) direct incubation of anti-PSA modified MP@silica@Au with affinity bound PSA to the lectin modified electrode surface. The electrochemical impedance spectroscopy (EIS) signal was enhanced 43 times integrating Au nanoshells with a magnetic core compared to the biosensor without them. This proof-of-concept study shows that the biosensor could detect PSA down to 1.2 fM and at the same time to glycoprofile such low PSA concentration using a lectin patterned biosensor device. The biosensor offers a recovery index of 108%, when serum sample was spiked with a physiological concentration of PSA (3.5 ng mL-1).

A crosslinked inclusion body process for sialic acid synthesis.

The propensity of a recombinant protein produced in bacteria to aggregate has been assumed to be unpredictable, and inclusion bodies have been thought of as wasted cell material. However, a target protein can be purposely driven to inclusion bodies, which demonstrate full cell tolerable activity. Sialic acid aldolase, N-terminally fused with the cellulose-binding module of Clostridium cellulovorans, was almost quantitatively physiologically aggregated into active inclusion bodies. These inclusion bodies were entrapped in alginate beads and crosslinked by glutaraldehyde. The immobilized biocatalyst generated by this crosslinked inclusion bodies (CLIB) technology was used in a repetitive batch protocol for sialic acid production that was monitored on-line by flow calorimetry. The required substrate, N-acetyl-D-mannosamine, was obtained by partially improved alkaline epimerization.

Glycomics meets artificial intelligence - Potential of glycan analysis for identification of seropositive and seronegative rheumatoid arthritis patients revealed.

In this study, one hundred serum samples from healthy people and patients with rheumatoid arthritis (RA) were analyzed. Standard immunoassays for detection of 10 different RA markers and analysis of glycan markers on antibodies in 10 different assay formats with several lectins were applied for each serum sample. A dataset containing 2000 data points was data mined using artificial neural networks (ANN). We identified key RA markers, which can discriminate between healthy people and seropositive RA patients (serum containing autoantibodies) with accuracy of 83.3%. Combination of RA markers with glycan analysis provided much better discrimination accuracy of 92.5%. Immunoassays completely failed to identify seronegative RA patients (serum not containing autoantibodies), while glycan analysis correctly identified 43.8% of these patients. Further, we revealed other critical parameters for successful glycan analysis such as type of a sample, format of analysis and orientation of captured antibodies for glycan analysis.

Immobilization of bilirubin oxidase on graphene oxide flakes with different negative charge density for oxygen reduction. The effect of GO charge density on enzyme coverage, electron transfer rate and current density.

Previously we showed that an effective bilirubin oxidase (BOD)-based biocathode using graphene oxide (GO) could be prepared in 2 steps: 1. electrostatic adsorption of BOD on GO; 2. electrochemical reduction of the BOD-GO composite to form a BOD-ErGO (electrochemically reduced GO) film on the electrode. In order to identify an optimal charge density of GO for BOD-ErGO composite preparation, several GO fractions differing in an average flake size and ζ-potential were prepared using centrifugation and consequently employed for BOD-ErGO biocathode preparation. A simple way to express surface charge density of these particular GO nanosheets was developed. The values obtained were then correlated with biocatalytic and electrochemical parameters of the prepared biocathodes, i.e. electrocatalytically active BOD surface coverage (Γ), heterogeneous electron transfer rate (kS) and a maximum biocatalytic current density. The highest bioelectrocatalytic current density of (597±25)µAcm-2 and the highest Γ of (23.6±0.9)pmolcm-2 were obtained on BOD-GO composite having the same moderate negative charge density, but the highest kS of (79.4±4.6)s-1 was observed on BOD-GO composite having different negative charge density. This study is a solid foundation for others to consider the influence of a charge density of GO on direct bioelectrochemistry/bioelectrocatalysis of other redox enzymes applicable for construction of biosensors, bioanodes, biocathodes or biofuel cells.