Biosensor Surface Research Articles

Direct electron transfer of fructose dehydrogenase immobilized on thiol-gold electrodes

Direct electrochemical electron transfer (DET) of oxidoreductases has attracted increasing attention in pure and applied bioelectrochemistry (e.g. biosensors and biofuel cells) over the last decades. We report here a systematic study of DET-type bioelectrocatalysis of the membrane-bound redox enzyme fructose dehydrogenase (FDH, Gluconobacter sp.), on variable-length and variably terminated thiol self-assembled monolayers (SAMs) both on Au(111) and nanoporous gold (NPG) electrodes. FDH on Au(111) modified by short-chain moderately hydrophilic 2-mercaptoethanol (BME) SAMs exhibits the highest DET activities and largest DET-capable fraction. Fitting of theoretical polarization curves to the data and homology modeling/docking of FDH offer further mechanistic insight. The dependence of the DET efficiency of FDH on the length and differently terminated carbon chain is systematically presented. The decreased DET rate with increasing chain length is associated with increasingly unfavourable long-range electron tunnelling, and not with lowered enzyme loading. The porous structure of NPG is favorable for FDH bioelectrocatalysis by improving both efficient enzyme orientation and operational stability. Overall, our study maps systematically the controlled local environmental structural flexibility of the Au/SAM/enzyme/solution interface, a paradigm for thiol modified surfaces in biosensors and bioelectronics.

Development of a Bio-Layer Interferometry-Based Protease Assay Using HIV-1 Protease as a Model

Proteolytic enzymes have great significance in medicine and the pharmaceutical industry and are applied in multiple fields of life sciences. Therefore, cost-efficient, reliable and sensitive real-time monitoring methods are highly desirable to measure protease activity. In this paper, we describe the development of a new experimental approach for investigation of proteolytic enzymes. The method was designed by the combination of recombinant fusion protein substrates and bio-layer interferometry (BLI). The protease (PR) of human immunodeficiency virus type 1 (HIV-1) was applied as model enzyme to set up and test the method. The principle of the assay is that the recombinant protein substrates immobilized to the surface of biosensor are specifically cleaved by the PR, and the substrate processing can be followed by measuring change in the layer thickness by optical measurement. We successfully used this method to detect the HIV-1 PR activity in real time, and the initial rate of the signal decrease was found to be proportional to the enzyme activity. Substrates representing wild-type and modified cleavage sites were designed to study HIV-1 PR’s specificity, and the BLI-based measurements showed differential cleavage efficiency of the substrates, which was proven by enzyme kinetic measurements. We applied this BLI-based assay to experimentally confirm the existence of extended binding sites at the surface of HIV-1 PR. We found the measurements may be performed using lysates of cells expressing the fusion protein, without primary purification of the substrate. The designed BLI-based protease assay is high-throughput-compatible and enables real-time and small-volume measurements, thus providing a new and versatile approach to study proteolytic enzymes.

Interfacial Modeling of Fibrinogen Adsorption onto LiNbO3 Single Crystal-Single Domain Surfaces.

For the development of next-generation protein-based biosensor surfaces, it is important to understand how functional proteins, such as fibrinogen (FBG), interact with polar substrate surfaces in order to prepare highly sensitive points of medical care diagnostics. FBG, which is a fibrous protein with an extracellular matrix, has both positively and negatively charged regions on its 3-dimensional surface, which makes interpreting how it effectively binds to polarized surfaces challenging. In this study, single-crystal LiNbO3 (LNO) substrates that have surface charges were used to investigate the adsorption of FBG protruding polar fragments on the positively and negatively charged LNO surfaces. We performed a combination of experiments and multi-scale molecular modeling to understand the binding of FBG in vacuum and water-solvated surfaces of LNO. XPS measurements showed that the FBG adsorption on LNO increased with increment in solution concentration on surfaces independent of charges. Multi-scale molecular modeling employing Quantum Mechanics, Monte Carlo, and Molecular Mechanics addressed the phenomenon of FBG fragment bonding on LNO surfaces. The binding simulation validated the experimental observation using zeta potential measurements which showed presence of solvated medium influenced the adsorption phenomenon due to the negative surface potential.

Real-Time Viscoelasticity Monitoring of Cardiomyocytes Based on Love Wave Biosensor

Introduction In addition to electrophysiological signals [1-2], mechanical properties such as contraction and relaxation of cardiomyocytes are also important properties. They are essential for the evaluation of drug efficacy and toxicity, and they can change the viscoelasticity of cells, thus providing the possibility for quantitative detection. The acoustic wave sensors are able to detect this slight change in viscoelasticity, which may not be easily noticed under the microscope in the early stages. At present, it has been reported that QCM-D can be used to detect viscoelastic properties of pre-osteoblast cells [3]. Besides, shear-horizontal surface acoustic wave sensor has been applied for the monitoring of cell spreading and attachment [4]. However, no study has realized the label free, real-time and quantitative detection of cardiac viscoelasticity for drug evaluation based on acoustic wave sensors.Recently, the surface acoustic wave (SAW) sensor has been greatly developed as a well-performed mass and viscoelasticity sensing device for label-free immunoassay. In particular, the Love Wave sensor has shown its advantages in biosensor applications due to its high sensitivity and stability in the liquid phase [5]. Hence, it is a promising device capable of monitoring viscoelastic properties of a population of cells in a label free and real-time way.As shown in Fig.1a, a self-designed Love Wave biosensor was used to monitor the viscoelastic properties of HL-1 cardiomyocytes in this study. Our previous studies have shown that insertion loss (IL) of Love Wave biosensor is a more suitable parameter for the quantitative detection of viscoelasticity than phase. Then, two different drugs were added to observe the effects on the viscoelastic properties of HL-1 cells. Method In this study, the Love Wave biosensor is constructed based on a piezoelectric quartz substrate. Interdigitated transducers (IDTs) with Ti/Au (20/200 nm) are deposited to generate acoustic waves. The input and output IDTs electrodes consist of 50 split-finger pairs with a preset wavelength λ=28 μm, which determines the center frequency of the sensor to be about 152 MHz. The distance between IDTs centers is 200λ and the acoustic aperture is 75λ. Afterwards, the IDTs patterned substrate is guided with a 3 μm SiO2 film deposited by plasma enhanced chemical vapor deposition (PECVD). Au layer (200 nm) is deposited on top of guiding layer to improve the cell attachment on the sensor surface. A polydimethylsiloxane (PDMS) chip (Fig.1b) was designed and used as the cell culturing chamber. The height and volume of this cavity are about 10 mm and 110 μL, respectively. A portable multi-channel detection system was developed to collect both the IL and phase signals of Love Wave biosensors. Then the HL-1 cardiac cells with the density of 50,000 cells/well were seeded on the surface of Love Wave biosensor (Fig.1c). To test the function of the detection system, isoprenaline (ISO) and verapamil (VRP) with different concentrations were used to induce changes in the viscoelastic properties of HL-1 cells. Results and Conclusions In Fig.1c, HL-1 cells attach and grow well on the pre-coated Love Wave biosensor, which demonstrates the biocompatibility of the sensor. The insertion loss signals of sensor after treated by ISO and VRP are shown in Fig.1d. It can be found that the insertion loss of the control group decreases slightly, which may be due to cell proliferation. VRP is a calcium channel antagonist that blocks the L-type calcium channel and prevents calcium influx into the cell, thereby decreasing the cardiomyocyte contraction force. With the addition of VRP solutions, the insertion loss of Love Wave biosensors increases with the increase of VRP concentration within the dosage range tested. In contrast to VRP, ISO is positive inotropic drug. After ISO treatment, HL-1 cells tend to be contractive due to the traction forces. Therefore, the higher the ISO concentration, the tighter the contact of the cells with the sensor substrate. Thus, insertion loss decreases after ISO treatment which changed positively correlated with concentration. Moreover, the optical microscope photos also proved that the cells after ISO treatment seems to be smaller than control group, while the cells after VRP treatment seemed to be larger. According to our preliminary experimental results, the affection on cell viscoelasticity of different drugs can be monitored by recording the insertion loss value of the SAW biosensor. Our results demonstrated that the proposed cell-based SAW biosensor is a potential, convenient and quantitative platform for in vitro cardiac viscoelasticity evaluation, especially in the early stages. It has promising applications in cell monitoring, drug evaluation and many other fields.

Detection of Exosomes Using Total Internal Reflected Imaging Ellipsometry.

Exosomes are a kind of membrane-bound phospholipid nanovesicle that are secreted extensively in a variety of biological fluids. Accumulating evidence has indicated that exosomes not only communicate with cells, but also perform functional roles in physiology and pathology. In addition, exosomes have also elicited a great deal of excitement due to their potential as disease biomarkers. Therefore, requirements for sensitive methods capable of precisely and specifically determining exosomes were needed. Herein, we not only develop a sensing surface to capture exosomes but also compare two surface proteins on exosomes, which are appropriate for detecting exosome surface markers by total internal reflected imaging ellipsometry (TIRIE). Protein G and antibody were immobilized on a thin layer of golden substrate to form the biosensing surface. The bio-interaction between antibodies and exosomes was recorded by the TIRIE in real time. The distance between exosomes adhered on a surface was 44 nm ± 0.5 nm. The of anti-CD9 and exosome was lower than anti-CD63 and exosome by introducing pseudo-first-order interaction kinetics, which suggested that CD9 is more suitable for exosome surface markers than CD63. The limit of detection (LOD) of TIRIE was 0.4 μg/mL. In conclusion, we have proposed a surface for the detection of exosomes based on TIRIE, which can make the detection of exosomes convenient and efficient.

Scanning electrochemical microscopy and electrochemical impedance spectroscopy-based characterization of perforated polycarbonate membrane modified by carbon-nanomaterials and glucose oxidase

In this research, scanning electrochemical microscopy (SECM) and electrochemical impedance spectroscopy (EIS) were applied for the evaluation of surface characteristics of electrochemical (amperometric) biosensor based on two composite structures consisting of perforated polycarbonate membrane modified by carbon-nanomaterials and glucose oxidase. The first structure consisted of the polycarbonate filter membrane, punctured by 400 nm holes (PCM) consequently modified with single walled carbon nanotubes (SWCNT) and graphene oxide (GO) layer (PCM/SWCNT/GO); and the other structure consisted of the same PCM, consequently modified with SWCNT and reduced graphene oxide (rGO) layer (PCM/SWCNT/rGO). These two composite structures were developed and applied in biosensor design. In order to form a biosensing surfaces the PCM/SWCNT/GO and PCM/SWCNT/rGO composite structures were modified by immobilized glucose oxidase (GOx). The surface of GOx modified PCM/SWCNT/GO composite structure (PCM/SWCNT/GO/GOx) was evaluated by SECM redox-competition mode at different glucose concentrations. It was determined that PCM/SWCNT/GO/GOx composite structure is suitable for the design of amperometric glucose biosensors, because SWCNT/GO layer facilitates electron transfer from GOx via the oxidation of formed catalyzed reaction product H 2 O 2 . Therefore, the PCM/SWCNT/GO/GOx structure is a promising material for the design of amperometric glucose biosensors and biofuel cells, which are useful for a wide variety of environmental purposes and green energy production using food-industry waste. • Punctured polycarbonate membrane (PCM) was modified by single walled carbon nanotubes (SWCNT). • PCM/SWCNT-based structure was modified by graphene oxide (GO) or reduced graphene oxide (rGO). • On the surface of PCM/SWCNT/GO and PCM/SWCNT/rGO structures glucose oxidase (GOx) was immobilized. • Scanning electrochemical microscopy (SECM) and impedance spectroscopy (EIS) were applied. • It was determined that PCM/SWCNT/GO/GOx structure is suitable for the design of glucose biosensor.

The magnetic field effect on the improvement of the binding reaction of C-reactive protein at the microfluidic channel surface of an SPR biosensor

The magnetic field effect on the improvement of the binding reaction of C-reactive protein at the microfluidic channel surface of an SPR biosensor

Highly sensitive detection of malignant glioma cells using metamaterial-inspired THz biosensor based on electromagnetically induced transparency

Metamaterial-inspired biosensors have been extensively studied recently years for fast and low-cost THz detection. However, only the variation of the resonance frequency has been closely concerned in such sensors so far, whiles the magnitude variation, which also provide important information of the analyte, has not been sufficiently analyzed. In this paper, by the observation of two degree of variations, we propose a label-free biosensing approach for molecular classification of glioma cells. The metamaterial biosensor consisting of cut wires and split ring resonators are proposed to realize polarization-independent electromagnetic induced transparency (EIT) at THz frequencies. Simulated results show that the EIT-like resonance experiences both resonance frequency and magnitude variations when the properties of analyte change, which is further explained with coupled oscillators model theory. The theoretical sensitivity of the biosensor is evaluated up to 496.01 GHz/RIU. In experiments, two types of glioma cells (mutant and wild-type) are cultured on the biosensor surface. The dependences of frequency shifts and the peak magnitude variations on the cells concentrations for different types give new perspective for molecular classification of glioma cells. The measured results indicate that the mutant and wild-type glioma cells can be distinguished directly by observing both the variations of EIT resonance frequency and magnitude at any cells concentrations without antibody introduction. Our metamaterial-based biosensor shows a great potential in the recognition of molecule types of glioma cells, opening alternative way to sensitive biosensing technology.

Construction of a CRISPR-Biolayer Interferometry Platform for Real-Time, Sensitive, and Specific DNA Detection.

The clustered regularly interspaced short palindromic repeats (CRISPR) technology has been widely applied for nucleic acid detection because of its high specificity. By using the highly specific and irreversible bond between HaloTag and its alkane chlorine ligand, we modified dCas9 (deactivated CRISPR/Cas9) with biotin as a biosensor to detect nucleic acids. The CRISPR biosensor was facilely prepared to adequately maintain its DNA-recognition capability. Furthermore, by coupling biolayer interferometry (BLI) with the CRISPR biosensor, a real-time, sensitive, and rapid digital system called CRISPR-BLI was established for the detection of double-stranded DNA. The CRISPR biosensor immobilised on the biolayer could recruit the target DNA onto the biosensor surface and change its optical thickness, resulting in a shift in the interference pattern and responding signal of the BLI. The CRISPR-BLI system was further applied to detect the ALP gene of Escherichia coli DH5α combined with a polymerase chain reaction, which demonstrated a linear range from 20 to 20 000 pg and a low detection limit (1.34 pg). The CRISPR-BLI system is a promising approach for rapid and sensitive detection of target DNA analytes.

Screening and Identification of Herbal Urease Inhibitors Using Surface Plasmon Resonance Biosensor

Background and objectives: Urease, that catalyzes the hydrolysis of urea, has received substantial attention for its impact on living organisms’ health and human life quality. Urease inhibitors play important role in management of different diseases including gastritis and other gastrointestinal disorders. In the present study, a new surface plasmon resonance-based biosensor was designed to discover new urease inhibitors. Methods: The biosensor surface was prepared by the covalent immobilization of urease on carboxymethyldextran hydrogel (CMD 500D) via its primary amine groups. Results: The biosensor combined with an orthogonal enzyme inhibition assay was utilized for screening of 40 traditional medicinal plant extracts against Jack-bean urease. Among them, Laurus nobilis leaf extract displayed a high affinity with the immobilized urease; therefore, its active compound (quercetin) was isolated and identified as a urease inhibitor. The equilibrium constant (KD) and Gibbs free energy (ΔGbinding) values for the interaction of quercetin with urease were obtained to be 55 nM and -41.62 kJ/mol, respectively. The results of molecular docking analysis also confirmed our findings. Conclusion: This SPR-based biosensor represents a new, fast, reliable, and an accurate technique for the identification of new urease inhibitors or novel 'lead' compounds from natural resources.

A novel biosensor based on graphene/platinum nanoparticles/Nafion composites for determination of glucose

In this study, a new amperometric biosensor for the detection of glucose was developed. For this aim, the glassy carbon electrode surface was covered with a platinum nanoparticle graphene solution by dropping. The glucose oxidase enzyme was immobilized by cross-linking method on the modified electrode by graphene/platinum nanoparticles/Nafion (GR/PtNPs/NF) solution. Glucose determination was based on the oxidation of hydrogen peroxide at + 0.60 V produced by the enzymatic reaction on the glucose biosensor surface. Surface morphologies of film electrode and glucose biosensor were evaluated by atomic force microscopy (AFM), scanning electron microscopy (SEM), and surface contact angle analysis. The linear working range of biosensor for glucose was determined. The effects of temperature and pH on the response of the glucose biosensor were investigated. The Km and Vmax constants were calculated as 0.94 mM and 0.775 μA min-1, respectively. The determination limit for the glucose molecule of the prepared biosensor was found as 10 nM. Reproducibility and storage stability were determined for the glucose biosensor. The activity of glucose biosensor was maintained 94.5% at the end of 20 measurements. The effects of the interference occurring in biological environments on the response of the glucose biosensor were examined. The detection of glucose was carried out in synthetic blood sample by biosensor. Electrochemical studies show that graphene/platinum nanoparticles/Nafion (GR/PtNps/NF) film can present a promising biosensing platform and exhibits high sensitivity and selectivity for clinical diagnosis applications.

Ultra-sensitive nucleic acid detection based on target cycling of triple helix molecular switch and ATRP double signal amplification

We propose a novel electrochemical biosensor platform with triple helix structure as switch, Exonuclease III (Exo III)-mediated target cycle at 2-D level and electrochemical-mediated atom transfer radical polymerization (ATRP) at 3-D level as double signal amplification strategy for nucleic acid detection with ultra-sensitivity and high-selectivity. In this strategy, triple-helical DNA modified with N3 was first constructed on the gold electrode (AuE) surface. Exo III mediated target cycle was then triggered in the presence of Target DNA. N3 was released and exposed on the AuE surface, owing to the triple helix structure was destroyed in this process. propargyl-2-bromoisobutyrate (PBIB), the initiator of ATRP, was then introduced to the biosensor surface via click chemistry. Finally, electrochemically mediated ATRP polymerizes a large amount of electroactive monomer, ferrocene methacrylate (FMMA), onto the AuE surface. The target circulates to make target DNA reused at the 2-D level, and ATRP polymerizes a large number of electrochemical signal molecules at the 3-D level, making the biosensor detection limit as low as 1.954 aM. Moreover, the linear range of target DNA detection reaches 7 orders of magnitude (10 aM-10 fM, R2 = 0.993). In addition, the biosensor shows excellent anti-interference ability when analyzing DNA in serum samples. In short, the preparation method is rapid, simple, easy to operate, and has potential applications in biological analysis.

Phenylalanine-responsive fluorescent biosensor based on graphene oxide-chitosan nanocomposites catalytic film for non-destructive fish freshness grading

An optical enzymatic phenylalanine biosensor for non-invasive evaluation of fish freshness using biofluid from the fish body surface has been developed based on phenylalanine dehydrogenase (PDH) and nicotinamide adenine dinucleotide (NAD+) chemisorbed on the graphene oxide-chitosan (GO/CT) nanocomposites film. The immobilized PDH on the biosensor surface via EDC/NHS coupling strategy catalyzed the oxidative deamination of phenylalanine to phenylpyruvate, and concomitantly converted the cofactor NAD+ (oxidized form) to fluorophore NADH (reduced form), which can be quantified by using fluorescence spectrophotometer. The phenylalanine-responsive fluorescent biosensor showed a dynamic linear concentration range between 0.03 mM and 0.24 mM phenylalanine at 644 nm emission wavelength with an excitation wavelength of 320 nm. The limit of detection (LOD) of the fluorometric phenylalanine biosensor was estimated at 0.02 mM with a fast response time of 1 min. No significant interference by amines-synthesized amino acids, such as histamine, creatine, sarcosine, creatinine, tyrosine and uric acid at typical physiological levels commonly co-exist with phenylalanine in the biofluid metabolite of fish towards fresh fish quality grading. Validation study of the GO/CT catalytic film revealed excellent practicality of the biosensor in biological fluid sample of tuna fish (Euthynnus affinis) for detection of phenylalanine. The proposed GO/CT nanocomposites biofilm offers rapid test that can be assessed with an easy manner to rapidly analyze large number of samples in a short period of time.

Recent Advances in Nanotechnology-Based Biosensors Development for Detection of Arsenic, Lead, Mercury, and Cadmium.

Heavy metals cause considerable environmental pollution due to their extent and non-degradability in the environment. Analysis and trace levels of arsenic, lead, mercury, and cadmium as the most toxic heavy metals show that they can cause various hazards in humans’ health. To achieve rapid, high-sensitivity methods for analyzing ultra-trace amounts of heavy metals in different environmental and biological samples, novel biosensors have been designed with the participation of strategies applied in nanotechnology. This review attempted to investigate the novel, sensitive, efficient, cost-benefit, point of care, and user-friendly biosensors designed to detect these heavy metals based on functional mechanisms. The study’s search strategies included examining the primary databases from 2015 onwards and various keywords focusing on heavy metal biosensors’ performance and toxicity mechanisms. The use of aptamers and whole cells as two important bio-functional nanomaterials is remarkable in heavy metal diagnostic biosensors’ bioreceptor design. The application of hybridized nanomaterials containing a specific physicochemical function in the presence of a suitable transducer can improve the sensing performance to achieve an integrated detection system. Our study showed that in addition to both labeled and label-free detection strategies, a wide range of nanoparticles and nanocomposites were used to modify the biosensor surface platform in the detection of heavy metals. The detection limit and linear dynamic range as an essential characteristic of superior biosensors for the primary toxic metals are studied. Furthermore, the perspectives and challenges facing the design of heavy metal biosensors are outlined. The development of novel biosensors and the application of nanotechnology, especially in real samples, face challenges such as the capability to simultaneously detect multiple heavy metals, the interference process in complex matrices, the efficiency and stability of nanomaterials implemented in various laboratory conditions.

Cryopreservation of a cell-based biosensor chip modified with elastic polymer fibers enabling ready-to-use on-site applications

An efficient preservation of a cell-based biosensor chip to achieve a ready-to-use on-site system is still very challenging as the chip contains a living component such as adherent mammalian cells. Herein, we propose a strategy called on-sensor cryopreservation (OSC), which enables the adherent cells to be preserved by freezing (−80 °C) on a biosensor surface, such as the light-addressable potentiometric sensor (LAPS). Adherent cells on rigid surfaces are prone to cryo-injury; thus, the surface was modified to enhance the cell recovery for OSC. It relies on i) the integration of elastic electrospun fibers composed of polyethylene vinyl acetate (PEVA), which has a high thermal expansion coefficient and low glass-transition temperature, and ii) the treatment with O2 plasma. The modified sensor is integrated into a microfluidic chip system not only to decrease the thermal mass, which is critical for fast thawing, but also to provide a precisely controlled micro-environment. This novel cryo-chip system is effective for keeping cells viable during OSC. As a proof-of-concept for the applicability of a ready-to-use format, the extracellular acidification of cancer cells (CHO-K1) was evaluated by differential LAPS measurements after thawing. Results show, for the first time, that the OSC strategy using the cryo-chip allows label-free and quantitative measurements directly after thawing, which eliminates additional post-thaw culturing steps. The freezing of the chips containing cells at the manufacturing stage and sending them via a cold-chain transport could open up a new possibility for a ready-to-use on-site system.

In silico antibody engineering for SARS-CoV-2 detection

Engineered immunoglobulin-G molecules (IgGs) are of wide interest for the development of detection elements in protein-based biosensors with clinical applications. The strategy usually employed for the de novo design of such engineered IgGs consists on merging fragments of the three-dimensional structure of a native IgG, which is immobilized on the biosensor surface, and of an antibody with an exquisite target specificity and affinity. In this work conventional and accelerated classical molecular dynamics (cMD and aMD, respectively) simulations have been used to propose two IgG-like antibodies for COVID-19 detection. More specifically, the crystal structure of the IgG1 B12 antibody, which inactivates the human immunodeficiency virus-1, has been merged with the structure of the antibody CR3022 Fab tightly bounded to SARS-CoV-2 receptor-binding domain (RBD) and the structure of the S309 antibody Fab fragment complexed with SARS-CoV-2 RBD. The two constructed antibodies, named IgG1-CR3022 and IgG1-S309, respectively, have been immobilized on a stable gold surface through a linker. Analyses of the influence of both the merging strategy and the substrate on the stability of the two constructs indicate that the IgG1-S309 antibody better preserves the neutralizing structure than the IgG1-CR3022 one. Overall, results indicate that the IgG1-S309 is appropriated for the generation of antibody based sensors for COVID-19 diagnosis.

A β-Amyloid(1-42) Biosensor Based on Molecularly Imprinted Poly-Pyrrole for Early Diagnosis of Alzheimer's Disease.

Background: Alzheimer’s disease (AD) is a common form of dementia, characterized by production and deposition of β-amyloid peptide in the brain. Thus, β-amyloid peptide is a potentially promising biomarker used to diagnose and monitor the progression of AD. Objective: The study aims to develop a biosensor based on a molecularly imprinted poly-pyrrole for detection of β-amyloid.Material and Methods: In this experimental study, an imprinted poly-pyrrole was employed as an artificial receptor synthesized by electro-polymerization of pyrrole on screen-printed carbon electrodes in the presence of β-amyloid. β-amyloid acts as a molecular template within the polymer. The biosensor was evaluated by cyclic voltammetry using ferro/ferricyanide marker. The parameters influencing the biosensor performance, including electro-polymerization cycle umbers and β-amyloid binding time were optimized to achieve the best biosensor sensitivity. Results: The β-amyloid binding affinity with the biosensor surface was evaluated by the Freundlich isotherm, and Freundlich constant and exponent were obtained as 0.22 ng mL-1 and 10.60, respectively. The biosensor demonstrated a detection limit of 1.2 pg mL-1. The biosensor was applied for β-amyloid determination in artificial cerebrospinal fluid. Conclusion: The biosensor is applicable for early Alzheimer’s disease detection.

Electrochemiluminescence biosensor based on a 3D DNA walking nanomachine with a powerful payload capacity

Abstract Herein, we constructed a three-dimensional (3D) DNA walking nanomachine, just built from Watson–Crick base pairing, which had large payload capacity by using a functionalized DNA nanotube (NT) as track and was further applied to sensitive electrochemiluminescence (ECL) assay of biomarker. The NT as 3D track was composed of a long backbone chain, a triangular fixation unit (UB) and abundant triangular rung units (UA) that arranged along the backbone. Every UA with three footholds could immobilize three ECL emitter (ABEI) labeled-DNA-stators, so this track exhibited a powerful payload capacity on a planar surface compared to that of the existing 1D and 2D DNA walking nanomachines due to the formation of a linear array from UA. Moreover, an amplification strategy made tiny amounts of HIV DNA transform into numerous walkers, which then hybridized with DNA-stator chains. In the presence of Mg2+, the DNA walker initiated moving along the 3D nanotube track, causing a reduce of ECL signal intensity owing to the release of ABEI from the biosensing surface to achieve the assay for HIV DNA with a detection limit of 0.82 fM. Such a strategy and subsequent application for biosensing develop an effective signal amplification 3D DNA walking nanomachine for sensing assay.

An Allosteric Transcription Factor-Based Electrochemical Progesterone Sensor

There is an immense potential for using biosensors in medical diagnostics as well as industries like pharmaceutical, food, beverages, environmental, and agricultural. Today, there remains a significant challenge to meet low levels of detection without compromising simplicity and affordability. Hormone measurements play a central role in family planning programs benefiting millions of women, and in monitoring endocrine disorders. Progesterone is a steroid hormone secreted by the corpus luteum and tracking progesterone levels during an ovulation cycle is beneficial for increasing fertility odds. Furthermore, during pregnancy, progesterone balance helps nurture and develop the fetus. Currently, there are no convenient and cost effective point-of-care (POC) devices to detect progesterone: antibody based approaches are used for progesterone sensing but their use is limited by high cost, production duration, and unreliability. This work has led to the development of in vitro biosensors based on bacterial allosteric transcription factors (aTFs). Like antibodies, aTFs recognize analytes with high sensitivity and specificity. In contrast to antibodies, aTFs respond to analyte binding by changing their affinity for a cognate DNA sequence. Using a novel progesterone-specific aTF, and its binding affinity to a specific DNA sequence provides an intrinsic electrochemical transduction mechanism for the development of novel progesterone biosensor. An electrochemical transduction method offers higher sensitivity and specificity at low cost. The biosensor surface includes a gold electrode with a surface immobilized DNA-aTF complex. During electrochemical measurements using a negative redox mediator (ferrocyanide/ferricyanide complex), in the absence of progesterone the overall negative charge of DNA-aTF complex blocks the negative mediator, prevents exchange of electrons with gold electrode, and generates lower current output. Addition of analyte or progesterone alters the affinity binding of DNA-aTF, releases the aTF from the surface, and enables the mediator to reach the surface, thereby resulting in an amplified current output. Preliminary results using our biosensor yielded a limit of detection of 33 nM over a physiologically relevant range of 0-10 µM progesterone in buffer, and 44 nM in presence of interferents in artificial urine.

Impedimetric Biosensor Based on a Hechtia argentea Lectin for the Detection of Salmonella spp.

A sensitive electrochemical detection method for Salmonella spp. was described, based on the use of Hechtia argentea lectin immobilised on a screen-printed gold electrode. The lectin was extracted from Hechtia argentea, a plant belonging to the Bromeliaceae family. The lectin with molecular weight near 27.4 kDa showed selectivity towards D-mannose, contained on the lipopolysaccharide cell wall of Salmonella spp. Carbohydrate selectivity of the lectin was measured as a change in impedance with respect to concentration. The binding of the bacteria to the biosensor surface increased impedance with increasing concentrations of Salmonella spp., achieving a linear range of detection of 15–2.57 × 107 CFU mL−1, with a limit of detection of 5 CFU mL−1. Increases in impedance were measured using electrochemical impedance spectroscopy and analysed using Nyquist plots. The biosensor was applied in analysis of hen egg samples, and the results were consistent with those obtained using the official analysis methodology.