Redox features of hexaammineruthenium(III) on MXene modified interface: Three options for affinity biosensing

The effects of bovine serum albumin (BSA) on the mixed dipalmitoyl phosphatidylcholine (DPPC)/1-palmitoyl-2-oleoyl phosphatidylglycerol (POPG) (molar ratio 3:1) monolayer behavior at cyclic air/liquid interfaces were investigated by the fluorescence microscopy (FM) with the measurements of surface pressure-area isotherms. With spreading DPPC molecules onto the air/liquid interface with an adsorbed BSA layer, it was revealed from the surface pressure-relative area hysteresis curves and the morphology of mixed DPPC/BSA layers that BSA molecules were expelled from the interface by DPPC molecules as the mixed layer was compressed to a condensed state. The squeeze-out of BSA molecules from the interface would induce the loss of free DPPC molecules at the interface. The increased surface pressure during the following interface expansion stage was probably due to the readsorption of BSA molecules. Furthermore, during the compression-expansion process of mixed DPPC/BSA layers, FM images indicated that BSA might interact with DPPC to form complexes. The results suggested that under the condition of continuous interface compression-expansion, the presence of BSA would decrease the number of free DPPC molecules at the air/liquid interface, resulting in the inhibited dynamic surface activity of DPPC. After spreading POPG molecules onto the air/liquid interface with an adsorbed BSA layer, under the condition of continuous interface compression-expansion, it was found that part of POPG molecules would leave the air/liquid interface with BSA molecules, resulting in the loss of free POPG molecules at the interface and the inhibited dynamic surface activity of POPG. The loss of free POPG molecules was probably related to the electrostatic repulsion between negatively charged BSA molecules and headgroups of POPG molecules, or to the hydrophobic interactions between the hydrocarbon chains of POPG molecules and the hydrophobic part of BSA molecules. Therefore, under the condition of continuous interface compression-expansion, when BSA molecules left the interface, it also caused the loss of POPG molecules. After simultaneously spreading DPPC and POPG molecules onto the interface with an adsorbed BSA layer, it was found that the dynamic interface behavior of the mixed DPPC/POPG/BSA layer was very similar to that of the mixed DPPC/BSA layer, indicating that the POPG fraction at the interface was significantly decreased. This is probably caused by the stranger interaction between BSA and POPG. As a result, under the condition of continuously interface compression-expansion, POPG molecules were selectively removed from the interface by BSA and the amount of free POPG molecules at the interface was dramatically decreased, resulting in the dynamic interface behavior dominated by DPPC and BSA.

Electrochemical studies of bovine serum albumin immobilization onto the poly- o-phenylenediamine and carbon-coated nickel composite film and its interaction with papaverine

Highly sensitive electrochemical aptasensor for immunoglobulin E detection based on sandwich assay using enzyme-linked aptamer

In this study, a nanocomposite of zinc oxide (ZnO) nanoparticles and chitosan‐based carbon quantum dots (ZnO/chQDs) in the presence of bovine serum albumin (BSA) as a promising drug carrier candidate was synthesized and characterized. For this purpose, chitosan and citric acid were used for the synthesis of chQDs and donepezil (DNP) was selected as a model drug. The BSA binding properties of DNP were analyzed by FT‐IR and UV‐Vis techniques, electrochemical methods and molecular docking in the absence and presence of ZnO/chQDs. The thermodynamic parameters and binding constants of DNP with BSA were also evaluated by these methods. The changes in the structural properties of BSA in the presence of DNP, ZnO/chQDs and ZnO/chQDs‐DNP were monitored by ATR‐FTIR spectroscopy. Cyclic and differential pulse voltammetric techniques and electrochemical impedance spectroscopy were applied to investigate the interactions, using methylene blue in electrolyte solution as redox probe, and its redox behaviour was observed to explore the interactions. Molecular docking was carried out on the interaction of DNP with BSA in the absence and presence of the synthesized ZnO/chQDs, and the results were compared with those of the experimental observations. The experimental results were confirmed by molecular docking studies.

The authors describe an electrochemical aptamer based assay for the determination of the serine protease lysozyme in very low (pM) concentrations. The method is based on the formation of a complex between anti-lysozyme aptamer fragments and lysozyme, and on electrochemical detection by differential pulse voltammetry (DPV) and electrochemical impedance spectroscopy (EIS). The surface of a glassy carbon electrode was modified with a nanocomposite consisting of gold nanoparticles and electrochemically reduced graphene oxide nanosheets (AuNPs/erGO), and the thiolated aptamer was then linked to the AuNPs by self-assembly through Au-S bonds. The interaction of immobilized aptamers with lysozyme leads to the decreased peak current in DPV and increased charge transfer resistance (Rct) in EIS when using hexacyanoferrate or Methylene Blue as a redox probe. The calibration plot, when applying EIS and working at a typical voltage of −0.22 V (vs. SCE), is linear over 1.0 to 104.3 pM concentration range, with a detection limit of 0.06 pM (at a signal-to-noise ratio of 3). The respective data for DPV are a 9.6–205.5 pM linear range with a detection limit of 0.24 pM. Depending on the redox marker applied, the method works in the “signal-off” or “signal-on” mode in DPV and EIS protocols, respectively. The sensing interface is high specific for lysozyme and not affected by other proteins. The method was applied to the determination of lysozyme in spiked diluted human serum, and the results agreed well with data obtained with a standard ELISA.

Objective A label-free electrochemical immunosensor was developed for the detection of nuclear matrix protein-22 (NMP22) as a biomarker of bladder cancer. Methods The study was based on the establishment and validation of the methodology. Urine samples were collected from 20 patients with bladder cancer and 20 controls in the affiliated Hongqi hospital of Mudanjiang medical university from September in 2017 to July in 2019 to validate the developed method. A screen-printed electrode (SPE) was modified with a film of a composite made from the reduced graphene oxide-tetraethylene pentamine (rGO-TEPA) immobilized Zn-based-Metal-organic frameworks deposited with Au nanoparticles (rGO-TEPA@Au-ZIF8). Primary antibody against NMP22 was immobilized on the Au nanoparticles on the surface of the modified SPE, which then was blocked with bovine serum albumin to elimiate nonspecific binding sites. The process of the construction of the proposed sensorwas characterized by cyclic voltammetry and electrochemical impedance spectroscopy. Differential pulse voltammetry was used to evaluate the linear range, recovery, precision, selectivity and stability. The data were analyzed by Mann-Whitney U test. Results Under optimal conditions, the immunosensor exhibited a linear range of 0.01-1000 ng/mlwith a detection limit of 3.33 pg/ml (S/N=3) and a standard recovery of 97.65%-107.05%. The levels of NMP22 in urine samples from patients with bladder cancer [66.03 (4.34, 91.74)]ng/ml determined by the proposed sensor were significantly higher than those of controls 0.54(0.06, 8.84) ng/ml(P=0.001). Conclusion The immunosensor can achieve sensitive, rapid and acucurate detection of NMP22, and has potential application prospects in monitoring tumor markers. Key words: Urinary bladder neoplasms; Nuclear proteins; Biosensing techniques; Electrochemical techniques; Immunoassay; Graphite; Polyamines; Biomarkers, tumor

In this paper, sodium alginate (NaAlg) was used as functional monomers, bovine serum albumin (BSA) was used as template molecules, and calcium chloride (CaCl2) aqueous solution was used as a cross-linking agent to prepare BSA molecularly imprinted carboxylated multi-wall carbon nanotubes (CMWCNT)/CaAlg hydrogel films (MIPs) and non-imprinted hydrogel films (NIPs). The adsorption capacity of the MIP film for BSA was 27.23 mg/g and the imprinting efficiency was 2.73. The MIP and NIP hydrogel film were loaded on the surface of the printed electrode, and electrochemical performance tests were carried out by electrochemical impedance spectroscopy (EIS) and differential pulse voltammetry (DPV) using the electrochemical workstation. The loaded MIP film and NIP film effectively improved the electrochemical signal of the bare carbon electrode. When the pH value of the Tris HCl elution solution was 7.4, the elution time was 15 min and the adsorption time was 15 min, and the peak currents of MIP-modified electrodes and NIP-modified electrodes reached their maximum values. There was a specific interaction between MIP-modified electrodes and BSA, exhibiting specific recognition for BSA. In addition, the MIP-modified electrodes had good anti-interference, reusability, stability, and reproducibility. The detection limit (LOD) was 5.6 × 10-6 mg mL-1.

Electrochemical study of bovine serum albumin damage induced by Fenton reaction using tris (2,2′-bipyridyl) cobalt (III) perchlorate as the electroactive indicator

This paper describes the development of a simple voltammetric biosensor for the stereoselective discrimination of myo-inositol (myo-Ins) and D-chiro-inositol (D-chiro-Ins) by means of bovine serum albumin (BSA) adsorption onto a multi-walled carbon nanotube (MWCNT) graphite screen-printed electrode (MWCNT-GSPE), previously functionalized by the electropolymerization of methylene blue (MB). After a morphological characterization, the enantioselective biosensor platform was electrochemically characterized after each modification step by differential pulse voltammetry (DPV) and electrochemical impedance spectroscopy (EIS). The results show that the binding affinity between myo-Ins and BSA was higher than that between D-chiro-Ins and BSA, confirming the different interactions exhibited by the novel BSA/MB/MWCNT/GSPE platform towards the two diastereoisomers. The biosensor showed a linear response towards both stereoisomers in the range of 2–100 μM, with LODs of 0.5 and 1 μM for myo-Ins and D-chiro-Ins, respectively. Moreover, a stereoselectivity coefficient α of 1.6 was found, with association constants of 0.90 and 0.79, for the two stereoisomers, respectively. Lastly, the proposed biosensor allowed for the determination of the stereoisomeric composition of myo-/D-chiro-Ins mixtures in commercial pharmaceutical preparations, and thus, it is expected to be successfully applied in the chiral analysis of pharmaceuticals and illicit drugs of forensic interest.

Molecularly imprinted polymers (MIPs) belong to the illustrious examples of bio-mimicking recognizing materials.1 They have found numerous applications in the fabrication of selective chemosensors.2 Their analytical parameters, such as sensitivity, selectivity, and detectability, are almost as high as those of biosensors. Additionally, MIP based chemosensors are superior to biosensors concerning their ease of fabrication, durability, and tolerance to harsh conditions, including elevated or decreased temperature, high ionic strength, extreme pH values, the presence of heavy metal ions and organic solvents in the samples. Conductive MIPs have recently become more frequently applied. That is mainly due to the easy control of MIPs deposition as thin films by electropolymerization.3 For the electrochemical determination of non-electroactive analytes, some external redox probe is usually added to the test solution. It is assumed that target analyte molecules' binding into molecular cavities causes MIP film swelling or shrinking. According to the so-called "gate effect" mechanism, this polymer "breathing" causes changes in the redox probe permeability through an MIP film, thus changing faradaic current corresponding to the redox probe's reduction or oxidation in cyclic voltammetry (CV) and differential pulse voltammetry (DPV) determinations.4-5 This mechanism is operative for nonconductive MIP films. Another mechanism may be considered for surface imprinted macromolecular compounds, e.g., proteins. A drop in the faradaic current of the redox probe accompanying protein adsorption originates from physical blocking of the electrode surface by their bulky nonconductive molecules.6 But both of these mechanisms seem to be invalid in case of electrochemical sensors based on conductive MIP films. In our previous studies, we demonstrated that a drop in the DPV current, caused by the appearance in a solution of an analyte, at conductive MIP film-coated electrodes might originate not from hindering the diffusion of the redox probe through the film but from changes in electrochemical properties of the film itself 7. Suppose the redox probe diffusion through the MIP film is not a decisive parameter for the faradaic current involving. Then, in the, e.g., DPV, determinations of electroinactive analytes at conductive MIP film-coated electrodes, this diffusion may be eliminated. For that the redox probe could be immobilized inside the MIP film matrix. Herein, we propose to deposit a self-reporting MIP film and apply it for fabrication of the selective electrochemical sensor determining the target analyte in the redox probe free test solutions. For that purpose, a ferrocene redox probe was covalently immobilized in a bis-bithiophene polymer molecularly imprinted with the p-synephrine template. Simultaneously, this polymer was deposited on the Pt electrode as a thin film. After the template extraction from the film, the analyte was determined with differential pulse voltammetry (DPV) in a redox probe free solution. That was possible because the internal ferrocene redox probe generated the DPV analytical signal. The thickness and morphology of the film were crucial for the sensor's performance. The mechanism of this redox self-reporting MIP film-based chemosensor was examined with electrochemical methods, simultaneous piezomicrogravimetry and electrochemistry at an electrochemical quartz crystal microbalance, and surface plasmon resonance spectroscopy. The devised chemosensor was applied for selective p-synephrine determination in a concentration range of 2.0 to 75 nM. References Cieplak, M.; Kutner, W., Artificial biosensors: How can molecular imprinting mimic biorecognition? Trends Biotechnol. 2016, 34 (11), 922-941. Uzun, L.; Turner, A. P. F., Molecularly-imprinted polymer sensors: realising their potential. Biosens. Bioelectron. 2016, 76, 131-144. Huynh, T.-P.; Sharma, P. S.; Sosnowska, M.; D'Souza, F.; Kutner, W., Functionalized polythiophenes: Recognition materials for chemosensors and biosensors of superior sensitivity, selectivity, and detectability. Prog. Polym. Sci. 2015, 47, 1-25. Yoshimi, Y.; Narimatsu, A.; Nakayama, K.; Sekine, S.; Hattori, K.; Sakai, K., Development of an enzyme-free glucose sensor using the gate effect of a molecularly imprinted polymer. J. Artif. Organs 2009, 12 (4), 264-270. Sharma, P. S.; Garcia-Cruz, A.; Cieplak, M.; Noworyta, K. R.; Kutner, W., 'Gate effect' in molecularly imprinted polymers: the current state of understanding. Curr. Opin. Electroche. 2019, 16, 50-56. Moreira, F. T. C.; Dutra, R. A. F.; Noronha, J. P. C.; Fernandes, J. C. S.; Sales, M. G. F., Novel biosensing device for point-of-care applications with plastic antibodies grown on Au-screen printed electrodes. Sens. Actuators, B 2013, 182, 733-740. Lach, P.; Cieplak, M.; Majewska, M.; Noworyta, K. R.; Sharma, P. S.; Kutner, W., "Gate Effect" in p-Synephrine Electrochemical Sensing with a Molecularly Imprinted Polymer and Redox Probes. Anal. Chem. 2019, 91 (12), 7546-7553. Figure 1

Biowaste-derived gold nanoparticles on reduced graphene oxide: An innovative nanoplatform for the label-free immunosensing of dengue NS1.

The "gate effect" mechanism for conductive molecularly imprinted polymer (MIP) film coated electrodes was investigated in detail. It was demonstrated that the decrease of the DPV signal for the Fe(CN)64-/Fe(CN)63- redox probe with the increase of the p-synephrine target analyte concentration in solution at the polythiophene MIP-film coated electrode did not originate from swelling or shrinking of the MIP film, as it was previously postulated, but from changes in the electrochemical process kinetics. The MIP-film coated electrode was examined with cyclic voltammetry (CV), differential pulse voltammetry (DPV), electrochemical impedance spectroscopy (EIS), and surface plasmon resonance (SPR). The MIP-film thickness in the absence and in the presence of the p-synephrine analyte was examined with in situ AFM imaging. Moreover, it was demonstrated that doping of the MIP film was not affected by p-synephrine binding in MIP-film molecular cavities. It was concluded that the "gate effect" was most likely caused by changes in radical cation (polaron) mobility in the film.

Biosensor for electrochemical detection of biomarkers of osteoarthritis

An aptamer is a single-stranded DNA (or an RNA)-based oligonucleotide used as a synthetic molecular recognition element in biosensors. Its many advantages include high affinity and binding efficiency, chemical stability, resistance to harsh environment without losing its bioactivity, mass producibility and reusability which make aptamers superior to the natural receptors such as antibodies and enzymes. Aptamers have found their use in the selective detection of a broad range analytes including proteins, peptides, amino acids, drugs, metal ions, and even whole cells. One of the most promising uses of aptamers is in the electrochemical detection of biomarkers for point-of-care (POC) diagnostics for diseases. In order to utilize such aptamers as molecular receptors in the electrochemical sensors, they must be securely immobilized to the surface of the working electrode so that the specific binding between the target and the aptamer can be transduced into a measurable electrical signal. Sophisticated surface chemistries such as thiol-gold binding or carbodiimide cross-linker chemistry (i.e. EDC/NHS coupling) are often used to covalently attach aptamers to the electrodes. However, such surface chemistries make the fabrication of the aptamer-based sensors difficult in terms of mass production or microscale aptamer array development. In our approach, a simpler immobilization technique is proposed to functionalize the electrode with aptamers. In this work, we make use of the pi-pi stacking forces between carbon nanotubes (CNTs) and aptamers to immobilize the aptamers onto the electrode surface. The fabrication procedure is illustrated in Figure 1. First, CNT-aptamer liquid suspension is prepared by mixing CNT and aptamers in deionized water and sonicating until the mixture is fully dispersed. A small droplet of the prepared solution is then deposited on the working electrode of the screen-printed carbon electrode (SPCE) and is left to dry a hot plate. The detection principle of the biosensor is illustrated in Figure 2 where the lysozyme-binding aptamers were used. Due to the strong affinity between a CNT and a DNA, aptamers tend to wrap around the CNTs as the hydrophobic DNA bases of the aptamer interact with the sidewall of the CNTs via pi-stacking. Hence, as shown in Figure 2(a), the charge transfer from the redox probe to the electrode is hindered due to the negatively charged phosphate backbone of the oligonucleotide wrapping around the CNT, hence causing electrical insulation. As the SPCEs are exposed to the target protein (lysozyme), the aptamer unbinds itself (partially or entirely) from the CNT due to the conformational changes caused by the specific recognition of lysozyme as illustrated in Figure 2(b). This enhances the charge transfer rate from the redox probe in the solution to the electrode. Figure 3 shows the Nyquist plots obtained from the electrochemical impedance spectroscopy (EIS) measurements before and after incubation with the solution containing 0, 1, 5, and 10 µg/mL of lysozyme. The radius of the semicircle in the plot indicates the charge transfer resistance of the redox probe (ferro/ferricyanide) at the electrode. The results show that the aptamer-lysozyme binding causes a decrease in the charge transfer resistance (Rct) because of the conformational change of the aptamers. Figure 4(a) shows the post-lysozyme exposure Nyquist curves for different lysozyme concentrations which represents that the degree of Rct change correlates with the lysozyme concentration. Figure 4 (b) shows the calibration curve of the EIS-based lysozyme sensor under exposure to various concentrations of 3 different types of proteins. The plot shows that Rct change is proportional to the lysozyme concentration whereas the other two proteins does not cause significant Rct change. We also characterized the selectivity of our lysozyme biosensor against other protein biomarkers such as thrombin and bovine serum albumin (BSA). Figure 5 presents the relative response (charge transfer resistance) for the three types of proteins with concentrations of 1, 5, and 10 µg/mL. The sensor response is most pronounced for the lysozyme detection while the other two proteins yield minimal responses. This high specificity arises from the high affinity of the anti-lysozyme aptamer. An increase in the charge transfer resistance for BSA at 10 µg/mL can be attributed to the non-specific adsorption to the electrode which further increases the charge transfer resistance of the redox probe at the electrode. The proposed aptamer immobilization technique in conjunction with EIS can be an effective method in developing a simple and low-cost biomarker sensor for point-of-care testing. Figure 1

Precise revealing and early detection of 3-Nitro-L-Tyrosine (3-NLT), a biomarker of oxidative stress in biological media is critical for the early treatment of cancer tumorigenic cells and immunologic disorders. In this study, zinc tungstate (ZnWO4) was incorporated with functionalized carbon nanofibers (f-CNF) to form a ZnWO4/f-CNF composite. The composite improves detection of 3-NLT by increasing the electrical conductivity, electrocatalytic activity, and rapid electron transfer kinetics. Various physical characterization techniques were employed to confirm the ZnWO4/f-CNF composite. Electrochemical impedance spectroscopy, cyclic voltammetry, and differential pulse voltammetry were utilized to detect 3-NLT after modifying ZnWO4/f-CNF on glassy carbon electrode (GCE). The ZnWO4/f-CNF/GCE achieved an elevated electrochemically active surface area (0.08 cm2), a linear range of 1.0–117.0 μM, and a low detection limit of 0.07 μM. Finally, the ZnWO4/f-CNF/GCE was tested with bovine serum albumin and tap water in the real sample investigation.