A new impedimetric biosensor utilizing VEGF receptor-1 (Flt-1): early diagnosis of vascular endothelial growth factor in breast cancer

A new impedimetric biosensor, based on the use of vascular endothelial growth factor receptor-1 (VEGF-R1), was developed for the determination of vascular endothelial growth factor (VEGF). VEGF-R1 was immobilized through covalent coupling with 3-mercaptopropionic acid which formed a self-assembled monolayer on gold electrodes. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy techniques were employed to characterize the immobilization process and to detect VEGF. To successfully construct the biosensor current, experimental parameters were optimized. Kramers-Kronig Transform was performed on the experimental impedance data. The obtained results provided a linear response range from 10 to 70 pg/mL human VEGF. The applicability of the developed biosensor in the determination of VEGF in a spiked artificial human serum sample was experienced, yielding average recovery of 101%, in that order, with an average relative deviation value less than 5%.     

An impedimetric vascular endothelial growth factor biosensor-based PAMAM/cysteamine-modified gold electrode for monitoring of tumor growth

In the present work, we have developed a new biosensor based on fourth-generation (G4) PAMAM dendrimers for the analysis of vascular endothelial growth factor (VEGF). First, the PAMAM dendrimers were covalently attached to a cysteamine-modified Au electrode by glutaraldehyde. With the help of the amino groups located on its surface, vascular endothelial growth factor receptor-1 (VEGF-R1) was immobilized via glutaraldehyde cross-linking. VEGF-R1 loading was investigated to identify the optimal VEGF-R1 immobilization conditions for the best sensitivity of the new biosensor. In addition, Kramers-Kronig transforms were also analyzed for immobilization and measurement processes. The biosensor had a linear range of 5 to 125 pg/mL VEGF. The fabricated biosensor had good repeatability and reproducibility. Finally, the results for artificial serum samples measured by the present biosensor showed a good recovery for VEGF detection.     

Detection of Vascular Endothelial Growth Factor Based on Gold Nanoparticles and Immunoreaction Using Resonance Light Scattering

In the study, a new assay of vascular endothelial growth factor (VEGF) has been developed by the use of gold nanoparticles (GNPs)–anti-VEGF conjugates. The immunoreaction between GNPs–anti-VEGF conjugates and VEGF took place in pH?7.5 PBS buffer solution after the addition of VEGF. The formation of GNPs modified VEGF immunocomplex resulted in the enhanced resonance light scattering (RLS) intensity at 388.0 nm. Under the optimal conditions, the magnitude of enhanced RLS intensity (ΔI _RLS) was proportional to the VEGF concentration in the range from 100 to 1,500 pg?mL^?1, with a detection limit of 60 pg?mL^?1. The surface plasma resonance absorption spectrum, the characteristics of RLS, the VEGF immunocomplex, and the optimum conditions of the immunoreaction have all been investigated. The VEGF concentrations of 20 serum specimens detected by the developed assay showed consistent results in comparison with those obtained by commercially available enzyme-linked immunosorbent assay kit.     

Electrochemical impedimetric immunosensor for insulin like growth factor-1 using specific monoclonal antibody-nanogold modified electrode

An electrochemical impedimetric immunosensor was developed for ultrasensitive determination of insulin-like growth factor-1 (IGF-1) based on immobilization of a specific monoclonal antibody on gold nanoparticles (GNPs) modified gold electrode. Self-assembly of colloidal gold nanoparticles on the gold electrode was conducted through the thiol groups of 1,6-hexanedithiol (HDT) monolayer as a cross linker. The redox reactions of [Fe(CN)(6)](4-)/[Fe(CN)(6)](3-) on the electrode surface was probed for studying the immobilization and determination processes, using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The interaction of antigen with grafted antibody recognition layer was carried out by soaking the modified electrode into antigen solution at 37°C for 3 h. The immunosensor showed linearity over 1.0-180.0 pg mL(-1) and the limit of detection was 0.15 pg mL(-1). The association constant between IGF-1 and immobilized antibody was calculated to be 9.17×10(11) M(-1). The proposed method is a useful tool for screening picogram amounts of IGF-1 in clinical laboratory as a diagnostic test.     

Circulating vascular endothelial growth factor and its soluble receptors in patients with liver cirrhosis: possible association with hepatic function impairment

Recent studies provided in vivo evidences of an increased angiogenesis in animal model of portal hypertension and cirrhosis which was linked to increased expression of vascular endothelial growth factor. The aim of study was to evaluate the plasma concentration of VEGF and its receptors in liver cirrhosis and the possible association with the degree of liver insufficiency. Methods. Vascular endothelial growth factor (VEGF) and its soluble receptors: sVEGF-R1, sVEGF-R2 were measured in plasma of 78 patients with liver cirrhosis by ELISA. Results. The significant increase of plasma VEGF and sVEGF-R1 was observed in liver cirrhosis compared to healthy individuals (153.1+/-51.9 vs. 46.8+/-4.1pg/mL, P<0.05; 279.8+/-34.3 vs. 105.1+/-5.9pg/mL, P<0.001, respectively). Plasma VEGF and foremost sVEGF R1 showed significant associations with biochemical indices of liver function. Among clinical parameters, only ascites revealed significant association with plasma VEGR and sVEGF-R1. VEGF and sVEGF-R1 were increased respectively to the degree of liver insufficiency. It was demonstrated through a significant positive correlation with Child-Pugh score and MELD classification. In conclusion, our study suggests that serum VEGF and VEGF-R1 may reflect the hepatic function impairment in liver cirrhosis and seems to be associated with portal hypertension symptoms.     

A label-free immunosensor for determination of salbutamol based on localized surface plasmon resonance biosensing

We developed a localized surface plasmon resonance (LSPR)-based label-free optical biosensor for detection of salbutamol (Sal). Hollow gold nanoparticles (HGNs) which deposited on transparent indium tin oxide (ITO) film coated glass was used to sensing platform. Antibody against Sal was immobilized on HGN surface to recognize the target Sal molecules. Thus, the change of LSPR peak was proportional to the concentration of Sal in the solution. The experimental results demonstrated that the LSPR immunosensor possessed a good sensitivity and a high selectivity for Sal. The detection range for Sal was from 0.05 to 0.8 μg/mL with a correlation coefficient of 0.996. The biosensor was applied for the detection for Sal in spiked animal feed and pork liver samples, and the recoveries were in the range of 97–105 %. Therefore, it is expected that this approach may offer a new method in designing label-free LSPR immunosensor for detection of small molecules.     

A folding-based electrochemical aptasensor for detection of vascular endothelial growth factor in human whole blood

We herein report a folding-based electrochemical DNA aptasensor for the detection of vascular endothelial growth factor (VEGF) directly in complex biological samples, including blood serum and whole blood. The electrochemical signal generation is coupled to a large, target-induced conformational change in a methylene blue-modified and surface immobilized anti-VEGF aptamer. The sensor is sensitive, selective and essentially reagentless: we can readily detect VEGF down to 5 pM (190 pg/mL) directly in 50% blood serum. Similar to other aptasensors of this class, the VEGF sensor is also regenerable and reusable. In addition, the sensor performs comparably well even when fabricated on a gold-plated screen-printed carbon electrode and can potentially be implemented as a cost-effective, single-use biosensor for diseases diagnosis and therapy monitoring. The exceptional sensitivity, selectivity, and reusability of this electrochemical aptasensor platform suggest it may be a promising strategy for a wide variety of sensing applications.     

Highly stable electrochemical immunosensor for carcinoembryonic antigen

The long-term stability of sensing interfaces is an important issue in biosensor fabrication. A novel stable gold nanoparticle (AuNP)-modified glassy carbon (GC) electrode interface (GC-Ph-AuNP)-based biosensor for detecting carcinoembryonic antigen (CEA) was developed. GC electrodes were modified with 1,4-phenylenediamine to form a stable layer, and then AuNPs were bound onto the GC electrodes through CAu bonds. Anti-CEA was directly adsorbed on AuNPs fixed on the GC electrode. The linear range of the immunosensor was from 10 fg to 100 ng mL(-1) with a detection limit of 3 fg mL(-1) (S/N=3). The current of the immunosensor was increased by 4% after one month. The GC-Ph-AuNP immunosensor showed high sensitivity, a wide linear range, low detection limit, and good selectivity and stability. The immobilization method of the immunosensor could be widely applied to construct other immunosensors.     

Newly identified biologically active and proteolysis-resistant VEGF-A isoform VEGF111 is induced by genotoxic agents

Ultraviolet B and genotoxic drugs induce the expression of a vascular endothelial growth factor A (VEGF-A) splice variant (VEGF111) encoded by exons 1–4 and 8 in many cultured cells. Although not detected in a series of normal human and mouse tissue, VEGF111 expression is induced in MCF-7 xenografts in nude mice upon treatment by camptothecin. The skipping of exons that contain proteolytic cleavage sites and extracellular matrix–binding domains makes VEGF111 diffusible and resistant to proteolysis. Recombinant VEGF111 activates VEGF receptor 2 (VEGF-R2) and extracellularly regulated kinase 1/2 in human umbilical vascular endothelial cells and porcine aortic endothelial cells expressing VEGF-R2. The mitogenic and chemotactic activity and VEGF111's ability to promote vascular network formation during embyonic stem cell differentiation are similar to those of VEGF121 and 165. Tumors in nude mice formed by HEK293 cells expressing VEGF111 develop a more widespread network of numerous small vessels in the peritumoral tissue than those expressing other isoforms. Its potent angiogenic activity and remarkable resistance to proteolysis makes VEGF111 a potential adverse factor during chemotherapy but a beneficial therapeutic tool for ischemic diseases.     

Vascular endothelial growth factor, vascular endothelial growth factor receptor-3 and cyclooxygenase-2 expression in psoriasis

OBJECTIVE: To investigate expression patterns and relationship of vascular endothelial growth factor (VEGF), vascular endothelial receptor-3 (VEGF-R3) (FLT-4) and cyclooxygenase-2 (COX-2) in psoriasis. STUDY DESIGN: Forty-three patients were included in this study. The clinical severity of psoriasis was assessed using the psoriasis area and severity index (PASI). Punch biopsy samples both from psoriatic and nonlesional skin were taken and VEGF, VEGF-R3 and COX-2 expressions determined. RESULTS: VEGF, VEGF-R3 and COX-2 expressions were detected in 90.9%, 78.0% and 86.4% of psoriatic and 84.1%, 71.8%, and 84.1% of nonlesional skin, respectively. Epidermal VEGF, VEGF-R3 and COX-2 expressions were detected in 56.8%, 77.8% and 34.1 of psoriatic and 75%, 78.1% and 65.9% of nonlesional skin, respectively. In dermis, VEGF, VEGF-R3 and COX-2 expression was observed in 88.6%, 77.5% and 84.1% of psoriatic and 81.8%, 64.1% and 77.3% of nonlesional skin, respectively. Among the PASI subgroups no statistically significant differences were detected for VEGF, VEGF-R3 and COX-2 expression. CONCLUSION: Our study demonstrated that VEGF, VEGF-R3 and COX-2 expression in psoriatic and nonlesional skin is significantly high in epidermis and dermis. Although there was significant concordance between VEGF and VEGF-R3 expressions in psoriatic lesions, there seems to be no concordance between the others.     

Electrochemiluminescence of peroxydisulfate enhanced by L-cysteine film for sensitive immunoassay

A novel label free electrochemiluminescence (ECL) immunosensor based on the ECL of peroxydisulfate solution for detection of α-1-fetoprotein (AFP) has been developed. For this proposed immunosensor, L-cysteine was firstly electrodeposited on the gold electrode surface, which promoted the electron transfer and largely enhanced the ECL of peroxydisulfate solution. Subsequently, gold nanoparticles (nano-Au) were assembled onto the L-cysteine film modified electrode to improve the absorption capacity of antibody and further amplify the ECL signal. Then, antibody was immobilized onto the electrode through nano-Au. At last bovine serum albumin (BSA) was employed to block the nonspecific binding sites. As a result, a novel ECL immunosensor was firstly obtained by applying the ECL of peroxydisulfate solution without conventional luminescent reagents. The AFP was determined in the range of 0.01-100 ng mL(-1), with a low detection limit of 3.3 pg mL(-1) (S/N=3). The proposed ECL immunosensor provides a rapid, simple, and sensitive immunoassay protocol for protein detection, which might hold a promise for clinical application. Moreover, this work would open up a new field in the application of peroxydisulfate solution ECL for highly sensitive bioassays.     

Label-free impedimetric immunosensor for ultrasensitive detection of cancer marker Murine double minute 2 in brain tissue

The detection of cancer biomarkers is as important tool for the diagnosis and prognosis of cancer such as brain cancer. Murine double minute 2 (MDM2) has been widely studied as prognostic marker for brain tumor. Here we describe development of a new sensitive label free impedimetric immunosensor for the detection of MDM2 based on cysteamine self assembled monolayers on a clean polycrystalline Au electrode surface. The amine-modified electrodes were further functionalized with antibody using homobifunctional 1,4-phenylene diisothiocyanate (PDITC) linker. The assembly processes of the immunosensor had been monitored with cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) techniques using Fe(CN)(6)(3-/4-) solution as redox probe. The impedance changes upon binding of MDM2 protein to the sensor surface was utilized for the detection of MDM2. The increase in relative electron-transfer resistance (ΔR/R(0)%) values was linearly proportional to the concentration of tumor marker MDM2 in the wide dynamic range of 1pg/ml-1μg/ml. The limit of detection was 0.29pg/ml in phosphate buffer saline (PBS) and 1.3pg/ml in mouse brain tissue homogenate, respectively. The immunosensor showed a good performance in comparison with ELISA for the analysis of the MDM2 in the cancerous mouse brain tissue homogenates. Moreover, the immunosensor had a good selectivity against epidermal growth factor receptor (EGFR) protein, long-storage stability and reproducibility. It might be become a promising assay for clinical diagnosis and early detection of tumors.     

Detection of estradiol at an electrochemical immunosensor with a Cu UPD|DTBP-Protein G scaffold

A copper monolayer was formed on a gold electrode surface via underpotential deposition (UPD) method to construct a Cu UPD|DTBP-Protein G immunosensor for the sensitive detection of 17β-estradiol. Copper UPD monolayer can minimize the non-specific adsorption of biological molecules on the immunosensor surface and enhance the binding efficiency between immunosensor surface and thiolated Protein G. The crosslinker DTBP (Dimethyl 3,3'-dithiobispropionimidate · 2HCl) has strong ability to immobilize Protein G molecules on the electrode surface and the immobilized Protein G provides an orientation-controlled binding of antibodies. A monolayer of propanethiol was firstly self-assembled on the gold electrode surface, and a copper monolayer was deposited via UPD on the propanethiol modified electrode. Propanethiol monolayer helps to stabilize the copper monolayer by pushing the formation and stripping potentials of the copper UPD monolayer outside the potential range in which copper monolayer can be damaged easily by oxygen in air. A droplet DTBP-Protein G was then applied on the modified electrode surface followed by the immobilization of estradiol antibody. Finally, a competitive immunoassay was conducted between estradiol-BSA (bovine serum albumin) conjugate and free estradiol for the limited binding sites of estradiol antibody. Square wave voltammetry (SWV) was employed to monitor the electrochemical reduction current of ferrocenemethanol and the SWV current decreased with the increase of estradiol-BSA conjugate concentration at the immunosensor surface. Calibration of immunosensors in waste water samples spiked with 17β-estradiol yielded a linear response up to ≈ 2200 pg mL(-1), a sensitivity of 3.20 μA/pg mL(-1) and a detection limit of 12 pg mL(-1). The favorable characteristics of the immunosensors such as high selectivity, sensitivity and low detection limit can be attributed to the Cu UPD|DTBP-Protein G scaffold.     

Direct immobilisation of antibodies on a bioinspired architecture as a sensing platform

A sensitive and selective immunosensor for the nonlabeled detection of sulfate-reducing bacteria (SRB) is constructed using a self-polymerised polydopamine film as the immobilisation platform. Self-polymerisation of dopamine is used as a powerful approach for applying multifunctional coatings onto the surface of a gold electrode. The polydopamine film is used not only as the immobilisation platform, but also as a cross-linker reagent for the immobilisation of the anti-SRB antibody. The polydopamine film is loaded with a high density of anti-SRB antibodies linked to the substrate to obtain high response signals. The formation and fabrication of the biosensor and the quantification of antibody anchoring are monitored, and SRB detection is performed by either quartz crystal microbalance (QCM) or electrochemical impedance spectroscopy (EIS). After modeling the impedance Nyquist plots of the SRB/anti-SRB/polydopamine/gold electrode for increasing concentrations of SRB, the electron transfer resistance (R(ct)) is used as a measure of immunocomplex binding. The R(ct) is correlated with the concentration of bacterial cells in the range of 1.8×10(2) to 1.8×10(6) CFU mL(-1); the detection limit is 50 CFU mL(-1). This work demonstrates a new immobilisation platform for the development of a sensitive and label-less impedimetric and piezoelectric immunosensor. This immunosensor may be broadly applied in clinical diagnoses and the monitoring of water environmental pollution. The method proposed is distinct in its ease of application, use of a simple protocol, and mild reaction conditions. These allow it to be applied to a wide variety of materials.     

Angiotensin II increases the permeability and PV-1 expression of endothelial cells

Angiotensin II (ANG II), the major effector molecule of the renin-angiotensin system (RAS), is a powerful vasoactive mediator associated with hypertension and renal failure. In this study the permeability changes and its morphological attributes in endothelial cells of human umbilical vein (HUVECs) were studied considering the potential regulatory role of ANG II. The effects of ANG II were compared with those of vascular endothelial growth factor (VEGF). Permeability was determined by 40 kDa FITC-Dextran and electrical impedance measurements. Plasmalemmal vesicle-1 (PV-1) mRNA levels were measured by PCR. Endothelial cell surface was studied by atomic force microscopy (AFM), and caveolae were visualized by transmission electron microscopy (TEM) in HUVEC monolayers. ANG II (10(-7) M), similarly to VEGF (100 ng/ml), increased the endothelial permeability parallel with an increase in the number of cell surface openings and caveolae. AT1 and VEGF-R2 receptor blockers (candesartan and ZM-323881, respectively) blunted these effects. ANG II and VEGF increased the expression of PV-1, which could be blocked by candesartan or ZM-323881 pretreatments and by the p38 mitogem-activated protein (MAP) kinase inhibitor SB-203580. Additionally, SB-203580 blocked the increase in endothelial permeability and the number of surface openings and caveolae. In conclusion, we have demonstrated that ANG II plays a role in regulation of permeability and formation of cell surface openings through AT1 receptor and PV-1 protein synthesis in a p38 MAP kinase-dependent manner in endothelial cells. The surface openings that increase in parallel with permeability may represent transcellular channels, caveolae, or both. These morphological and permeability changes may be involved in (patho-) physiological effects of ANG II.     

A high density microelectrode array biosensor for detection of E. coli O157:H7

A high density microelectrode array biosensor was developed for the detection of Escherichia coli O157:H7. The biosensor was fabricated from (100) silicon with a 2 microm layer of thermal oxide as an insulating layer, an active area of 9.6 mm2 and consists of an interdigitated gold electrode array. The sensor surface was functionalised for bacterial detection using heterobifunctional crosslinkers and immobilised polyclonal antibodies to create a biological sensing surface. Bacteria suspended in solution became attached to the immobilised antibodies when the biosensor was tested in liquid samples. The change in impedance caused by the bacteria was measured over a frequency range of 100 Hz-10 M Hz. The biosensor was evaluated for E. coli O157:H7 detection in pure culture and inoculated food samples. The biosensor was able to discriminate between cellular concentrations of 10(4)-10(7)CFU/mL and has applications in detecting pathogens in food samples.     

Increased H2O2, vascular endothelial growth factor and receptors in the retina of the BBZ/Wor diabetic rat

Hyperglycemia in diabetes induces increased levels of hydrogen peroxide (H2O2), a reactive oxygen species generated by reduced nicotinamide adenine dinucleotide (NADH) oxidase. Nontoxic levels of H2O2 increase endothelial cell permeability. Using a model of non-insulin-dependent diabetes, the BBZ/Wor rat, we investigated retinal levels of H2O2, vascular endothelial growth factor (VEGF) and its receptors, VEGF-R1 and VEGF-R2 by transmission electron microscopy at sites of the blood-retinal barrier (BRB). H2O2 localization was done by the cerium NADH oxidase method, and extravasation of endogenous serum albumin was used to document disruption of the BRB. Higher levels of H2O2 were detected in blood vessels of diabetic (78.7 +/- 4.84%) as compared with vessels from nondiabetic rats (39.0 +/- 4.47%). VEGF immunoreactivity was statistically higher in the inner BRB (24.67 +/- 0.33 colloidal gold particles/63 microm2 vs. 21.52 +/- 0.43 colloidal gold particles/63 microm2, p = .0001) and outer BRB (42.56 +/- 0.45 colloidal gold particles/63 microm2 vs. 15.51 +/- 0.51 colloidal gold particles/63 microm2, p = .0001) of diabetic rats as compared with age matched nondiabetic control rats. VEGF-R1 immunoreactivity was significantly higher in diabetic retinas in both the inner BRB (21.66 +/- 0.75 colloidal gold particles/63 microm2 vs. 12.69 +/- 0.61 colloidal gold particles/63 microm2, p = .0001) and outer BRB (22.76 +/- 2.36 colloidal gold particles/63 microm2 vs. 8.53 +/- 2.67 colloidal gold particles/63 microm2, p = .0013). VEGF-R2 was statistically higher in the inner BRB (8.97 +/- 0.57 colloidal gold particles/63 microm2 versus 7.03 +/- 0.65 colloidal gold particles/63 microm2, p = .0419) but not in the outer BRB (29.42 +/- 1.25 colloidal gold particles/63 microm2 vs. 28.07 +/- 1.42 colloidal gold particles/63 microm2, p = .4889). H2O2 levels correlated with increased VEGF (correlation coefficient = 0.82, p = .001) in this model of nonproliferative diabetic retinopathy. These results support that hyperglycemia is one factor that induces retinal endothelial cells in vivo to increase H2O2 via NADH oxidase and stimulates increases in VEGF resulting in disruption of the BRB.     

Electrochemical impedance immunosensor based on gold nanoparticles and aryl diazonium salt functionalized gold electrodes for the detection of antibody

Electrodes modified with passivating organic layers have been shown to, here and previously, to exhibit good Faradaic electrochemistry upon attachment of gold nanoparticles (AuNP). Due to their low background capacitances these constructs have good potential in electrochemical sensing. Herein is reported the application of these electrode constructs for impedance based immunosensing. The immunosensor was constructed by modifying a gold electrode with 4-thiophenol (4-TP) passivating layers by diazonium salt chemistry. Subsequently, the attachment of AuNP and then a biotin derivative as a model epitope to detect anti-biotin IgG were carried out. The interfacial properties of the modified electrodes were evaluated in the presence of Fe(CN)(6)(4-/3-) redox couple as a probe by cyclic voltammetry and electrochemical impedance spectroscopy. The impedance change, due to the specific immuno-interaction at the immunosensor surface was utilized to detect anti-biotin IgG. The increase in charge-transfer resistance (R(ct)) was linearly proportional to the concentration of anti-biotin IgG in the range of 5-500 ng mL(-1), with a detection limit of 5 ng mL(-1).