Bladder carcinoma (BC) is evaluated as the ninth most common cancer worldwide and the sixth most common cancer among men. The determination of the occurrence and stage of the disease is a significant diagnostic task. An alternative to an invasive biopsy may be the determination of biomarkers in patient samples such as bladder tissue, blood serum, plasma, or urine samples. The aim of this paper is to review reports on the role of proteases in bladder cancer and their determination in cancerous samples. Proteases can be classified in several groups depending on their catalytic residue, most commonly aspartic, cysteine, serine, metalloproteinases, and others. A review was made of papers reporting cysteine cathepsins: B, L, H, V, S, aspartyl cathepsin D, and metalloproteinases MMP 1, 2, 3, 7, 9, 10, 14, and 15, as well as ubiquitin-specific proteases USP 1, 2a and 5. The majority of the reviewed papers show an increase in marker concentration in bladder cancer samples versus a control. Only a few of them provide quantitative information about MMP biomarkers in bladder tissue (cancerous and control tissue), and none give such information about cathepsins. Levels of the order of µg/g protein are characteristic of MMP biomarkers in urinary bladder tissue. Most reported concentrations of proteases in blood serum/plasma and urine are at levels of ng/mL, both cancerous and control samples. It is concluded that the reviewed papers do not provide a clear picture concerning the use of proteases as bladder cancer biomarkers or concerning the levels of particular proteases in control samples. Potential new analytical tools for protease determination are discussed. More work in this area is necessary, especially by scientists equipped with new analytical tools.

Diagnostics based on the determination of biomarkers in body fluids will be more successful when several biomarkers are determined. A multiple-array SPRi biosensor for the simultaneous determination of CA125, HE4, CEA, IL-6 and aromatase has been developed. Five individual biosensors were placed on the same chip. Each of them consisted of a suitable antibody covalently immobilized onto a gold chip surface via a cysteamine linker by means of the NHS/EDC protocol. The biosensor for IL-6 works in the pg mL−1 range, that for CA125 in the µg mL−1 range, and the other three within the ng mL−1 range; these are ranges suitable for the determination of biomarkers in real samples. The results obtained with the multiple-array biosensor are very similar to those obtained with a single biosensor. The applicability of the multiple biosensor was demonstrated using several examples of plasma from patients suffering from ovarian cancer and endometrial cyst. The average precision was 3.4% for the determination of CA125, 3.5% for HE4, 5.0% for CEA and IL-6, and 7.6% for aromatase. The simultaneous determination of several biomarkers may be an excellent tool for the screening of the population for earlier detection of diseases.

Vascular endothelial growth factor receptor 2 (VEGF-R2) is a marker of angiogenesis and metastasis of cancer. Two biosensors for the determination of VEGF-R2 in plasma have been developed. One of them is based on a pure gold chip, and the other on a silver/gold bimetallic chip; both have the receptor, monoclonal rabbit antibody specific for human VEGF-R2, attached to the chip via a cysteamine linker. The biosensor with the gold chip exhibits linearity of the analytical signal between 0.03 and 2 ng/mL, a precision of 1.4% and recovery between 99% and 102%. The biosensor with the bimetallic chip exhibits linearity between 0.03 and 1 ng/mL, a precision of 2.2% and recovery between 99% and 103%. Both biosensors tolerate a 1:100 excess of VEGF, VEGF-R1 and VEGF-R3. Both biosensors were validated by parallel determination of VEGF-R2 in 27 different plasma samples using the ELISA immunosensor assay, with very good agreement of the results. Thermodynamic parameters of the interaction of VEGF-R2 with the antibody were determined by QCM (Quartz Crystal Microbalance) and SPRi (Surface Plasmon Resonance imaging) measurements.

A new method for the determination of cadherin 12 (CDH12)—an adhesive protein that has a significant impact on the development, growth, and movement of cancer cells—was developed and validated. The method is based on a biosensor using surface plasmon resonance imaging (SPRi) detection. A quartz crystal microbalance was used to analyze the characteristics of the formation of successive layers of the biosensor, from the linker monolayer to the final capture of CDH12 from solution. The association equilibrium constant (KA = 1.66 × 1011 dm3 mol−1) and the dissociation equilibrium constant (KD = 7.52 × 10−12 mol dm−3) of the anti-CDH12 antibody–CDH12 protein complex were determined. The determined analytical parameters, namely the values determining the accuracy, precision, and repeatability of the method, do not exceed the permissible 20% deviations specified by the aforementioned institutions. The proposed method is also selective with respect to possible potential interferents, occurring in up to 100-fold excess concentration relative to the CDH12 concentration. The determined Limit of Quantification (LOQ = 4.92 pg mL−1) indicates the possibility of performing quantitative analysis in human plasma or peritoneal fluid without the need to concentrate the samples; however, particular attention should be paid to their storage conditions, as the analyte does not exhibit high stability. The Passing–Bablok regression model revealed good agreement between the reference method and the SPRi biosensor, with ρSpearman values of 0.961 and 0.925.

Circulating body fluids such as blood, urea, saliva, cerebrospinal fluid, etc [...].

Follicle-stimulating hormone (FSH) regulates the development, growth, pubertal maturation and reproductive processes of the human body. The determination of serous FSH concentration is significant as an alternative to testicular biopsy in the case of boys suffering from cryptorchidism after orchidopexy, and as a means of determining the menopausal stage in women. The aim of this investigation is to develop a specific array surface plasmon resonance imaging (SPRi) biosensor for the determination of FSH in body liquids such as blood plasma, obtaining sufficient sensitivity to determine FSH at levels characteristic for that hormone in blood plasma, without any signal enhancement. The biosensor consists of a mouse monoclonal anti-FSH antibody attached to the gold surface of a chip via a cysteamine linker. Its linear response range is from 0.08 mIU mL-1 (LOQ) to 20 mIU mL-1, and well covers most of the range of FSH activities found in blood without dilution. The precision of measurement is between 3.2% and 13.1% for model samples, and between 3.7% and 5.6% for spiked plasma samples. Recoveries are in the range from 94% to 108%. The biosensor has good selectivity, and is validated by comparison with ECLE, with good agreement of the results

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.

Cortisol is a hormone which plays an essential role in the immune, endocrine, cardiovascular, renal and skeletal systems. Its level increases in response to stress, illness, injury or exhaustion, and it is therefore a significant diagnostic biomarker of stress. An immunosensor for the determination of cortisol by SPRi array was developed. The receptive part of the immunosensor is mouse monoclonal antibody against cortisol, immobilized via cysteamine linker. The optimum pH of the immunosensor is 7.4, and the optimum concentration of the antibody is 50 ng mL−1. The immunosensor is specific for cortisol, and its linear response ranges from 0.20 ng mL−1 (LOQ) to 8 ng mL−1. The precision of the determination was between 3.1% and 3.3%, and the recovery between 99% and 102%. The immunosensor was validated by simultaneous determination of cortisol in serum and saliva samples by a standard method, with good agreement between the results.

A new method was developed for the simultaneous determination of vascular endothelial growth factor (VEGF-A) and fibroblast growth factor-2 (FGF-2) in blood serum, using biosensors with array Surface Plasmon Resonance imaging (SPRi) detection. It can be applied as a single method for simultaneous VEGF-A and FGF-2 determination or as two separate methods for testing only one selected protein in each case. Validation was carried out for each method. Limit of detection (LOD) and limit of quantification (LOQ) values were determined and were found not to differ significantly from the parameters obtained in comparisons with commercial enzyme-linked immunosorbent assay (ELISA) tests. Tests were carried out to check the robustness of the method. The results indicate a lack of robustness of the analytical method to elevated temperature and pH values other than those recommended by the manufacturers of the reagents (recommended pH = 7.40). The values of recoveries were determined and confirmed the reliability of the results obtained with the use of the newly developed method. The selectivity studies showed no negative influence of other proteins present in the matrix of the tested samples on the results of the VEGF-A and FGF-2 concentration measurements. The developed method is also characterized by high reproducibility of the results obtained and agreement with the VEGF-A and FGF-2 concentration values obtained with commercial ELISA tests. The proposed method offers fast, reproducible, and accurate simultaneous quantification of VEGF-A and FGF-2 in human body fluids. Only 4 µL of test sample are required for simultaneous analysis. The total time for simultaneous analysis of both biomarkers does not exceed 20 min. The developed analytical method is superior to ELISA in terms of analysis time and sample volume for analysis, and it offers lower LOD and LOQ values and allows for the simultaneous analysis of two biomarkers. There is also no need to collect a large number of samples. Standard ELISAs usually have 96 reaction wells. The proposed biosensor can be used to analyse only one sample, without the need to waste reagents on unused reaction sites. In addition, it is possible to regenerate the biosensor and reuse it.

Surface Plasmon Resonance Imaging (SPRi) and the Quartz Crystal Microbalance (QCM) technique were used to characterize the interactions between fibronectin and a specific monoclonal antibody against fibronectin. These techniques were used to investigate the formation of successive layers of the biosensor and how they affect the biosensor's response. The analytical response of both detectors to fibronectin concentration is linear in the range 5-100 ng/mL. The changes in mass on the surface of a quartz crystal covered with a layer of gold during biosensor formation were investigated with a QCM. The equilibrium constant was determined to be KC = 1.22 center dot 108 dm3/mol and the dissociation constant to be KD= 8.20 center dot 10-9 mol/dm3.

Non-fluidic array SPR imaging (SPRi) with appropriate biosensors is a new tool for the determination of various biomarkers in body fluids. Numerous biomarkers can be determined without signal enhancement or preliminarily preconcentration. The introduction of a new material solution of the chip may increase the scope of the application of this technique. Solutions with adhesive separating foil and an Ag/Au chip were compared with the previously used two-paint separating polymer and pure gold chip. These solutions were tested using the example of a biosensor for cathepsin D (Cath D), which consisted of pepstatin A (a Cath D inhibitor) immobilized via a cysteamine linker using the NHS/EDC protocol. Four material versions of the Cath D biosensor proved adequate in terms of range of linearity, LOQ, precision and recovery. All four versions of the biosensor were used for the determination of Cath D in the blood serum patients with glioblastoma and control samples, producing very similar results and showing an elevated biomarker concentration in the case of cancer. Therefore, the problem of determining the correct level of Cath D in the serum of healthy individuals has been resolved, correcting literature data which ranged over three orders of magnitude.

Cathepsin S is an emerging marker for ovarian cancer. Two 'analytically specific' SPRi biosensors for the determination of Cath S have been developed. The reception part of one of the biosensors consists of the rat monoclonal antibody specific for cathepsin S attached to the gold surface via covalent bonds with cysteamine linker, while the second biosensor consists of the inhibitor LY3000328 attached via hydrophobic interaction with the 1-octadecanothiol linker. Under optimized conditions, in terms of pH and receptor concentration, both biosensors have linear response ranges between LOQ (0.14 ng mL(-1)) and 2.5 ng mL(-1), which is suitable for the determination of Cath S in blood plasma samples of ovarian cancer patients and healthy individuals, after corresponding dilution with 0.15 M PBS buffer. Precision and recoveries are quite acceptable: below 7% and 98-101% respectively for the biosensor with antibody, and below 12% and 101-103% for the biosensor with inhibitor. The biosensors were validated by the determination of Cath S in series of plasma from ovarian cancer patients and healthy volunteers using both biosensors and ELISA, giving Pearson coefficients close to 1. Plasma Cath S concentration can be used as an ovarian cancer marker, in view of the highly elevated concentrations detected.

Leptin is a hormone that has a fundamental role in the regulation of feeding and energy balance. A developed specific SPRi immunosensor for leptin may be a new tool for leptin determination in blood plasma. The immunosensor consists of rabbit anti-leptin antibody immobilized on a gold chip via cysteamine linker, using the EDC/NHS protocol. Non-fluidic array SPRi is used for analytical signal formation. Under optimized conditions, the linear response range of the immunosensor covers concentrations from 0.23 to 5 ng mL(-1). The LOD of the immunosensor is 0.07 ng mL(-1), and the LOQ is 0.23 ng mL(-1). The precision of measurement depends on leptin concentration, and is between 9.1% and 2.2%. Recoveries of the leptin spike are between 97% and 110%. The immunosensor and related analytical method were validated by parallel determination of leptin in series of plasma from children suffering from malnutrition and a control group, using Enzyme-Linked Immunosorbent Assay (ELISA) and SPRi. Pearson's correlation coefficient was equal to 0.991. The developed immunosensor and related method are more direct, faster and much simpler than ELISA.

Human epididymis protein 4 (HE4) is an ovarian cancer marker. Various cut-off values of the marker in blood are recommended, depending on the method used for its determination. An alternative biosensor for HE4 determination in blood plasma has been developed. It consists of rabbit polyclonal antibody against HE4, covalently attached to a gold chip via cysteamine linker. The biosensor is used with the non-fluidic array SPRi technique. The linear range of the analytical signal response was found to be 2–120 pM, and the biosensor can be used for the determination of the HE4 marker in the plasma of both healthy subjects and ovarian cancer patients after suitable dilution with a PBS buffer. Precision (6–10%) and recovery (101.8–103.5%) were found to be acceptable, and the LOD was equal to 2 pM. The biosensor was validated by the parallel determination of a series of plasma samples from ovarian cancer patients using the Elecsys HE4 test and the developed biosensor, with a good agreement of the results (a Pearson coefficient of 0.989). An example of the diagnostic application of the developed biosensor is given—the influence of ovarian tumor resection on the level of HE4 in blood serum.

A review is made of 71 papers on surface plasmon resonance biosensors, published between 2005 and 2020, mostly in the last decade. The reviewed papers are divided into two groups, depending on the validation of the developed biosensor. Validated biosensors are briefly characterized, while those that are not validated are listed in a table. Focus is placed on applications of SPR biosensors in testing the effectiveness of cancer markers and in the discovery of new cancer markers. Seven new markers are proposed, two of them having high sensitivity and diagnostic selectivity as determined by ROC curves. Papers concerning the determination of micro RNA and large particles such as vesicles, exosomes and cancer cells are also reviewed.

The array SPR imaging (SPRi) technique is well suited to the determination of biomarkers in body fluids, called liquid biopsy. No signal enhancement or analyte preconcentration is required. With the aim of achieving signal enhancement and lowering the cost of a single determination, the replacement of gold-covered chips by silver–gold chips was investigated. The aim of this work was to investigate the analytical characteristics of a biosensor formed on a Ag/Au chip and to compare them with those of a biosensor formed on a gold chip. A biosensor for the determination of cathepsin S (Cath S) was chosen as an example. The biosensor consisted of the linker cysteamine and an immobilized rat monoclonal antibody specific for cathepsin S. Both biosensors exhibited a Langmuirian response to Cath S concentration, with linear response ranging from LOQ to 1.5 ng mL−1. The LOQ is 0.1 ng mL−1 for the biosensor formed on the Ag/Au chip, and 0.22 ng mL−1 for that formed on the gold chip. Recoveries and precision for medium and high Cath S concentrations were acceptable for both biosensors, i.e., precision better than 10% and recoveries within the range 102–105%. However, the results for the lowest Cath S concentration were better for the biosensor formed on the Ag/Au chip (9.4 and 106% for precision and recovery, respectively). Generally, no significant differences in analytical characteristics were observed between the Ag/Au and Au chips. The two biosensors were also compared in the determination of Cath S in real samples. Nine plasma samples from healthy donors and nine from patients with ovarian cancer were analyzed for Cath S concentration with the biosensors formed on Ag/Au and Au chips. The results obtained with the two biosensors were very similar and show no significant differences on the Bland–Altman plot. The Cath S concentration in the blood plasma of ovarian cancer patients was elevated by one order of magnitude as compared with the control (12.6 ± 3.6 vs. 1.6 ± 1.2 ng mL−1).

Carcinoembryonic antigen (CEA) is one of the biomarkers most commonly used to determine tumor activity. In this work, a Surface Plasmon Resonance imaging (SPRi) immunosensor was developed. The immunosensor consists of a cysteamine linker attached to a gold chip and mouse monoclonal anti-CEA antibody bonded by the "EDC/NHS protocol". The formation of successive immunosensor layers was confirmed by AFM measurements. The concentration of the antibody was optimized. The linear response range of the developed immunosensor is between 0.40 and 20 ng mL(-1), and it is suitable for CEA measurement in both blood cancer patients and healthy individuals. Only 3 mu L of serum or plasma sample is required, and no preconcentration is used. The method has a precision of 2-16%, a recovery of 101-104% depending on CEA concentration, a detection limit of 0.12 ng mL(-1) and a quantification limit of 0.40 ng mL(-1). The method is selective (with respect to albumin, leptin, interleukin 6, metallopmteinase-1, metallopeptidase inhibitor 1 and CA 125/MUC16) and it was validated by comparison with the standard electrochemiluminescence method on a series of colorectal cancer blood samples.

CA125/MUC16 is an ovarian tumor cell marker widely used as a biomarker in epithelial ovarian carcinoma. CA125/MUC16 is also used for evaluation of the ROMA (Risk of Ovarian Malignancy Algorithm) value. In this work, a Surface Plasmon Resonance Imaging (SPRI) biosensor for circulating CA125/MUC16 has been developed. The anti-MUC16 antibody was attached to a gold chip via a cysteamine linker. The EDS/NHS protocol was used for the covalent attachment of the antibody. The developed biosensor is specific for CA125/MUC16, and. exhibits good recovery and acceptable precision. Its linear response range (2.2-150 U/ml) is well suited to determination of the marker in the blood serum of a healthy control group and, after appropriate dilution, of patients with ovarian cancer. CA125/MUC16 was determined in two series of real samples: blood serum from patients with ovarian cancer and endometrial cysts. The method was validated by parallel determination of the samples using the chemiluminescent Architect i2000 method.

Bladder cancer (BCa) is the ninth most common cancer in the world and its early detection is crucial for successful therapy. Unfortunately, there are no satisfactory tools to detect BCa at early stages and BCa's confirmation muscle-invasive. The search for a suitable biomarker is therefore necessary and aromatase is a potential candidate. The purpose of the current study was to determine if aromatase serves as a biomarker of BCa. A Surface Plasmon Resonance Imaging biosensor was applied for the quantification and determination of aromatase. A total of 3 mu l blood plasma was used for a single measurement. The results revealed that the aromatase concentration in the plasma of patients with BCa (n=78) ranged from 17.41-57.44 ng/ml. The range determined in healthy donors (n=18) was 2.59-7.74 ng/ml. Additionally, it was revealed that muscle invasive BCa samples exhibited elevated, statistically significant (P=0.01) average aromatase concentrations in blood plasma (38.64 ng/ml) when compared with non-muscle invasive samples (29.83 ng/ml). The results demonstrated that plasma aromatase may serve as an excellent bimarker of BCa with 100% sensitivity, 100% selectivity and an area under the curve value of the reciever operating characteristic curve equal to 1.0. Furthermore, the marker differenciated between muscle-invasive and non muscle-invasive BCa with a sensitivity of 60% and a specificity of 81%. In conclusion, aromatase may serve a role in bladder tumorigenesis.