A simple and reliable assay for detecting specific nucleotide sequences in plants using optical thin-film biosensor chips

Here we report the adaptation and optimization of an efficient, accurate and inexpensive assay that employs custom-designed silicon-based optical thin-film biosensor chips to detect unique transgenes in genetically modified (GM) crops and SNP markers in model plant genomes. Briefly, aldehyde-attached sequence-specific single-stranded oligonucleotide probes are arrayed and covalently attached to a hydrazine-derivatized biosensor chip surface. Unique DNA sequences (or genes) are detected by hybridizing biotinylated PCR amplicons of the DNA sequences to probes on the chip surface. In the SNP assay, target sequences (PCR amplicons) are hybridized in the presence of a mixture of biotinylated detector probes and a thermostable DNA ligase. Only perfect matches between the probe and target sequences, but not those with even a single nucleotide mismatch, can be covalently fixed on the chip surface. In both cases, the presence of specific target sequences is signified by a color change on the chip surface (gold to blue/purple) after brief incubation with an anti-biotin IgG horseradish peroxidase (HRP) to generate a precipitable product from an HRP substrate. Highly sensitive and accurate identification of PCR targets can be completed within 30 min. This assay is extremely robust, exhibits high sensitivity and specificity, and is flexible from low to high throughput and very economical. This technology can be customized for any nucleotide sequence-based identification assay and widely applied in crop breeding, trait mapping, and other work requiring positive detection of specific nucleotide sequences.     

An aptamer-based fluorescent biosensor for potassium ion detection using a pyrene-labeled molecular beacon

A novel and sensitive biosensor based on aptamer and pyrene-labeled fluorescent probes for the determination of K⁺ was developed. The aptamer was used as a molecular recognition element and a partially complementary oligonucleotide with the aptamer was labeled by pyrene moieties at both ends to transduce the binding event of K⁺ with aptamer. In the presence of K⁺, the complementary oligonucleotides were displaced from aptamers, which was accompanied by excimer fluorescence of pyrenes because the self-hairpin structure of the complementary oligonucleotide brought pyrene moieties into close proximity. However, it gave only monomer emission in the absence of K⁺. Under optimum conditions, the relative fluorescence intensity of pyrene was proportional to the concentration of K⁺ in the range of 6.0 × 10⁻⁴ to 2.0 × 10⁻² M. A detection limit of 4.0 × 10⁻⁴ M was achieved. Moreover, this method was able to detect K⁺ with high selectivity in the presence of Na⁺, , Mg²⁺, and Ca²⁺ ions of biological fluids. In brief, the assay may have great potential applications, especially in a biological environment because of its simplicity, sensitivity, and specificity.     

A copper‐specific microbial fuel cell biosensor based on riboflavin biosynthesis of engineered Escherichia coli

Copper pollution poses a serious threat to the aquatic environment; however, in situ analytical methods for copper monitoring are still scarce. In the current study, Escherichia coli Rosetta was genetically modified to express OprF and ribB with promoter P_t7 and P_cusC, respectively, which could synthesize porin and senses Cu²⁺ to produce riboflavin. The cell membrane permeability of this engineered strain was increased and its riboflavin production (1.45–3.56 μM) was positively correlated to Cu²⁺ (0–0.5 mM). The biosynthetic strain was then employed in microbial fuel cell (MFC) based biosensor. Under optimal operating parameters of pH 7.1 and 37°C, the maximum voltage (248, 295, 333, 352, and 407 mV) of the constructed MFC biosensor showed a linear correlation with Cu²⁺ concentration (0.1, 0.2, 0.3, 0.4, 0.5 mM, respectively; R² = 0.977). The continuous mode testing demonstrated that the MFC biosensor specifically senses Cu²⁺ with calculated detection limit of 28 μM, which conforms to the common Cu²⁺ safety standard (32 μM). The results obtained with the developed biosensor system were consistent with the existing analytical methods such as colorimetry, flame atomic absorption spectrometry, and inductively coupled plasma optical emission spectrometry. In conclusion, this MFC‐based biosensor overcomes the signal conversion and transmission problems of conventional approaches, providing a fast and economic analytical alternative for in situ monitoring of Cu²⁺ in water.     

RNA‐based fluorescent biosensors for live cell detection of bacterial sRNA

Bacteria contain a diverse set of RNAs to provide tight regulation of gene expression in response to environmental stimuli. Bacterial small RNAs (sRNAs) work in conjunction with protein cofactors to bind complementary mRNA sequences in the cell, leading to up‐ or downregulation of protein synthesis. In vivo imaging of sRNAs can aid in understanding their spatiotemporal dynamics in real time, which inspires new ways to manipulate these systems for a variety of applications including synthetic biology and therapeutics. Current methods for sRNA imaging are quite limited in vivo and do not provide real‐time information about fluctuations in sRNA levels. Herein, we describe our efforts toward the development of an RNA‐based fluorescent biosensor for bacterial sRNA both in vitro and in vivo. We validated these sensors for three different bacterial sRNAs in Escherichia coli and demonstrated that the designs provide a bright, sequence‐specific signal output in response to exogenous and endogenous RNA targets.     

DNA‐based probes for flow cytometry analysis of endocytosis and recycling

The internalization of proteins plays a key role in cell development, cell signaling and immunity. We have previously developed a specific hybridization internalization probe (SHIP) to quantitate the internalization of proteins and particles into cells. Herein, we extend the utility of SHIP to examine both the endocytosis and recycling of surface receptors using flow cytometry. SHIP was used to monitor endocytosis of membrane‐bound transferrin receptor (TFR) and its soluble ligand transferrin (TF). SHIP enabled measurements of the proportion of surface molecules internalized, the internalization kinetics and the proportion and rate of internalized molecules that recycle to the cell surface with time. Using this method, we have demonstrated the internalization and recycling of holo‐TF and an antibody against the TFR behave differently. This assay therefore highlights the implications of receptor internalization and recycling, where the internalization of the receptor‐antibody complex behaves differently to the receptor‐ligand complex. In addition, we observe distinct internalization patterns for these molecules expressed by different subpopulations of primary cells. SHIP provides a convenient and high throughput technique for analysis of trafficking parameters for both cell surface receptors and their ligands.      

Taste receptor cell-based biosensor for taste specific recognition based on temporal firing

Taste receptor cells are the taste sensation elements expressing sour, salty, sweet, bitter and umami receptors, respectively. There are cell-to-cell communications between different types of cells. Nevertheless, the mechanism of taste sensation and taste information coded by taste receptor cell is not well understood at present and it is a long-standing issue. In order to explore taste sensation and analyze taste-firing responses from another point of view, we present a promising biomimetic taste receptor cell-based biosensor. The temporal firing responses to different tastants are recorded. Meanwhile, we investigate the firing rate and temporal firing of taste receptor cells. The experimental results are consistent with that from patch clamp and molecular biology experiment. Firing rate is dependent on the concentration of stimulus. PCA analysis (principal component analysis) of the temporal firing responses shows that the responses from different types of taste receptor cells can be distinguished. Furthermore, exogenous ATP is applied to mimic the effects of transmitter ATP (adenosine triphosphate) released from type II cells onto type III cells. Both enhanced and inhibitory effects on spontaneous firing are observed. This novel biomimetic hybrid biosensor provides a potential solution to investigate the taste sensation and coding mechanisms in a non-invasive way.     

Novel biosensor system model based on fluorescence quenching by a fluorescent streptavidin and carbazole‐labeled biotin

In the present study, a novel molecular biosensor system model was designed by using a couple of the fluorescent unnatural mutant streptavidin and the carbazole‐labeled biotin. BODIPY‐FL‐aminophenylalanine (BFLAF), a fluorescent unnatural amino acid was position‐specifically incorporated into Trp120 position of streptavidin by four‐base codon method. On the other hand, carbazole‐labeled biotin was synthesized as a quencher for the fluorescent Trp120BFLAF mutant streptavidin. The fluorescence of fluorescent Trp120BFLAF mutant streptavidin was decreased as we expected when carbazole‐labeled biotin was added into the mutant streptavidin solution. Furthermore, the fluorescence decrease of Trp120BFLAF mutant streptavidin with carbazole‐labeled biotin (100 nM) was recovered by the competitive addition of natural biotin. This result demonstrated that by measuring the fluorescence quenching and recovery, a couple of the fluorescent Trp120BFLAF mutant streptavidin and the carbazole‐labeled biotin were successfully applicable for quantification of free biotin as a molecular biosensor system. Copyright © 2016 John Wiley & Sons, Ltd.     

SERS‐based biosensor for Alzheimer disease evaluation through the fast analysis of human serum

Alzheimer disease (AD) is the most common form of dementia in the elderly, progressively affecting the cognitive functions with a complex diagnostic procedure that limits the time for a prompt intervention. In this study we optimized a reliable protocol for the analysis of AD patients and healthy subjects' serum using the Surface Enhanced Raman Spectroscopy (SERS), taking into consideration the effect of different variables on the final spectra, analyzed and compared through multivariate analysis and correlated with hippocampus volume. As results, we demonstrated a statistical difference between the spectra collected from the two investigated groups, with an accuracy, precision and specificity of respectively 83%, 86%, and 86%. The correlation of these data with those obtained from MRI, demonstrated a direct correlation between Raman spectra and hippocampus degeneration showing the Raman Spectroscopy (RS) as a potential tool for the monitoring of AD progression and rehabilitation treatments.     

Quantum dots‐based fluorescence resonance energy transfer biosensor for monitoring cell apoptosis

The development of advanced methods for accurately monitoring cell apoptosis has extensive significance in the diagnostic and pharmaceutical fields. In this study, we developed a rapid, sensitive and selective approach for the detection of cell apoptosis by combining the site‐specific recognition and cleavage of the DEVD–peptide with quantum dots (QDs)‐based fluorescence resonance energy transfer (FRET). Firstly, biotin‐peptide was conjugated on the surface of AuNPs to form AuNPs‐pep through the formation of an Au‐S bond. Then, AuNPs–pep–QDs nanoprobe was obtained through the connection between AuNPs–pep and QDs. FRET is on and the fluorescence of QDs is quenched at this point. The evidence of UV–vis spectra, transmission electron microscopy (TEM), and Fourier transform infrared (FT‐IR) spectroscopy revealed that the connection was successful. Upon the addition of apoptosis cell lysis solution, peptide was cleaved by caspase‐3, and AuNPs was dissociated from the QDs. At this time, FRET is off, and thus the QDs fluorescence was recovered. The experimental conditions were optimized in terms of ratio of peptide to AuNPs, buffer solution, and the temperature of conjugation and enzyme reaction. The biosensor was successfully applied to distinguishing apoptosis cells and normal cells within 2 h. This study demonstrated that the biosensor could be utilized to evaluate anticancer drugs.     

A T7exonuclease‐assisted target recycling amplification with graphene oxide acting as the signal amplifier for fluorescence polarization detection of human immunodeficiency virus (HIV) DNA

We report a fluorescence polarization (FP) platform for human immunodeficiency virus (HIV) DNA detection based on T7exonuclease‐assisted target recycling amplification with graphene oxide (GO) acting as a FP signal amplifier. In the sensing method, the presence of the target DNA leads to target recycling with the assistance of T7exonuclease, furthermore, the amplification products are absorbed onto the surface of GO, so the all FP values are enhanced by GO. More importantly, this FP sensor exhibits high detection sensitivity; under optimal conditions, the change in FP is linear with the concentration of the target DNA within a concentration range of 50–2000 pmol/L, and the detection limit of this method is as low as 38.6 pmol/L. This FP sensor also exhibits high selectivity, even single‐base mismatched DNA can be effectively discriminated from complementary target DNA. Above all, the proposed FP sensor may serve as a general platform for the sensitive assay of disease‐related genes. Copyright © 2015 John Wiley & Sons, Ltd.     

Novel device for quick measurement of glucose in POCT areas without pre‐analytical steps based on a multi‐way glucose biosensor

Professional Point of Care testing demands rapid analysis and professional quality. To assure rapid analysis of high quality the analytical tool ideally should be able to work without sample pre‐treatment and should offer the opportunity to calibrate and/or control the analytical performance of the tool. In contrast to an enormous number of different disposable‐strips used for patient self monitoring today and based on an extended knowledge with respect to multi‐way biosensors used in laboratory analyzers we decided to develop a professional Point of Care Testing system for glucose analysis based on a multi‐way biosensor. The multi‐way glucose biosensor placed in the instrument for 30 days did reduce the lag time between blood withdrawal and availability of a result of lab quality in a bedside area to about 10 seconds. No pre‐analytical steps are necessary for measuring capillary whole blood, no crossing over was observed, and the data could be transferred into a laboratory information system or a hospital information system. Thus, we were able to realize tools for professional health control able to measure glucose values in laboratory quality at places outside laboratories: e.g., in doctor's offices, hospital wards, critical care units, and training units of athletes. By combining the advantages of laboratory analyzers (high quality and low sample price) and the advantages of disposable strips (simple procedure and immediate results after sample withdrawal) with the Glukometer 3000 and LactatProfi 3000 we did start to fill the gap between the two basic technologies available on the market for diagnosis today. Glukometer 3000 and LactatProfi 3000 are worldwide the first and only mobile glucose and lactate measuring instruments for decentralized locations based on multi‐way biosensors.     

Structural and functional study of a simple,rapid, and label‐free DNAzyme‐based DNA biosensor for optimization activity

Peroxidase‐mimicking DNAzyme has a potential to self‐assemble into a G‐quadruplex and shows peroxidase activity. In comparison to proteins, peroxidase‐mimicking DNAzyme is less expensive and more stable. Herein, it is used in fabricating non‐labeling biosensors. This paper investigates the structural and functional properties of a DNA biosensor based on split DNAzyme with a detection limit in nM range (9.48 nM). Two halves of DNAzyme were linked by a complementary sequence of DNA target. Hybridization of the DNA target pulled two DNAzyme halves apart and peroxidase activity decreased. This study can be divided into 3 stages. First, the characteristics of DNAzyme were studied by Circular Dichroism technique and UV–Vis spectroscopy to find out DNAzyme's optimum activity. It is worth to note that some divalent cations were used to form G‐quadruplex, in addition to common monovalent cations. Furthermore, the hemin incubation was also optimized. Secondly, the structural and functional properties of two types of split DNAzyme were compared with DNAzyme. Thirdly, the hybridization of DNA target was monitored. The results revealed that peroxidase activities of split types decreased by half without any specific conformational changes. Interestingly, the catalytic activities of split DNAzymes could be promoted by adding Mg²⁺. Besides, it was demonstrated that the structure, peroxidation reaction, and DNA target hybridization of 2:2 and 3:1 split modes were almost alike. It was also illustrated that magnesium promoted the possibility of hybridization.     

A new biosensor for specific determination of sucrose using an oxidoreductase of Zymomonas mobilis and invertase

A new biosensor for specific determination of sucrose was developed using an oxidoreductase of Zymomonas mobilis and invertase. Cells of Z. mobilis were permeabilized with toluene in order to utilize the enzymes of glucose-fructose oxidoreductase and gluconolactonase inside the intact cells. Permeabilized cells and invertase were coimmobilized in a gelatin membrane, and a whole cell enzyme electrode was constructed by fixing the membrane on a pH electrode. The production of hydrogen ion was detected using the biosensor-connected microcomputer, and the concentration of sucrose was determined by using both the initial rate and the steady-state methods. Optimum conditions for biosensor response were pH 6.2 and temperature 35 degrees C. The effect of interfering compounds on the electrode response was investigated, and the interference by various sugars was eliminated by determining sucrose concentration using the steady-state method. The biosensor developed is simple and reproducible, and the calibration curve for sucrose is linear up to 70 g/L.     

Application of a fluorescent biosensor based‐on magneto‐γ‐Fe2O3–methyldopa nanoparticles for adsorption of human serum albumin

Understanding and controlling the interaction between the polymer methyldopa (2‐amino‐3‐(3,4‐dihydroxyphenyl)‐2‐methyl‐propanoic acid) (PMDP)–γ‐Fe₂O₃ nanoparticles and biological fluids is important if the potential of nanoparticles (NPs) in biomedicine is to be realized. Physicochemical studies on the interactions between proteins and NPs are influenced by the surface properties of the NPs. To identify the effects of the NP surface, interactions between human serum albumin (HSA) and PMDP–γ‐Fe₂O₃ NPs were investigated. Here, the adsorption of HSA onto small (10–30 nm diameter) PMDP–γ‐Fe₂O₃ NPs was quantitatively analyzed using spectroscopic methods. The fluorescence quenching data were checked for the inner‐filter effect, the main confounding factor in the observed quenching. The binding constants, K_a, were calculated at different temperatures, using a nonlinear fit to the experimental data, and the thermodynamic parameters ?H, ?S and ?G were given. The obtained thermodynamic signature suggests that hydrophobic interactions at least are present. This result indicates that the structure of the protein turns from a structureless denatured state at pH 3 into an ordered biologically active native state on addition of PMDP–γ‐Fe₂O₃ NPs. Copyright © 2015 John Wiley & Sons, Ltd.     

Development of a biosensor for urea assay based on amidase inhibition,using an ion-selective electrode

Abstract

A biosensor for urea has been developed based on the observation that urea is a powerful active-site inhibitor of amidase, which catalyzes the hydrolysis of amides such as acetamide to produce ammonia and the corresponding organic acid. Cell-free extract from Pseudomonas aeruginosa was the source of amidase (acylamide hydrolase, EC 3.5.1.4) which was immobilized on a polyethersulfone membrane in the presence of glutaraldehyde; an ion-selective electrode for ammonium ions was used for biosensor development. Analysis of variance was used for optimization of the biosensor response and showed that 30 μL of cell-free extract containing 7.47 mg protein mL^?1, 2 μL of glutaraldehyde (5%, v/v) and 10 μL of gelatin (15%, w/v) exhibited the highest response. Optimization of other parameters showed that pH 7.2 and 30 min incubation time were optimum for incubation of membranes in urea. The biosensor exhibited a linear response in the range of 4.0–10.0 μM urea, a detection limit of 2.0 μM for urea, a response time of 20 s, a sensitivity of 58.245 % per μM urea and a storage stability of over 4 months. It was successfully used for quantification of urea in samples such as wine and milk; recovery experiments were carried out which revealed an average substrate recovery of 94.9%. The urea analogs hydroxyurea, methylurea and thiourea inhibited amidase activity by about 90%, 10% and 0%, respectively, compared with urea inhibition.