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.

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.

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.

Electrochemical Investigation of Interfacial Properties of Ti3C2T x MXene Modified by Aryldiazonium Betaine Derivatives.

For efficient and effective utilization of MXene such as biosensing or advanced applications, interfacial modification of MXene needs to be considered. To this end, we describe modification of Ti3C2Tx MXene by aryldiazonium-based grafting with derivatives bearing a sulfo- (SB) or carboxy- (CB) betaine pendant moiety. Since MXene contains free electrons, betaine derivatives could be grafted to MXene spontaneously. Kinetics of spontaneous grafting of SB and CB toward MXene was electrochemically examined in two different ways, and such experiments confirmed much quicker spontaneous SB grafting compared to spontaneous CB grafting. Moreover, a wide range of electrochemical methods investigating non-Faradaic and Faradaic redox behavior also in the presence of two redox probes together with contact-angle measurements and secondary ion mass spectrometry (SIMS) confirmed substantial differences in formation and interfacial presentation of betaine layers, when spontaneously grafted on MXene. Besides spontaneous grafting of CB and SB toward MXene, also electrochemical grafting by a redox trigger was performed. Results suggest that electrochemical grafting provides a denser layer of SB and CB on the MXene interface compared to spontaneous grafting of SB and CB. Moreover, an electrochemically grafted SB layer offers much lower interfacial resistance and an electrochemically active surface area compared to an electrochemically grafted CB layer. Thus, by adjusting the SB/CB ratio in the solution during electrochemical grafting, it is possible to effectively tune the redox behavior of an MXene-modified interface. Finally, electrochemically grafted CB and SB layers on MXene were evaluated against non-specific protein binding and compared to the anti-fouling behavior of an unmodified MXene interface.

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.

Prostate-specific antigen glycoprofiling as diagnostic and prognostic biomarker of prostate cancer.

The initial part of this review details the controversy behind the use of a serological level of prostate-specific antigen (PSA) for the diagnostics of prostate cancer (PCa). Novel biomarkers are in demand for PCa diagnostics, outperforming traditional PSA tests. The review provides a detailed and comprehensive summary that PSA glycoprofiling can effectively solve this problem, thereby considerably reducing the number of unnecessary biopsies. In addition, PSA glycoprofiling can serve as a prognostic PCa biomarker to identify PCa patients with an aggressive form of PCa, avoiding unnecessary further treatments which are significantly life altering (incontinence or impotence).

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).

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.