Anna Sankiewicz

Development of surface plasmon resonance imaging biosensors for detection of ubiquitin carboxyl-terminal hydrolase L1.

We have developed a new method for highly selective determination of the ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) concentration using a surface plasmon resonance imaging (SPRI) technique and two different biosensors. UCH-L1 was captured from a solution by immobilized specific rabbit monoclonal antibody or specific LDN-57444 inhibitor due to formation of receptor-UCH-L1 complex on the biosensor surface. The analytically useful dynamic response range of both biosensors is between 0.1 and 2.5ng/ml. The detection limit is 0.06ng/ml for the biosensor with antibody and 0.08ng/ml for the biosensor with inhibitor. Biosensors based on both antibody and inhibitor were found to be suitable for quantitative determination of the UCH-L1 and exhibit good tolerance to the potential interferents. Both biosensors gave comparable results in the range of 0 to 0.20ng/ml for plasma samples and 0.30 to 0.49ng/ml for cerebrospinal fluid samples. To validate the new methods, comparative determination of UCH-L1 by the commercial enzyme-linked immunosorbent assay (ELISA) kit was performed. In general, in terms of UCH-L1 concentration, a good correlation between SPRI and ELISA was found. The developed biosensors can be used successfully for the determination of UCH-L1 in body fluids.

Surface plasmon resonance imaging biosensors for aromatase based on a potent inhibitor and a specific antibody: Sensor development and application for biological material

AbstractAromatase (ARO) is an enzyme with potential diagnostic significance. Aberrant expression of aromatase in tissues is associated with a number of pathological conditions, including tumor of the breast, ovary, testes, liver, adrenal cortex and uterus, as well as endometriosis.Two methods for the highly selective determination of ARO concentration in human tissues by using of two different biosensors co-operating with the surface plasmon resonance imaging technique (SPRI) have been developed. One of the developed biosensors contains immobilised rabbit polyclonal antibody specific for aromatase (Y-ARO), while the other contains immobilized ARO inhibitor-exemestane (E-ARO). Both biosensors specifically bound ARO from analyzed samples. The analytically useful dynamic response range of both biosensors is between 0.3 and 5.0 ng mL−1. The detection limit (3S.D.) of both biosensors is 90 pg mL−1. Standard deviation of both biosensors is 1%. Recoveries of ARO spikes are between 97 and 108% for both biosensors under model conditions and for real samples. Albumin and alkaline phosphatase are tolerated for both biosensors up to 10,000 fold excess.

Podoplanin serum and urine concentration in transitional bladder cancer

BACKGROUND Podoplanin (PDP) is a mucin - a type of transmembrane protein expressed in numerous tissues during ontogeny and in adult animals, including the brain, heart, kidney, osteoblasts and lymphoid organs. OBJECTIVE The aim of this study was to determine podoplanin concentration in the blood serum and urine of patients with bladder cancer. Quantifying podoplanin concentration and its correlation with various clinicopathological parameters may be useful for more accurate predictions and identifying high-risk patients. METHODS The present study included 82 patients with bladder cancer confirmed by transurethral resection or cystectomy and 27 healthy volunteers. The Surface Plasmon Resonance Imaging biosensor was applied for the detection of podoplanin in the serum and urine samples. RESULTS Significant differences in serum and urine podoplanin concentration levels were observed between bladder cancer patients. The statistically significant higher values of PDP were detected in serum of patients with invasive, more aggressive, larger, multifocal tumors. CONCLUSIONS The association between podoplanin concentration and clinicopathological features indicates that it might be useful while making therapeutic decisions.

Overexpression of ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) in serum of children after thermal injury

PURPOSE The study aims to determinate concentrations of ubiquitin C-terminal hydrolase 1 (UCHL1), which hydrolyzes amino acids from ubiquitin and cleave di-ubiquitins, in serum of children after thermal injury. PATIENTS/METHODS 42 children scalded by hot water, managed at the Department of Pediatric Surgery, with burns in 4-20% TBSA were included into the study (age 9 months up to 14 years, mean age 2.5±1 years). Blood plasma UCHL1 concentration was assessed in 2-6h, 12-16h, 3d, 5d, and 7d after injury using surface plasmon resonance imaging biosensor. 18 healthy subjects admitted for planned surgeries served as controls. RESULTS The UCHL1 concentration in the blood plasma of patients with thermal injuries reached its peak 12-16h after thermal injury and slowly decreased over time, and still did not reach the normal range on the 7th day after thermal injury. Mean concentrations of UCHL1 after thermal injury were above the range measured in controls (0.12ng/ml): 2-6h after injury - 5.59ng/dl, 12-16h after injury - 9.16ng/dl, 3 days after injury - 6.94ng/dl, 5 days after 5.41ng/dl, 7 days after injury - 4.09ng/dl. CONCLUSIONS We observed sudden increase in the concentration of UCHL1 2-16h after thermal injury with the slow decrease in the UCHL1 concentration over the time. UCHL1 concentration was proportional to the severity of the burn. Further studies are needed to determine the mechanisms by which UCHL1 contributes to metabolic response following thermal injury.

Analytical applications of electrode sensitive to labetalol in pharmaceuticals

The analytical properties of an ion-selective electrode sensitive to labetalol with a liquid membrane, based on ion-pair complexes with sodium tetraphenylborate (TPB-Na+) are described. The studied electrode can be used for the determination of labetalol hydrochloride as a protonated form of labetalol in pharmaceuticals. The calibration curve, e.g. EMF=f(pCLabHCl) is linear in the range from 10−5 to 10−2 mol L−1 with a correlation coefficient of 0.9992 and slope of 61.13 mV/decade, which is close to the Nernstian slope. The detection limit of the examined electrode is 7.20×10−6 mol L−1. The influence of pH of the tested solutions on the formulation of the electrode is not as considerable since the electrode works correctly in the pH range 3.0–8.0. The main attributes of the developed electrode are: stability, good reproducibility of EMF and short response time, close to 30 seconds depending on labetalol concentration in the solution. The electrode shows good selectivity for many inorganic ions. The selectivity for drug cations is weaker due to the structural similarity of the interfering cations to labetalol. The results of labetalol determination using direct potentiometry in drugs such as Pressocard (Polpharma) and Trandate (GlaxoWellcome) were compatible with the quantity of labetalol declared by the manufacturer, and with parallel UV spectrophotometric and HPLC determinations.

Surface Plasmon Resonance Imaging biosensor for cystatin determination based on the application of bromelain, ficin and chymopapain

A Surface Plasmon Resonance Imaging (SPRI) sensor based on bromelain or chymopapain or ficin has been developed for specific cystatin determination. Cystatin was captured from a solution by immobilized bromelain or chymopapain or ficin due to the formation of an enzyme-inhibitor complex on the biosensor surface. The influence of bromelain, chymopapain or ficin concentration, as well as the pH of the interaction on the SPRI signal, was investigated and optimized. Sensor dynamic response range is between 0–0.6 μg/ml and the detection limit is equal to 0.1 μg/ml. In order to demonstrate the sensor potential, cystatin was determined in blood plasma, urine and saliva, showing good agreement with the data reported in the literature.

In Vitro Interaction of Lithium on Phospholipids in Human Erythrocytes

ABSTRACT Lithium salts are used in the treatment of mania and as prophylaxis against manic depressive disorder. The aim of these studies was the in vitro investigation of the effect of lithium on phospholipids of human erythrocyte membranes. Erythrocytes were treated with lithium for 1 h. Phospholipids phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylethanolamine (PE), and phosphatidylocholine (PC) were separated from erythrocyte ghosts and determined by HPLC. Blood samples from healthy adults were investigated. A very strong decrease in PC content in erythrocyte membranes due to lithium in vitro treatment was found, as well as a statistically significant increase in PI content.

SPR imaging biosensor for the quantitation of fibronectin concentration in blood samples

The purpose of this study was presentation of a new biosensor capable of determination of fibronectin. This biosensor was based on the specific interaction of anti-fibronectin antibody produced in rabbit with fibronectin. The surface plasmon resonance imaging (SPRI) technique was used as a detecting method. Optimization and characterization properties of the biosensor were studied. The determination of fibronectin concentration in natural samples was done. The results were compared with a reference method (Enzyme-Linked Immunosorbent Assay-ELISA). The analytically useful dynamic response range of biosensor is between 5 and 400ngmL-1. The detection limit is 1.5ngmL-1 and limit quantification is 5ngmL-1. The proposed SPRI biosensor showed good selectivity for potential interferences. It was applied to determine fibronectin concentrations in plasma of healthy donors and of patients after thermal injury. Good correlations between results obtained using the SPRI biosensor and ELISA test (correlation coefficients for healthy donors 0.996, for patients 0.984) were obtained. The average fibronectin concentration of healthy donors was 140.5±24.6μgmL -1 and the average fibronectin concentration of patients was 601.5±72.1μgmL -1, which was in agreement with results obtained by other investigators. The obtained results indicate that the developed biosensor may be a candidate for monitoring fibronectin concentration in blood samples.

Immunoproteasome in the Plasma of Pediatric Patients With Moderate and Major Burns, and Its Correlation With Proteasome and UCHL1 Measured by SPR Imaging Biosensors

The aim of this study was to determine the immunoproteasome concentration in blood plasma of pediatric patients with moderate and major burns and its correlation with circulating proteasome and ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) with surface plasmon resonance imaging biosensor. The study population comprised of 30 patients with moderate (n = 21) and severe burns (n = 9), aged 9 months to 14 years. The control group represented 18 healthy, age-matched patients, admitted for herniotomy. Exclusion criteria were as follows: admission to the hospital later than 6 hours after burn, cardiovascular or immunological diseases, and severe preexisting infections. Mean concentrations of immunoproteasome, 20S proteasome, and UCHL1 in the blood plasma of children with burns-4 to 6 hours, 12 hours, 3 days, 5 days, and 7 days after thermal injury-were above the levels measured in controls. The immunoproteasome, 20S proteasome, and UCHL1 concentrations in the blood plasma of their patients were highest 12 hours after burn, slowly decreased over time, and on the 5th day still were higher than in controls (P < .05). There was a strong correlation between immunoproteasome and 20S proteasome concentrations 6 hours and 5 days after burn, and moderate correlation 12 hours after burn (P < .05). The immunoproteasome concentration is elevated after burn injury and slowly reaches the normal range during the wound healing process. There is strong correlation between immunoproteasome and 20S proteasome concentrations in the serum of children with moderate and major burns. They did not find such correlation between immunoproteasome and UCHL1 concentrations. Immunoproteasome concentration do not correlate with age or sex.

Immunoproteasome in the of children with acute appendicitis, and its correlation with proteasome and UCHL1 measured by SPR imaging biosensors

The aim of this study was to determinate the immunoproteasome concentration in the blood plasma of children with appendicitis, and its correlation with circulating proteasome and ubiquitin carboxyl‐terminal hydrolase L1 (UCHL1). Twenty‐seven children with acute appendicitis, managed at the Paediatric Surgery Department, were included randomly into the study (age 2 years 9 months up to 14 years, mean age 9·5 ± 1 years). There were 10 girls and 17 boys; 18 healthy, age‐matched subjects, admitted for planned surgeries served as controls. Mean concentrations of immunoproteasome, 20S proteasome and UCHL1 in the blood plasma of children with appendicitis before surgery 24 h and 72 h after the appendectomy were higher than in the control group. The immunoproteasome, 20S proteasome and UCHL1 concentrations in the blood plasma of patients with acute appendicitis were highest before surgery. The immunoproteasome, 20S proteasome and UCHL1 concentration measured 24 and 72 h after the operation decreased slowly over time and still did not reach the normal range (P < 0·05). There was no statistical difference between immunoproteasome, 20S proteasome and UCHL1 concentrations in children operated on laparoscopically and children after classic appendectomy. The immunoproteasome concentration may reflect the metabolic response to acute state inflammation, and the process of gradual ebbing of the inflammation may thus be helpful in the assessment of the efficacy of treatment. The method of operation – classic open appendectomy or laparoscopic appendectomy – does not influence the general trend in immunoproteasome concentration in children with appendicitis.