Development of an SPRi Test for the Quantitative Detection of Cadherin 12 in Human Plasma and Peritoneal Fluid.

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.

An Array SPRi Biosensor for the Determination of Follicle-Stimulating Hormone in Blood Plasma.

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.

A Multiple-Array SPRi Biosensor as a Tool for Detection of Gynecological-Oncological Diseases.

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.

Biosensors for the Determination of Protein Biomarkers.

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

An Immunosensor for the Determination of Cortisol in Serum and Saliva by Array SPRi.

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.

Two Biosensors for the Determination of Interleukin-6 in Blood Plasma by Array SPRi.

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.

Studies of interactions between fibronectin and a specific antibody against fibronectin using SPRi and QCM.

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∙108 dm3/mol and the dissociation constant to be KD= 8.20 ∙ 10-9 mol/dm3.

Two Biosensors for the Determination of VEGF-R2 in Plasma by Array SPRi.

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.

An Immunosensor for the Determination of Cathepsin S in Blood Plasma by Array SPRi-A Comparison of Analytical Properties of Silver-Gold and Pure Gold Chips.

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).

An SPRi Biosensor for Determination of the Ovarian Cancer Marker HE4 in Human Plasma.

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.

Two SPRi biosensors for the determination of cathepsin S in blood plasma.

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.

An SPR imaging immunosensor for leptin determination in blood plasma.

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.

A Comparison of Various Chips Used for the Manufacture of Biosensors Applied in Non-Fluidic Array SPRi, Based on the Example of Determination of Cathepsin D.

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.

Potential of surface plasmon resonance biosensors in cancer detection.

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.

An immunosensor for the determination of carcinoembryonic antigen by Surface Plasmon Resonance imaging.

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 µL 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, metalloproteinase-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.

Plasma aromatase as a sensitive and selective potential biomarker of bladder cancer and its role in tumorigenesis.

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 µl 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.

Correction to: Methods for 20S Immunoproteasome and 20S Constitutive Proteasome Determination Based on SPRI Biosensors.

[This corrects the article DOI: 10.1007/s12195-017-0478-7.].

Recent Progress in Surface Plasmon Resonance Biosensors (2016 to Mid-2018).

: More than 50 papers on surface plasmon resonance biosensors, published between 2016 and mid-2018, are reviewed. Papers concerning the determination of large particles such as vesicles, exosomes, cancer cells, living cells, stem cells, and microRNA are excluded, as these are covered by a very recent review. The reviewed papers are categorized into five groups, depending on the degree of maturity of the reported solution; ranging from simple marker detection to clinical application of a previously developed biosensor. Instrumental solutions and details of biosensor construction are analyzed, including the chips, receptors, and linkers used, as well as calibration strategies. Biosensors with a sandwich structure containing different nanoparticles are considered separately, as are SPR (Surface Plasmon Resonance) applications for investigating the interactions of biomolecules. An analysis is also made of the markers determined using the biosensors. In conclusion, there is shown to be a growing number of SPR applications in the solution of real clinical problems.

Methods for 20S Immunoproteasome and 20S Constitutive Proteasome Determination Based on SPRI Biosensors.

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.

Bacterial strains isolated from river water having the ability to split alcohol ethoxylates by central fission.

Alcohol ethoxylates (AE) are a major component of the surfactant stream discharged into surface water. The "central fission" of AE with the formation of poly(ethylene glycols) (PEG) is considered to be the dominant biodegradation pathway. However, information as to which bacterial strains are able to perform this reaction is very limited. The aim of this work was to establish whether such an ability is unique or common, and which bacterial strains are able to split AE used as a sole source of organic carbon. Four bacterial strains were isolated from river water and were identified on the basis of phylogenetic trees as Enterobacter strain Z2, Enterobacter strain Z3, Citrobacter freundii strain Z4, and Stenotrophomonas strain Z5. Sterilized river water and "artificial sewage" were used for augmentation of the isolated bacteria. The test was performed in bottles filled with a mineral salt medium spiked with surfactant C12E10 (10 mg L(-1)) and an inoculating suspension of the investigated bacterial strain. Sequential extraction of the tested samples by ethyl acetate and chloroform was used for separation of PEG from the water matrix. LC-MS was used for PEG determination on the basis of single-ion chromatograms. All four selected and investigated bacterial strains exhibit the ability to split fatty alcohol ethoxylates with the production of PEG, which is evidence that this property is a common one rather than specific to certain bacterial strains. However, this ability increases in the sequence: Stenotrophomonas strain Z5 < Enterobacter strain Z2 < Enterobacter strain Z3 = Citrobacter freundii strain Z4. Graphical Abstract Biodegradation by central fission of alcohol ethoxylates by bacterial strains isolated from river water.