Impedimetric Single Carbon Fiber Electrode for Ultrasensitive Detection of Staphylococcus aureus Pathogen DNAs in Breast Milk by CRISPR Technology.

This study introduces a novel biosensing approach for the detection of pathogen DNA in breast milk utilizing single carbon fiber electrodes (SCFE) enhanced with MXene nanomaterial layers. The primary innovation lies in the modification of SCFE with MXenes to increase the electrode's surface area, followed by surface activation for the immobilization of dCas9-sgRNA complexes. This modification aims to leverage the unique properties of MXenes and the selective binding capability of the CRISPR technology for efficient and specific pathogen detection. Electrochemical impedance spectroscopy (EIS) and scanning electron microscopy (SEM) analyses were employed to characterize the electrode modifications and the immobilization process, demonstrating the successful enhancement of biosensor performance. This study further optimized the chronoimpedimetric detection method to achieve rapid, sensitive, and selective detection of Staphylococcus aureus (SAu) DNA in breast milk, with a notable detection time of 60 s in real samples. The biosensor demonstrated high selectivity and sensitivity, with a linear detection range between 50 and 6000 fM and a limit of detection (LOD) of 14.5 fM. The reproducibility and stability of the biosensor were also confirmed through multiple tests, showing promising potential for clinical and public health applications.

Carbon-Based Nanomaterials Decorated Electrospun Nanofibers in Biosensors: A Review.

Nanomaterials have revolutionized scientific research due to their exceptional physical and chemical capabilities. Carbon-based nanomaterials such as graphene and its derivates have excellent electrical, optical, thermal, physical, and chemical properties that have made them indispensable in several industries worldwide, including medicine, electronics, and energy. By incorporating carbon-based nanomaterials as nanofillers in electrospun nanofibers (ESNFs), smoother and highly conductive nanofibers can be achieved that possess a large surface area and porosity. This approach provides a superior alternative to traditional materials in the development of improved biosensors. Carbon-based ESNFs, among the most exciting new-generation materials, have many applications, including filtration, pharmaceuticals, biosensors, and membranes. The electrospinning technique is a highly efficient and cost-effective method for producing desired nanofibers compared to other methods. Various types of natural and synthetic organic polymers have been successfully utilized in solution electrospinning to produce nanofibers directly. To create diagnostics devices, various biomolecules like antibodies, enzymes, aptamers, ligands, and even cells can be bound to the surface of nanofibers. Electrospun nanofibers can serve as an immobilization matrix to create a biofunctional surface. Thus, biosensors with desired features can be produced in this way. This study comprehensively reviews biosensors that integrate nanodiamonds, fullerenes, carbon nanotubes, graphene oxide, and carbon dots into electrospun nanofibers.

Electrospun Nanofibers including Organic/Inorganic Nanohybrids: Polystyrene- and Clay-Based Architectures in Immunosensor Preparation for Serum Amyloid A.

Diagnostic techniques based on biomolecules have application potential that can be realized in many fields, such as disease diagnosis, bioprocess imaging, food/beverage industries, and environmental pollutant imaging. Successful surface immobilization of biomolecules is critical to increasing the stabilization, sensitivity, and selectivity of biomolecules used in bioassay systems. Nanofibers are good candidates for the immobilization of biomolecules owing to many advantages such as morphology and pore size. In this study, montmorillonite (MMT) clay is modified with poly(amidoamine) (PAMAM) generation 3 (PAMAMG3) and added to polystyrene (PS) solutions, following which PS/MMT-PAMAMG3 nanofibers are obtained using the electrospinning method. The nanofibers are obtained by testing PS% (wt%) and MMT-PAMAMG3% (wt%) ratios and characterized with scanning electron microscopy. Antiserum amyloid A antibody (Anti-SAA) is then conjugated to the nanofibers on the electrode surface via covalent bonds using a zero-length cross linker. Finally, the obtained selective surface is used for electrochemical determination of serum amyloid A (SAA) levels. The linear range of PS/MMT-PAMAM/Anti-SAA is between 1 and 200 ng/mL SAA, and the detection limit is 0.57 ng/mL SAA. The applicability of PS/MMT-PAMAMG3/Anti-SAA is investigated by taking measurements in synthetic saliva and serum both containing SAA.

Novel Approaches to Enzyme-Based Electrochemical Nanobiosensors.

Electrochemistry is a genuinely interdisciplinary science that may be used in various physical, chemical, and biological domains. Moreover, using biosensors to quantify biological or biochemical processes is critical in medical, biological, and biotechnological applications. Nowadays, there are several electrochemical biosensors for various healthcare applications, such as for the determination of glucose, lactate, catecholamines, nucleic acid, uric acid, and so on. Enzyme-based analytical techniques rely on detecting the co-substrate or, more precisely, the products of a catalyzed reaction. The glucose oxidase enzyme is generally used in enzyme-based biosensors to measure glucose in tears, blood, etc. Moreover, among all nanomaterials, carbon-based nanomaterials have generally been utilized thanks to the unique properties of carbon. The sensitivity can be up to pM levels using enzyme-based nanobiosensor, and these sensors are very selective, as all enzymes are specific for their substrates. Furthermore, enzyme-based biosensors frequently have fast reaction times, allowing for real-time monitoring and analyses. These biosensors, however, have several drawbacks. Changes in temperature, pH, and other environmental factors can influence the stability and activity of the enzymes, affecting the reliability and repeatability of the readings. Additionally, the cost of the enzymes and their immobilization onto appropriate transducer surfaces might be prohibitively expensive, impeding the large-scale commercialization and widespread use of biosensors. This review discusses the design, detection, and immobilization techniques for enzyme-based electrochemical nanobiosensors, and recent applications in enzyme-based electrochemical studies are evaluated and tabulated.

Antibody-Conjugated Electrospun Nanofibers for Electrochemical Detection of Methamphetamine.

Multifunctional electrospun nanofibers (ENs) with improved properties have increased attention nowadays. Their insoluble forms in water with decreased hydrophobicity are desired for the immobilization of biological molecules. Also, the addition of functional groups on the backbone provides the conjugation of biomolecules onto the surface of ENs via covalent bonds to increase their stability. Here, poly(vinylidene fluoride) (PVDF) was chosen to prepare a platform, which is insoluble in water, and polyethylenimine (PEI) was used to add amine groups on the surface of ENs to bind biological molecules via covalent conjugation. So, PVDF-PEI nanofibers were prepared on a glassy carbon electrode to immobilize an antimethamphetamine antibody (Anti-METH) as a model biomolecule. The obtained PVDF-PEI/Anti-METH was used for the bioelectrochemical detection of methamphetamine (METH), a common illicit drug. Bioelectrochemical detection of METH on PVDF-PEI/Anti-METH-coated electrodes was carried out by voltammetry in the range of 2.0-50 ng/mL METH. Moreover, the effect of dansyl chloride (DNC) derivatization of METH on the sensitivity of PVDF-PEI/Anti-METH was tested. Finally, METH analysis was carried out in synthetic body fluids. The obtained results showed that PVDF-PEI ENs can be adopted as an immobilization matrix for the biorecognition elements of biobased detection systems, and the derivative of METH (METH-DNC) increased the sensitivity of PVDF-PEI/Anti-METH.

Development of an Enzymatic Biosensor Using Glutamate Oxidase on Organic-Inorganic-Structured, Electrospun Nanofiber-Modified Electrodes for Monosodium Glutamate Detection.

Herein, dendrimer-modified montmorillonite (Mt)-decorated poly-Ɛ-caprolactone (PCL) and chitosan (CHIT)-based nanofibers were prepared. Mt was modified with a poly(amidoamine) generation 1 (PAMAMG1) dendrimer, and the obtained PAMAMG1-Mt was incorporated into the PCL-CHIT nanofiber's structure. The PCL-CHIT/PAMAMG1-Mt nanofibers were conjugated with glutamate oxidase (GluOx) to design a bio-based detection system for monosodium glutamate (MSG). PAMAMG1-Mt was added to the PCL-CHIT backbone to provide a multipoint binding side to immobilize GluOx via covalent bonds. After the characterization of PCL-CHIT/PAMAMG1-Mt/GluOx, it was calibrated for MSG. The linear ranges were determined from 0.025 to 0.25 mM MSG using PCL-CHIT/Mt/GluOx and from 0.0025 to 0.175 mM MSG using PCL-CHIT/PAMAMG1-Mt/GluOx (with a detection limit of 7.019 µM for PCL-CHIT/Mt/GluOx and 1.045 µM for PCL-CHIT/PAMAMG1-Mt/GluOx). Finally, PCL-CHIT/PAMAMG1-Mt/GluOx was applied to analyze MSG content in tomato soup without interfering with the sample matrix, giving a recovery percentage of 103.125%. Hence, the nanofiber modification with dendrimer-intercalated Mt and GluOx conjugation onto the formed nanocomposite structures was performed, and the PCL-CHIT/PAMAMG1-Mt/GluOx system was successfully developed for MSG detection.

Carbon Nanotube-Incorporated Nanofibers for Immunosensor Preparation against CD36.

The increased serum concentration of CD36 is significantly associated with atherosclerosis, insulin resistance, and diabetes mellitus. Currently, there is no sensor system used for the detection of CD36 in the clinical field. Therefore, there is a need to develop a sensor system for the detection of CD36. The large surface area/volume ratio and controllable surface conformation of electrospun nanofibers (ENs) make them highly attractive for immunosensor applications. In the present study, PS/MWCNT-PAMAM ENs were produced and used as an immobilization matrix of Anti-CD36. Thus, the electrochemical behavior of the developed nanocomposite-based ENs and their usage potential were investigated for immunosensor applications. First, an oxidized multiwall carbon nanotube (MWCNT-OH) was synthesized and modified with a polyamidoamine generation 3 (PAMAM G3) dendrimer. The synthesized MWCNT-PAMAM nanocomposite was mixed with polystyrene (PS) solutions at different ratios to produce bead-free, smooth, and uniform PS/MWCNT-PAMAM ENs. PS/MWCNT-PAMAM ENs were accumulated on a screen-printed carbon electrode (SPCE) using the electrospinning technique. A biofunctional surface on the PS/MWCNT-PAMAM EN-coated SPCE was created using carbodiimide chemistry by covalent immobilization of Anti-CD36. The analytic performance characteristics of the developed PS/MWCNT-PAMAM/Anti-CD36 immunosensor were determined by performing electrochemical measurements in the presence of the CD36 protein. The linear detection range was found to be from 5 to 40 ng/mL, and the limit of detection was calculated as 3.94 ng/mL for CD36. The developed PS/MWCNT-PAMAM/Anti-CD36 immunosensor also displayed high tolerance to interference substances, good repeatability, and high recovery percent (recovery%) for artificial blood serum analysis.

Graphene Oxide-Magnetic Nanoparticles Loaded Polystyrene-Polydopamine Electrospun Nanofibers Based Nanocomposites for Immunosensing Application of C-Reactive Protein.

The large surface area/volume ratio and controllable surface conformation of electrospun nanofibers (ENFs) make them highly attractive in applications where a large surface area is desired, such as sensors and affinity membranes. In this study, nanocomposite-based ENFs were produced and immobilization of Anti-CRP was carried out for the non-invasive detection of C-reactive protein (CRP). Initially, the synthesis of graphene oxide (GO) was carried out and it was modified with magnetic nanoparticles (MNP, Fe3O4) and polydopamine (PDA). Catechol-containing and quinone-containing functional groups were created on the nanocomposite surface for the immobilization of Anti-CRP. Polystyrene (PS) solution was mixed with rGO-MNP-PDA nanocomposite and PS/rGO-MNP-PDA ENFs were produced with bead-free, smooth, and uniform. The surface of the screen-printed carbon electrode (SPCE) was covered with PS/rGO-MNP-PDA ENFs by using the electrospinning technique under the determined optimum conditions. Next, Anti-CRP immobilization was carried out and the biofunctional surface was created on the PS/rGO-MNP-PDA ENFs coated SPCE. Moreover, PS/rGO-PDA/Anti-CRP and PS/MNP-PDA/Anti-CRP immunosensors were also prepared and the effect of each component in the nanocomposite-based electrospun nanofiber (MNP, rGO) on the sensor response was investigated. The analytic performance of the developed PS/rGO-MNP-PDA/Anti-CRP, PS/rGO-PDA/Anti-CRP, and PS/MNP-PDA/Anti-CRP immunosensors were examined by performing electrochemical measurements in the presence of CRP. The linear detection range of PS/rGO-MNP-PDA/Anti-CRP immunosensor was found to be from 0.5 to 60 ng/mL and the limit of detection (LOD) was calculated as 0.33 ng/mL for CRP. The PS/rGO-MNP-PDA/Anti-CRP immunosensor also exhibited good repeatability with a low coefficient of variation.

In situ synthesis of biomolecule encapsulated gold-cross-linked poly(ethylene glycol) nanocomposite as biosensing platform: a model study.

In situ synthesis of poly(ethylene glycol) (PEG) hydrogels containing gold nanoparticles (AuNPs) and glucose oxidase (GOx) enzyme by photo-induced electron transfer process was reported here and applied in electrochemical glucose biosensing as the model system. Newly designed bionanocomposite matrix by simple one-step fabrication offered a good contact between the active site of the enzyme and AuNPs inside the network that caused the promotion in the electron transfer properties that was evidenced by cyclic voltammetry as well as higher amperometric biosensing responses in comparing with response signals obtained from the matrix without AuNPs. As well as some parameters important in the optimization studies such as optimum pH, enzyme loading and AuNP amount, the analytical characteristics of the biosensor (AuNP/GOx) were examined by the monitoring of chronoamperometric response due to the oxygen consumption through the enzymatic reaction at -0.7 V under optimized conditions at sodium acetate buffer (50 mM, pH 4.0) and the linear graph was obtained in the range of 0.1-1.0 mM glucose. The detection limit (LOD) of the biosensor was calculated as 0.06 mM by using the signal to noise ratio of 3. Moreover, the presence of AuNPs was visualized by TEM. Finally, the biosensor was applied for glucose analysis for some beverages and obtained data were compared with HPLC as the reference method to test the possible matrix effect due to the nature of the samples.

Construction and comparison of Trametes versicolor laccase biosensors capable of detecting xenobiotics.

Amperometric biosensors using laccase from Trametes versicolor as a bioelement were developed for 2,4-dichloro phenoxy acetic acid (2,4-D). Laccase enzyme was immobilized by gelatin and glutaraldehyde onto a Clark oxygen probe and screen printed electrodes (SPEs). Amperometric and chronoamperometric measurements were carried out with the biosensors. First, the effect of laccase activity on the biosensor performances was investigated for both biosensors, and then optimum pH and temperature and also thermal stability of the biosensors were tested. In addition, the detection ranges of some phenolic compounds were obtained by the help of calibration graphs of them. In repeatability studies, variation coefficients and standard deviations for both biosensors were also calculated by the studies done for this purposes. Finally, the biosensors were applied to the determination of 2,4-D in a real herbicide sample.

A new approach for in vitro imaging of breast cancer cells by anti-metadherin targeted PVA-pyrene.

Poly(vinyl alcohol)-pyrene-anti-metadherin (PVA-Py-(Anti-MTDH)), a novel antibody based water soluble probe containing both fluorescent and target sites in the structure for in vitro imaging of breast cancer cells is reported here. Since breast cancer cells have an excess of MDTH protein expressed on the surface, a PVA-Py prepared by "Click chemistry" approach is targeted by Anti-MTDH antibody and applied to the MCF-7 cell line. After characterization, the designed architecture was evaluated in terms of cell incorporation efficiency and compared with a non-targeted structure (PVA-Py). Atomic force microscopy (AFM) and fluorescence microscopy images of cells after incubation of the probe molecules were also obtained to monitor the interaction of the probes with the cancerous cells.

Maltose biosensing based on co-immobilization of alpha-glucosidase and pyranose oxidase.

A new bi-enzymatic system was designed by co-immobilization of alpha-glucosidase (AG) and pyranose oxidase (PyOx) for maltose analysis. The immobilization was carried out by cross-linking enzyme mixture, chitosan (CHIT) and carbon nanotube (CNT) via glutaraldehyde. The structure of biosensor including enzyme, CHIT, glutaraldehyde and CNT amount together with operational conditions like pH, temperature and applied potential were optimized. Then analytical characterization was performed. A fast linear response of the biosensor was observed for maltose in the concentration range from 0.25 to 2.0 mM at 35 degrees C and pH 6.0. The effect of CNT addition into the immobilization matrix was also investigated. The linear relationships between sensor response (y; microA/cm(2)) and substrate concentration (x; mM) were defined by the equations of y=0.844x+0.029 (R(2)=0.999) and y=0.882x+0.0625 (R(2)=0.996) for AG/PyOx/CHIT and AG/PyOx/CHIT-CNT biosensors, respectively. All other data were also given as comparison of two systems one with CNT-modified and CNT-free. Finally, for the sample application, maltose was analyzed in beer samples. As a result, it has been found that; complex matrix of natural beer samples had no influence on the biosensing response. Also the results were in good agreement with those obtained by spectrophotometric measurements.

Alcohol biosensing by polyamidoamine (PAMAM)/cysteamine/alcohol oxidase-modified gold electrode.

A highly stable and sensitive amperometric alcohol biosensor was developed by immobilizing alcohol oxidase (AOX) through Polyamidoamine (PAMAM) dendrimers on a cysteamine-modified gold electrode surface. Ethanol determination is based on the consumption of dissolved oxygen content due to the enzymatic reaction. The decrease in oxygen level was monitored at -0.7 V vs. Ag/AgCl and correlated with ethanol concentration. Optimization of variables affecting the system was performed. The optimized ethanol biosensor showed a wide linearity from 0.025 to 1.0 mM with 100 s response time and detection limit of (LOD) 0.016 mM. In the characterization studies, besides linearity some parameters such as operational and storage stability, reproducibility, repeatability, and substrate specificity were studied in detail. Stability studies showed a good preservation of the bioanalytical properties of the sensor, 67% of its initial sensitivity was kept after 1 month storage at 4 degrees C. The analytical characteristics of the system were also evaluated for alcohol determination in flow injection analysis (FIA) mode. Finally, proposed biosensor was applied for ethanol analysis in various alcoholic beverage as well as offline monitoring of alcohol production through the yeast cultivation.

Fluorescence sensing of glucose using glucose oxidase modified by PVA-pyrene prepared via "click" chemistry.

Poly(vinyl alcohol)-pyrene-glucose oxidase (PVA-Py-GOx), a water-soluble polymer possessing both fluorescent and oxidant sites in the structure, is synthesized by "click" chemistry and modification processes and characterized. The morphology of PVA-Py-GOx was characterized with atomic force microscopy (AFM), and a heterogeneous morphology due to the incorporation of GOx was observed. The capability of PVA-Py-GOx to act as a bioprobe for fluorescence sensing of glucose is examined. The postulated fluorescence mechanism for glucose analysis is based on the consumption of glucose by dissolved oxygen and GOx present in the structure. Thus, the fluorescence intensity of pyrene groups of the probe increases by the elimination of fluorescence quenching by oxygen. Glucose concentration was analyzed quantitatively from 0.25 to 3.0 mM by the fluorescence measurement. The effect of pH and amount of PVA-Py-GOx was also studied. The proposed system was applied to analyze glucose in real samples and compared with those obtained from commercial kits.

Electrochemical polymerization of 1-(4-nitrophenyl)-2,5-di(2-thienyl)-1 H-pyrrole as a novel immobilization platform for microbial sensing.

Two types of bacterial biosensor were constructed by immobilization of Gluconobacter oxydans and Pseudomonas fluorescens cells on graphite electrodes modified with the conducting polymer; poly(1-(4-nitrophenyl)-2,5-di(2-thienyl)-1 H-pyrrole) [SNS(NO(2))]. The measurement was based on the respiratory activity of cells estimated by the oxygen consumption at -0.7 V due to the metabolic activity in the presence of substrate. As well as analytical characterization, the linear detection ranges, effects of electropolymerization time, pH and cell amount were examined by using glucose as the substrate. The linear relationships were observed in the range of 0.25-4.0 mM and 0.2-1.0 mM for G. oxydans and P. fluorescens based sensors, respectively.

A microbial biosensor based on bacterial cells immobilized on chitosan matrix.

A bio-electrochemical system consisting of Gluconobacter oxydans DSM 2343 cells as a biological material and carbon nanotube (CNT)-free and CNT-modified chitosan as immobilizing matrices has been developed. The measurement was based on the respiratory activity of the cells estimated by the oxygen consumption at -0.7 V (versus the Ag|AgCl reference electrode) due to the metabolic activity in the presence of substrates. The system was calibrated and dependence of signal amplitude on the measuring conditions and cell amount was studied as well as the substrate specificity, pH, temperature and working potential. The biosensors (CNT-modified and unmodified) were demonstrated for the quantification of glucose in the range of 0.05-1.0 mM, at 30 degrees C and pH 7.0 with the 40 s of response time. The linear relationships between sensor response (y; microA/cm(2)) and substrate concentration (x; mM) were defined by the equations of y=1.160x+0.151 (R(2)=0.990) and y=1.261x+0.197 (R(2)=0.982), respectively. All other data were also given as comparison of two systems one with CNT-modified and CNT-free.

Pseudomonas putida based amperometric biosensors for 2,4-D detection.

Amperometric biosensors using Pseudomonas putida cells as a bioelement were developed for 2,4-dichloro phenoxy acetic acid (2,4-D). After the adaptation process of Pseudomonas putida to 2,4-D, cells were immobilized onto the screen printed graphite electrodes (SPG) as well as Clark oxygen probe by gelatin and glutaraldehyde. Optimum pH, temperature, and stability of the biosensor were investigated. Substrate specificities for various phenolic compounds were also searched. In repeatability studies, variation coefficients and standard deviations for both type of systems were calculated; SPG and Clark electrodes were calculated and results are given as a comparison of two systems. Finally, the biosensors were applied to 2,4-D determination in a real herbicide sample.

Biosensing approach for glutathione detection using glutathione reductase and sulfhydryl oxidase bienzymatic system.

Chitosan membrane with glutathione reductase and sulfhydryl oxidase (SOX) was subsequently integrated onto the surface of spectrographic graphite rods for obtaining a glutathione biosensor. The working principle was based on the monitoring of O(2) consumption that correlates the concentration of glutathione during the enzymatic reaction. A linear relationship between sensor response and concentration was obtained between 0.5 and 2.0 mM for oxidized glutathione (GSSG), and 0.2-1.0 mM for reduced glutathione (GSH) in the presence of 2 microM nicotinamide adenine dinucleotide phosphate (NADPH) under the optimum working conditions. Also, reduced/oxidized glutathione were separated by HPLC and utility of bienzymatic system was investigated as an electrochemical detector for the analysis of these compounds. All data were given as a comparison of two systems: biosensor and diode array detector (DAD).

Determination of phenolic acids using Trametes versicolor laccase.

Two biosensors based on Trametes versicolor laccase (TvL) were developed for the determination of phenolic compounds. Commercial oxygen electrode and ferrocene-modified screen-printed graphite electrodes were used for preparation of laccase biosensors. The systems were calibrated for three phenolic acids. Linearity was obtained in the concentration range 0.1-1.0muM caffeic acid, 0.05-0.2muM ferulic acid, 2.0-14.0muM syringic acid for laccase immobilised on a commercial oxygen electrode and 2.0-30.0muM caffeic acid, 2.0-10.0muM ferulic acid, 4.0-30.0muM syringic acid for laccase immobilised on ferrocene-modified screen-printed electrodes. Furthermore, optimal pH, temperature and thermal stability studies were performed with the commercial oxygen electrode. Both electrodes were used for determination of a class of phenolic acids, achieving a cheap and fast tool and an easy to be used procedure for screening real samples of human plasma.

Effects of mediators on the laccase biosensor response in paracetamol detection.

An enzyme electrode suitable for paracetamol detection was developed by immobilizing laccase on a dissolved-oxygen probe surface. The immobilization procedure was achieved by means of gelatin, which was then cross-linked with glutaraldehyde. The measurement was based on the detection of oxygen consumption in relation to analyte oxidation. The optimum experimental conditions for the biosensor were investigated and the system was calibrated for paracetamol. Also the effects of three different mediators, namely HBT (1-hydroxybenzotriazole), VLA [violuric acid (5-isonitrosobarbituric acid)] and TEMPO (2,2',6,6'-tetramethylpiperidine-N-oxyl radical) were tested for the biosensor's response. As a result, it was observed that HBT has a remarkable effect on the signal by providing more oxygen consumption during the enzymatic reaction. A linear relationship between sensor responses and analyte concentrations was obtained over the concentration range 2.0-15.0 microM, whereas, in the presence of the mediator HBT, this range became 0.5-3.0 microM.