Search Results (14)

A user-friendly, portable, low-cost readout system for the on-site or point-of-care characterization of chemo- and biosensors based on an electrolyte–insulator–semiconductor capacitor (EISCAP) has been developed using a thumb-drive-sized commercial impedance analyzer. The system is controlled by a custom Python script and allows to characterize EISCAP sensors with different methods (impedance spectra, capacitance-voltage, and constant-capacitance modes), which are selected in a user interface. The performance of the portable readout system was evaluated by pH measurements and the detection of the antibiotic penicillin, hereby using EISCAPs consisting of Al/p-Si/SiO₂/Ta₂O₅ structures and compared to the results obtained with a stationary commercial impedance analyzer. Both the portable and the commercial systems provide very similar results with almost perfectly overlapping recorded EISCAP signals. The new portable system can accelerate the transition of EISCAP sensors from research laboratories to commercial end-user devices. Full article

Using free microorganisms for industrial processes has some limitations, such as the extensive consumption of substrates for growth, significant sensitivity to the microenvironment, and the necessity of separation from the product and, therefore, the cyclic process. It is widely acknowledged that confining or immobilizing cells in a matrix or support structure enhances enzyme stability, facilitates recycling, enhances rheological resilience, lowers bioprocess costs, and serves as a fundamental prerequisite for large-scale applications. This report summarizes the various cell immobilization methods, including several synthetic (polyvinylalcohol, polyethylenimine, polyacrylates, and Eudragit) and natural (gelatin, chitosan, alginate, cellulose, agar–agar, carboxymethylcellulose, and other polysaccharides) polymeric materials in the form of thin films, hydrogels, and cryogels. Advancements in the production of well-known antibiotics like penicillin and cephalosporin by various strains were discussed. Additionally, we highlighted cutting-edge research related to strain producers of peptide-based antibiotics (polymyxin B, Subtilin, Tyrothricin, varigomycin, gramicidin S, friulimicin, and bacteriocin), glusoseamines, and polyene derivatives. Crosslinking agents, especially covalent linkers, significantly affect the activity and stability of biocatalysts (penicillin G acylase, penicillinase, deacetoxycephalosporinase, L-asparaginase, β-glucosidase, Xylanase, and urease). The molecular weight of polymers is an important parameter influencing oxygen and nutrient diffusion, the kinetics of hydrogel formation, rigidity, rheology, elastic moduli, and other mechanical properties crucial for long-term utilization. A comparison of stability and enzymatic activity between immobilized enzymes and their free native counterparts was explored. The discussion was not limited to recent advancements in the biopharmaceutical field, such as microorganism or enzyme immobilization, but also extended to methods used in sensor and biosensor applications. In this study, we present data on the advantages of cell and enzyme immobilization over microorganism (bacteria and fungi) suspension states to produce various bioproducts and metabolites—such as antibiotics, enzymes, and precursors—and determine the efficiency of immobilization processes and the optimal conditions and process parameters to maximize the yield of the target products. Full article

Surfaces with antifouling properties are critical for optimizing biosensors to improve the selectivity and specificity of analyte detection in complex biological samples. This work describes the four-step synthesis of 3-dithiothreitol propanoic acid (DTT_COOH), a new antifouling thiol linker that (a) significantly reduces fouling of raw human serum samples and (b) binds amino receptors via its terminal carboxylic acid group. DTT_COOH was successfully functionalized on quartz crystal microbalance (QCM) discs and used to anchor penicillin-binding aptamers. Relative to bare and coated (11-mercaptoundecanoic acid (MUA) and 1-undecanethiol (UDT)) QCM crystals, DTT_COOH’s antifouling improved by approximately 75–86%. Following aptamer/ethanolamine extension, the modified DTT_COOH layer reduced serum fouling by approximately 95–97% compared to bare and coated (MUA, UDT) crystals. QCM with dissipation (QCM-D) monitoring, contact goniometry, and cyclic voltammetry techniques were used to compare the DTT_COOH surfaces with quartz crystals functionalized with hydrophobic and hydrophilic molecules. Full article

Utilizing an appropriate enzyme immobilization strategy is crucial for designing enzyme-based biosensors. Plant virus-like particles represent ideal nanoscaffolds for an extremely dense and precise immobilization of enzymes, due to their regular shape, high surface-to-volume ratio and high density of surface binding sites. In the present work, tobacco mosaic virus (TMV) particles were applied for the co-immobilization of penicillinase and urease onto the gate surface of a field-effect electrolyte-insulator-semiconductor capacitor (EISCAP) with a p-Si-SiO₂-Ta₂O₅ layer structure for the sequential detection of penicillin and urea. The TMV-assisted bi-enzyme EISCAP biosensor exhibited a high urea and penicillin sensitivity of 54 and 85 mV/dec, respectively, in the concentration range of 0.1–3 mM. For comparison, the characteristics of single-enzyme EISCAP biosensors modified with TMV particles immobilized with either penicillinase or urease were also investigated. The surface morphology of the TMV-modified Ta₂O₅-gate was analyzed by scanning electron microscopy. Additionally, the bi-enzyme EISCAP was applied to mimic an XOR (Exclusive OR) enzyme logic gate. Full article

Currently, assays for rapid therapeutic drug monitoring (TDM) of β-lactam antibiotics in blood, which might be of benefit in optimizing doses for treatment of critically ill patients, remain challenging. Previously, we developed an assay for determining the penicillin-class antibiotics in blood using a thermometric penicillinase biosensor. The assay eliminates sample pretreatment, which makes it possible to perform semicontinuous penicillin determinations in blood. However, penicillinase has a narrow substrate specificity, which makes it unsuitable for detecting other classes of β-lactam antibiotics, such as cephalosporins and carbapenems. In order to assay these classes of clinically useful antibiotics, a novel biosensor was developed using New Delhi metallo-β-lactamase-1 (NDM-1) as the biological recognition layer. NDM-1 has a broad specificity range and is capable of hydrolyzing all classes of β-lactam antibiotics in high efficacy with the exception of monobactams. In this study, we demonstrated that the NDM-1 biosensor was able to quantify multiple classes of β-lactam antibiotics in blood plasma at concentrations ranging from 6.25 mg/L or 12.5 mg/L to 200 mg/L, which covered the therapeutic concentration windows of the tested antibiotics used to treat critically ill patients. The detection of ceftazidime and meropenem was not affected by the presence of the β-lactamase inhibitors avibactam and vaborbactam, respectively. Furthermore, both free and protein-bound β-lactams present in the antibiotic-spiked plasma samples were detected by the NDM-1 biosensor. These results indicated that the NDM-1 biosensor is a promising technique for rapid TDM of total β-lactam antibiotics present in the blood of critically ill patients. Full article

The feasibility of light-addressed detection and manipulation of pH gradients inside an electrochemical microfluidic cell was studied. Local pH changes, induced by a light-addressable electrode (LAE), were detected using a light-addressable potentiometric sensor (LAPS) with different measurement modes representing an actuator-sensor system. Biosensor functionality was examined depending on locally induced pH gradients with the help of the model enzyme penicillinase, which had been immobilized in the microfluidic channel. The surface morphology of the LAE and enzyme-functionalized LAPS was studied by scanning electron microscopy. Furthermore, the penicillin sensitivity of the LAPS inside the microfluidic channel was determined with regard to the analyte’s pH influence on the enzymatic reaction rate. In a final experiment, the LAE-controlled pH inhibition of the enzyme activity was monitored by the LAPS. Full article

Multi-enzyme immobilization onto a capacitive field-effect biosensor by nano-spotting technique is presented. The nano-spotting technique allows to immobilize different enzymes simultaneously on the sensor surface with high spatial resolution without additional photolithographical patterning. The amount of applied enzymatic cocktail on the sensor surface can be tailored. Capacitive electrolyte-insulator-semiconductor (EIS) field-effect sensors with Ta₂O₅ as pH-sensitive transducer layer have been chosen to immobilize the three different (pL droplets) enzymes penicillinase, urease, and glucose oxidase. Nano-spotting immobilization is compared to conventional drop-coating method by defining different geometrical layouts on the sensor surface (fully, half-, and quarter-spotted). The drop diameter is varying between 84 µm and 102 µm, depending on the number of applied drops (1 to 4) per spot. For multi-analyte detection, penicillinase and urease are simultaneously nano-spotted on the EIS sensor. Sensor characterization was performed by C/V (capacitance/voltage) and ConCap (constant capacitance) measurements. Average penicillin, glucose, and urea sensitivities for the spotted enzymes were 81.7 mV/dec, 40.5 mV/dec, and 68.9 mV/dec, respectively. Full article

Since the discovery and subsequent use of penicillin, antibiotics have been used to treat most bacterial infections in the U.S. Over time, the repeated prescription of many antibiotics has given rise to many antibiotic-resistant microbes. A bacterial strain becomes resistant by horizontal gene transfer, where surviving microbes acquire genetic material or DNA fragments from adjacent bacteria that encode for resistance. In order to avoid significant bacterial resistance, novel and target therapeutics are needed. Further advancement of diagnostic technologies could be used to develop novel treatment strategies. The use of biosensors to detect quorum-sensing signaling molecules has the potential to provide timely diagnostic information toward mitigating the multidrug-resistant bacteria epidemic. Resistance and pathogenesis are controlled by quorum-sensing (QS) circuits. QS systems secrete or passively release signaling molecules when the bacterial concentration reaches a certain threshold. Signaling molecules give an early indication of virulence. Detection of these compounds in vitro or in vivo can be used to identify the onset of infection. Whole-cell and cell-free biosensors have been developed to detect quorum-sensing signaling molecules. This review will give an overview of quorum networks in the most common pathogens found in chronic and acute infections. Additionally, the current state of research surrounding the detection of quorum-sensing molecules will be reviewed. Followed by a discussion of future works toward the advancement of technologies to quantify quorum signaling molecules in chronic and acute infections. Full article

This work aimed to develop accurate, quick, and practical tools for the detection of residues of penicillin G antibiotic in biological and non-biological samples. The assays were developed based on the binding mechanism of β-lactam to penicillin-binding proteins; samples of different concentrations of penicillin G were incubated with in vitro expressed 6X-Histidine-tagged soluble penicillin-binding protein (PBP2x*) of Streptococcus pneumoniae (S. pneumoniae), whereby penicillin G in samples specifically binds to PBP2x*. The fluorescent-labeled β-lactam analogue Bocillin FL was used as a competent substrate, and two different routes estimated the amounts of the penicillin G. The first route was established based on the differences in the concentration of non-bounded Bocillin FL molecules within the reactions while using a real-time polymerase chain reaction (PCR)-based method for fluorescence detection. The second route depended on the amount of the relative intensity of Bocillin FL bounded to Soluble PBP-2x*, being run on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-page), visualized by a ChemiDoc-It^®2 Imager, and quantified based on the fluorescence affinity of the competent substrate. While both of the methods gave a broad range of linearity and high sensitivity, the on column based real-time method is fast, non-time consuming, and highly sensitive. The method identified traces of antibiotic in the range 0.01–0.2 nM in addition to higher accuracy in comparison to the SDS-based detection method, while the sensitivity of the SDS-based method ranged between 0.015 and 2 µM). Thus, the on column based real time assay is a fast novel method, which was developed for the first time based on the binding inhibition of a fluorescence competitor material and it can be adapted to screen traces of penicillin G in any biological and environmental samples. Full article

Antibiotic resistance and, accordingly, their pollution because of uncontrolled usage has emerged as a serious problem in recent years. Hence, there is an increased demand to develop robust, easy, and sensitive methods for rapid evaluation of antibiotics and their residues. Among different analytical methods, the aptamer-based biosensors (aptasensors) have attracted considerable attention because of good selectivity, specificity, and sensitivity. This review gives an overview about recently-developed aptasensors for antibiotic detection. The use of various aptamer assays to determine different groups of antibiotics, like β-lactams, aminoglycosides, anthracyclines, chloramphenicol, (fluoro)quinolones, lincosamide, tetracyclines, and sulfonamides are presented in this paper. Full article

A new concept for the development of semiconductor field-effect biosensors by modification of a gate surface with tobacco mosaic virus (TMV) nanotubes, serving as enzyme nanocarrier, is presented. TMV nanotubes enable an immobilization of a high amount of enzymes without substantial loss of their activity, resulting in an enhanced biosensor performance. This approach has been experimentally demonstrated by realizing a capacitive field-effect penicillin biosensor using TMV nanotubes functionalized with the enzyme penicillinase as model system. Full article

In this study, we have successfully demonstrated the fabrication of a biosensor based on well aligned single-crystal zinc oxide (ZnO) nanorods which were grown on gold coated glass substrate using a low temperature aqueous chemical growth (ACG) method. The ZnO nanorods were immobilized with penicillinase enzyme using the physical adsorption approach in combination with N-5-azido-2-nitrobenzoyloxysuccinimide (ANB-NOS) as cross linking molecules. The potentiometric response of the sensor configuration revealed good linearity over a large logarithmic concentration range from 100 µM to 100 mM. During the investigations, the proposed sensor showed a good stability with high sensitivity of ~121 mV/decade for sensing of penicillin. A quick electrochemical response of less than 5 s with a good selectivity, repeatability, reproducibility and a negligible response to common interferents such as Na¹⁺, K¹⁺, d-glucose, l-glucose, ascorbic acid, uric acid, urea, sucrose, lactose, glycine, penicilloic acid and cephalosporins, was observed. Full article

A sensitive and reliable potentiometric biosensor for determination of penicillin has been developed by exploiting the self-limiting growth of the non-conducting polymer, polytyramine. Optimum polytyramine-penicillinase (PTy-PNCnase) films for potentiometric detection of penicillin were accomplished with monomer solutions which contained 0.03 M tyramine, 37 U/mL penicillinase, 0.01 M KNO₃,and 3 mM penicillin with an applied current density of 0.8 mA/cm² and an electropolymerisation time of 40 seconds. The potentiometric biosensor gave a linear concentration range of 3–283 µM for penicillin and achieved a minimum detectable concentration of 0.3 µM. The biosensor was successfully utilized for the detection of Amoxycillin and gave an average percentage recovery of 102 ± 6%. Satisfactory recoveries of penicillin G were also achieved in milk samples with the potentiometric biosensor when concentrations are ≥20 ppm. Full article

Macroporous silicon has been etched from n-type Si, using a vertical etching cell where no rear side contact on the silicon wafer is necessary. The resulting macropores have been characterised by means of Scanning Electron Microscopy (SEM). After etching, SiO₂ was thermally grown on the top of the porous silicon as an insulating layer and Si₃N₄ was deposited by means of Low Pressure Chemical Vapour Deposition (LPCVD) as transducer material to fabricate a capacitive pH sensor. In order to prepare porous biosensors, the enzyme penicillinase has been additionally immobilised inside the porous structure. Electrochemical measurements of the pH sensor and the biosensor with an Electrolyte/Insulator/Semiconductor (EIS) structure have been performed in the Capacitance/Voltage (C/V) and Constant capacitance (ConCap) mode. Full article