The 20S proteasome is a multicatalytic enzyme complex responsible for intracellular protein degradation in mammalian cells. Its antigen level or enzymatic activity in blood plasma are potentially useful markers for various malignant and nonmalignant diseases. We have developed a method for highly selective determination of the 20S proteasome using a Surface Plasmon Resonance Imaging (SPRI) technique. It is based on the highly selective interaction between the proteasome's catalytic β5 subunit and immobilized inhibitors (the synthetic peptide PSI and epoxomicin). Inhibitor concentration and pH were optimized. Analytical responses, linear ranges, accuracy, precision and interferences were investigated. Biosensors based on either PSI and epoxomicin were found to be suitable for quantitative determination of the proteasome, with a precision of ±10% for each, and recoveries of 102% and 113%, respectively, and with little interference by albumin, trypsin, chymotrypsin, cathepsin B and papain. The proteasome also was determined in plasma of healthy subjects and of patients suffering from acute leukemia. Both biosensors gave comparable results (2860 ng·mL-1 on average for control, and 42300 ng·mL-1 on average for leukemia patients).FigureThe synthetic peptide aldehyde Z-Ile-Glu(OBut)-Ala-Leu-H (PSI) and a microbial α',β' epoxyketone peptide epoxomicin was used to develop SPRI biosensor for the highly selective determination of the 20S proteasome concentration, and to evaluate the sensor applicability for the determination of 20S proteasome in human blood plasma.

The 20S proteasome, released into the circulation, is a novel cancer biomarker. It exists in two forms: the constitutive proteasome (20Sc) and the immunoproteasome (20Si), which both have separate diagnostic significance. The aim of this work was to develop new methods for 20Si and 20Sc determination. Five alternative specific biosensors usable with the surface plasmon resonance imaging (SPRI) technique for 20Si determination have been developed. Specific 20Si entrapment on the biosensor surface from an analyzed solution was achieved by means of an immobilized specific 20Si receptor. Four of the biosensors contain newly synthesized specific 20Si receptors, while the fifth contains the inhibitor ONX 0914. A method for 20Sc determination using an SPRI biosensor containing PSI inhibitor has been developed. By the introduction of an inhibitor blocking 20Si, 20Sc is selectively determined. All of the methods developed for 20Si and 20Sc determination exhibit good selectivity and satisfactory precision, recoveries and dynamic response ranges. 20Si and 20Sc were determined in blood plasma samples from healthy donors and patients with acute leukemia. In the case of these patients 20Si was the major component, and its level was more than one order of magnitude higher than in the healthy donors.

Alzheimer's disease (AD) is the most common form of dementia in older people. Its prevalence is expected to increase, and therefore it poses a serious challenge to the healthcare system. The aim of the study was to assess the concentration of leptin, cystatin C, neuropilin-1 and tau protein, as well as the influence of dietary habits on these parameters, in a group of AD patients (n = 110) compared to 60 healthy people (n = 60). It has been shown that AD patients, compared to healthy people, are characterized by significantly higher median concentrations of leptin (9.97 vs. 3.08), cystatin c (1.53 vs. 0.56) and tau protein (8.46 vs. 4.19), but significantly lower median neuropilin-1 (69.94 vs. 167.28). Multiple regression analyses showed that leptin levels could be explained by dietary habits in 27%, cystatin C in 51%, neuropilin-1 in 41% and tau protein in 25% of cases. Modification of eating habits may contribute to improving the values of the discussed parameters.

Tripeptides of the general X-SO2-d-Ser-AA-Arg-CO-Y formula, where X = α-tolyl, p-tolyl, 2,4,6-triisopropylphenyl; AA = alanine, glycine, norvaline and Y = OH, NH-(CH2)5NH2 were obtained and tested for their effect on the amidolytic activities of urokinase, thrombin, trypsin, plasmin, t-PA and kallikrein. The most active compound towards urokinase was PhCH2SO2-d-Ser-Gly-Arg-OH with Ki value 5.4 μM and the most active compound toward thrombin was PhCH2SO2-d-Ser-NVa-Arg-OH with Ki value 0.82 μM. The peptides were nontoxic against porcine erythrocytes in vitro. PhCH2SO2-d-Ser-Gly-Arg-OH showed cytotoxic effect against DLD cell lines with IC50 values of 5 μM. For the highly selective determination of the interaction of some of the synthesised acids of tripeptides with urokinase and plasmin the Surface Plasmon Resonance Imaging sensor has been applied. These compounds bind to urokinase and plasmin in 0.05 mM concentration.

Alzheimer's disease (AD) is a neurodegenerative disease and the most common form of dementia in the elderly. In recent years, markers of this disease have been researched, with an emphasis on prophylaxis. The aim of this study is to evaluate the concentration of fibronectin and MMP-1 in serums in relation to levels of antioxidant elements, as well as eating habits in the group of patients with AD (n = 110). The control group consisted of 60 healthy people. The conducted studies showed that patients with AD are characterized by a significantly higher median concentration of fibronectin compared to healthy subjects (652.06 vs. 268.31 µg/mL), but a significantly lower median of MMP-1 (4.62 vs. 18.09 ng/mL). Significant inverse correlations between MMP-1 and the concentration of antioxidant elements, as well as positive correlations between MMP-1 vs. Total Antioxidant Status (TAS) and MMSE, were observed. Multiple regressions showed that the concentration of fibronectin can be explained in 28% cases by eating habits, and by MMP-1 in 25%. Nutritional modifications to reduce the consumption of fruit, meat and processed products can be part of AD prevention.

Cathepsin G (CatG) is an endopeptidase that is associated with the early immune response. The synthetic compound cathepsin G inhibitor I (CGI-I) was tested for its ability to inhibit the activity of CatG via a new surface plasmon resonance imaging assay. CGI-I was immobilized on the gold surface of an SPR sensor that was first modified with 1-octadecanethiol. A concentration of CGI-I equal to 4.0 μg·mL-1 and a pH of 8.0 were found to give the best results. The dynamic response of the sensor ranges from 0.25 to 1.5 ng·mL-1, and the detection limit is 0.12 ng·mL-1. The sensor was applied to detect CatG in human saliva and white blood cells.FigureThe synthetic compound cathepsin G inhibitor I (CGI-I) was tested for its ability to inhibit the activity of cathepsin G via a newly developed surface plasmon resonance imaging assay. The sensor was applied to detect cathepsin G in human saliva and white blood cells.

Research on the markers of immunoregulatory response in multiple sclerosis (MS) is still of great importance. The aim of our study was the evaluation of leptin, fibronectin, and UCHL1 concentrations as potential biomarkers of a relapsing-remitting type of MS (RRMS). Surface Plasmon Resonance Imaging (SPRI) biosensors were used for the evaluation of proteins concentrations in 100 RRMS patients and 46 healthy volunteers. Plasma leptin, fibronectin, and UCHL1 concentrations were significantly higher in RRMS patients compared to the control group (p < 0.001, respectively). UCHL1 concentration evaluation revealed the highest diagnostic sensitivity (100%) and negative predictive value (100%) in differentiating MS patients from healthy individuals. There was no significant difference in the UCHL1 concentrations depending on the patient's sex, the presence of relapse within the last 24 months, and the EDSS value (p > 0.05, respectively). In RRMS patients UCHL1 concentration positively correlated with fibronectin levels (r = 0.3928; p < 0.001). In the current cohort of patients plasma UCHL1 concentration was independent of the time of MS relapse and the severity of neurological symptoms. Thus current study may indicate that plasma UCHL1, besides leptin and fibronectin, also could be a promising high-sensitive potential biomarker of relapsing-remitting type of MS. However, these results should be validated with a larger group of patients, taking into account neuroimaging and cerebrospinal fluid analysis data, and by comparing them to patients with other neurological diseases as a control group.

Interleukin-6 (IL-6) is a biomarker of inflammation, the advanced stage of COVID-19, and several cancers, including ovarian cancer. Two biosensors for the determination of IL-6 in blood plasma by array SPRi have been developed. One of these biosensors consists of the mouse monoclonal anti-IL-6 antibody as the receptor immobilized via the cysteamine linker. The second contains galiellalactone as the receptor, being an inhibitor specific for IL-6, immobilized via octadecanethiol (ODM) as the linker. Both biosensors are specific for IL-6. The biosensor with the antibody as the receptor gives a linear analytical response between 3 (LOQ) and 20 pg mL-1 and has a precision between 8% and 9.8% and recovery between 97% and 107%, depending on the IL-6 concentration. The biosensor with galiellalactone as the receptor gives a linear analytical response between 1.1 (LOQ) and 20 pg mL-1, and has a precision between 3.5% and 9.3% and recovery between 101% and 105%, depending on IL-6 concentration. Both biosensors were validated. Changes in IL-6 concentration in blood plasma before and after resection of ovarian tumor and endometrial cyst, as determined by the two developed biosensors, are given as an example of a real clinical application.