Search Results (19)

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 Full article

A new biosensor based on the “surface plasmon resonance imaging (SPRi)” detection technique for the quantification of “fibroblast growth factor 23 (FGF23)” has been developed. FGF23 is mainly produced in bone tissues as a phosphaturic hormone that forms a trimeric complex with “fibroblast growth factor receptor 1 (FGFR1)” and αKlotho upon secretion. FGF23 stimulates phosphate excretion and inhibits the formation of active vitamin D in the kidneys. FGF23 has been shown to play a role in bone carcinogenesis and metastasis. The newly developed method, based on the array SPRi biosensor, was validated—the precision, accuracy, and selectivity were acceptable, and yielded less than ±10% recovery. The rectilinear response of the biosensor ranges from 1 to 75 pg/mL. The limit of detection was 0.033 pg/mL, and the limit of quantification was 0.107 pg/mL. The biosensor was used to determine FGF23 concentrations in the blood plasma of healthy subjects and patients with “clear cell” renal cell carcinoma (ccRCC). The obtained results were compared with those measured through an “enzyme-linked immunosorbent assay (ELISA)”. The determined Pearson correlation coefficients were 0.994 and 0.989, demonstrating that the newly developed biosensor can be used as a competitive method for the ELISA. Full article

A biosensor was developed for the quantification of poly(ADP-ribose) polymerase-1 (PARP-1) in body fluids. An antibody specific for PARP-1 was placed on a chip with cysteamine (linker) and a gold layer. This biosensor has a linear response range (10–1000 pg∙mL⁻¹) under appropriate pH conditions and with an antibody ligand concentration of 5 ng∙mL⁻¹. Plasma samples were diluted with PBS buffer in appropriate quantities so that they fell within the linear range of the calibration curve. The biosensor exhibited suitable precision and accuracy, and good recovery (at levels from 95% to 105%). The method was validated by means of PARP-1 determinations in plasma samples from patients with endometriosis and a control group, using surface plasmon resonance imaging (SPRi) biosensors and an enzyme-linked immunosorbent assay (ELISA) test. The Spearman correlation coefficient was close to 1. PARP-1 may be a marker providing information about pathological changes in the body during endometriosis. Full article

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. Full article

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. Full article

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. Full article

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. Full article

A functional cure of hepatitis B virus (HBV) infection or HB antigen loss is rarely achieved by nucleos(t)ide analogs which target viral polymerase. HBx protein is a regulatory protein associated with HBV replication. We thought to identify antiviral compounds targeting HBx protein by analyzing HBx binding activity. Recombinant GST-tagged HBx protein was applied on an FDA-approved drug library chip including 1018 compounds to determine binding affinity by surface plasmon resonance imaging (SPRi) using a PlexArray HT system. GST protein alone was used for control experiments. Candidate compounds were tested for anti-HBV activity as well as cell viability using HepG2.2.15.7 cells and HBV-infected human hepatocytes. Of the 1018 compounds screened, 24 compounds showed binding to HBx protein. Of the top 6 compounds with high affinity to HBx protein, tranilast was found to inhibit HBV replication without affecting cell viability using HepG2.2.15.7 cells. Tranilast also inhibited HBV infection using cultured human hepatocytes. Tranilast reduced HB antigen level dose-dependently. Overall, theSPRi screening assay identified novel drug candidates targeting HBx protein. Tranilast and its related compounds warrant further investigation for the treatment of HBV infection. Full article

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. Full article

In the context of bacteriophage (phage) therapy, there is an urgent need for a method permitting multiplexed, parallel phage susceptibility testing (PST) prior to the formulation of personalized phage cocktails for administration to patients suffering from antimicrobial-resistant bacterial infections. Methods based on surface plasmon resonance imaging (SPRi) and phase imaging were demonstrated as candidates for very rapid (<2 h) PST in the broth phase. Biosensing layers composed of arrays of phages 44AHJD, P68, and gh-1 were covalently immobilized on the surface of an SPRi prism and exposed to liquid culture of either Pseudomonas putida or methicillin-resistant Staphylococcus aureus (i.e., either the phages’ host or non-host bacteria). Monitoring of reflectivity reveals susceptibility of the challenge bacteria to the immobilized phage strains. Investigation of phase imaging of lytic replication of gh-1 demonstrates PST at the single-cell scale, without requiring phage immobilization. SPRi sensorgrams show that on-target regions increase in reflectivity more slowly, stabilizing later and to a lower level compared to off-target regions. Phage susceptibility can be revealed in as little as 30 min in both the SPRi and phase imaging methods. Full article

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. Full article

Understanding transient protein interactions biochemically at the proteome scale remains a long-standing challenge. Current tools developed to study protein interactions in high-throughput measure stable protein complexes and provide binary readouts; they do not elucidate dynamic and weak protein interactions in a proteome. The majority of protein interactions are transient and cover a wide range of affinities. Nucleic acid programmable protein arrays (NAPPA) are self-assembling protein microarrays produced by freshly translating full-length proteins in situ on the array surface. Herein, we have coupled NAPPA to surface plasmon resonance imaging (SPRi) to produce a novel label-free platform that measures many protein interactions in real-time allowing the determination of the KDs and rate constants. The developed novel NAPPA-SPRi technique showed excellent ability to study protein-protein interactions of clinical mutants of p53 with its regulator MDM2. Furthermore, this method was employed to identify mutant p53 proteins insensitive to the drug nutlin-3, currently in clinical practice, which usually disrupts the p53-MDM2 interactions. Thus, significant differences in the interactions were observed for p53 mutants on the DNA binding domain (Arg-273-Cys, Arg-273-His, Arg-248-Glu, Arg-280-Lys), on the structural domain (His-179-Tyr, Cys-176-Phe), on hydrophobic moieties in the DNA binding domain (Arg-280-Thr, Pro-151-Ser, Cys-176-Phe) and hot spot mutants (Gly-245-Cys, Arg-273-Leu, Arg-248-Glu, Arg-248-Gly), which signifies the importance of point mutations on the MDM2 interaction and nutlin3 effect, even in molecular locations related to other protein activities. Full article

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). Full article

This review aims to summarize the recent advances and progress of plasmonic biosensors based on patterned plasmonic nanostructure arrays that are integrated with microfluidic chips for various biomedical detection applications. The plasmonic biosensors have made rapid progress in miniaturization sensors with greatly enhanced performance through the continuous advances in plasmon resonance techniques such as surface plasmon resonance (SPR) and localized SPR (LSPR)-based refractive index sensing, SPR imaging (SPRi), and surface-enhanced Raman scattering (SERS). Meanwhile, microfluidic integration promotes multiplexing opportunities for the plasmonic biosensors in the simultaneous detection of multiple analytes. Particularly, different types of microfluidic-integrated plasmonic biosensor systems based on versatile patterned plasmonic nanostructured arrays were reviewed comprehensively, including their methods and relevant typical works. The microfluidics-based plasmonic biosensors provide a high-throughput platform for the biochemical molecular analysis with the advantages such as ultra-high sensitivity, label-free, and real time performance; thus, they continue to benefit the existing and emerging applications of biomedical studies, chemical analyses, and point-of-care diagnostics. Full article

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. Full article