Bacterial Membrane Depolarization-Linked Fuel Cell Potential Burst as Signal for Selective Detection of Alcohol

The biosensing application of microbial fuel cell (MFC) is hampered by its long response time, poor selectivity, and technical difficulty in developing portable devices. Herein, a novel signal form for rapid detection of ethanol was generated in a photosynthetic MFC (PMFC). First, a dual chambered (100 mL each) PMFC was fabricated by using cyanobacteria-based anode and abiotic cathode, and its performance was examined for detection of alcohols. A graphene-based nanobiocomposite matrix was layered over graphite anode to support cyanobacterial biofilm growth and to facilitate electron transfer. Injection of alcohols into the anodic chamber caused a transient potential burst of the PMFC within 60 s (load 1000 Ω), and the magnitude of potential could be correlated to the ethanol concentrations in the range 0.001–20% with a limit of detection (LOD) of 0.13% (<i>R</i>² = 0.96). The device exhibited higher selectivity toward ethanol than methanol as discerned from the corresponding cell–alcohol interaction constant (<i>K</i>_i) of 780 and 1250 mM. The concept was then translated to a paper-based PMFC (p-PMFC) (size ∼20 cm²) wherein, the cells were merely immobilized over the anode. The device with a shelf life of ∼3 months detected ethanol within 10 s with a dynamic range of 0.005–10% and LOD of 0.02% (<i>R</i>² = 0.99). The fast response time was attributed to the higher wettability of ethanol on the immobilized cell surface as validated by the contact angle data. Alcohols degraded the cell membrane on the order of ethanol > methanol, enhanced the redox current of the membrane-bound electron carrier proteins, and pushed the anodic band gap toward more negative value. The consequence was the potential burst, the magnitude of which was correlated to the ethanol concentrations. This novel approach has a great application potential for selective, sensitive, rapid, and portable detection of ethanol

Multifaceted Interaction Studies between Carbon Dots and Proteins of Clinical Importance for Optical Sensing Signals

Carbon dots (CDs) are emerging as efficient optical probes. However, their application potential for clinical diagnosis has not been adequately explored. Herein, we examined the suitability of pyroglutamate CDs for detecting glucose, cholesterol, and alcohol in blood serum through their peroxidative activity in the respective enzyme-catalyzed reactions following fluorometric and colorimetric approaches. In buffer, the CD’s fluorescence intensity (λex 354nm) enhanced over 115% after interaction with the enzyme proteins due to different lifetime components on its surface. The enhancement was also linked to FRET with the proteins (λex 274nm for TRP/TYR). The electrostatic interactions, as revealed from the zeta potential study, generated binding energy (ΔG, kcal/mol) in the range of −5.8 to −6.3 and greatly shifted the protein’s secondary structure to β-strand contents. The CD’s fluorescence in the blood serum medium was also enhanced where serum’s particulate components contributed to the emission. All these subvert fluorescence emissions could be substantially cleaned for detection of peroxide generated in the enzymatic reaction by filtering the serum particulates and redox proteins prior to the addition of CDs to the reaction systems. The CD, however, could complement well in ABTS-based (absorbance at λmax 414nm) colorimetric reaction in blood serum without introducing protein or particle separation steps for sensitive detection of peroxide. The limit of detection, dynamic range, and sensitivity discerned for peroxide in the glucose oxidase-catalyzed reaction system were 183 μM, 0.02–0.10 mM (R2 = 0.98), and 0.2482 AU mM–1, respectively. Overall, these findings will guide clinical application of the peroxidatic CDs to detect various analytes in blood serum following fluorometric- and colorimetric-based principles

Dye Coupled Aptamer-Captured Enzyme Catalyzed Reaction for Detection of Pan Malaria and <i>P. falciparum</i> Species in Laboratory Settings and Instrument-Free Paper-Based Platform

Malaria diagnosis methods offering species-specific information on the causative parasites, along with their flexibility to use in different resource settings, have great demand for precise treatment and management of the disease. Herein, we report the detection of pan malaria and P. falciparum species using a dye-based reaction catalyzed by the biomarker enzymes Plasmodium lactate dehydrogenase (PLDH) and Plasmodium falciparum glutamate dehydrogenase (PfGDH), respectively, through instrument-based and instrument-free approaches. For the detection, two ssDNA aptamers specific to the corresponding PLDH and PfGDH were used. The aptamer-captured enzymes were detected through a substrate-dependent reaction coupled with the conversion of resazurin (blue, ∼λ605nm) to resorufin (pink, ∼λ570nm) dye. The reaction was monitored by measuring the fluorescence intensity at λ660nm for resorufin, absorbance ratio (λ570nm/λ605nm), and change in color (blue to pink). The detection approach could be customized to a spectrophotometer-based method and an instrument-free device. For both the approaches, the biomarkers were captured from the serum samples with the help of aptamer-coated magnetic beads prior to the analysis to exclude potential interferences from the serum. In the instrument-free device, a medical syringe (5 mL) prefabricated with a magnet was used for in situ separation of the enzyme-captured beads from the reaction supernatant. The converted dye in the supernatant was then efficiently adsorbed over a DEAE cellulose-treated paper wick assembled in the syringe hose. The biomarkers could be detected by both qualitative and quantitative format following the color and pixel intensity, respectively, developed on the paper surface. The developed method and technique offered detection of the biomarkers within a clinically relevant dynamic range, with the limit of detection values in the picomolar level. Flexible detection capability, low cost, interference-free detections, and portable nature (for instrument-free devices) are the major advantages offered by the developed approaches

Protein-Induced Fluorescence Enhancement Based Detection of <i>Plasmodium falciparum</i> Glutamate Dehydrogenase Using Carbon Dot Coupled Specific Aptamer

A novel 90-mer long ssDNA aptamer (NG3) covering a 40-mer random region targeting <i>Plasmodium falciparum</i> glutamate dehydrogenase (<i>Pf</i>GDH) developed through systematic evolution of ligands by exponential enrichment (SELEX) technique. The binding affinity of the aptamer to <i>Pf</i>GDH discerned by circular dichroism (CD) was 0.5 ± 0.04 μM. The specificity of the aptamer toward the target was confirmed by gel electrophoresis and CD studies. The presence of two quadruplex forming regions, two big and four small stem loop structures with a δG of −7.99 kcal mol^–1 for NG3 were deduced by computational studies. The spherical carbon dots (Cdots) of size 2–4 nm, synthesized by pyrolysis method using l-glutamate as a substrate were covalently linked to the amine modified aptamer. The Cdot with a band gap of 2.8 eV and a quantum yield of 34% produced fluorescence at ∼ λ_410 nm when excited at λ_320nm. The quantum yield of Cdot-aptamer assembly was increased up to 40% in the presence of the <i>Pf</i>GDH in solution. A linear relationship with a dynamic range of 0.5 nM to 25 nM (R² = 0.98) and a limit of detection (LOD) of 0.48 nM was observed between the fluorescence intensity of the Cdots-aptamer conjugate and the concentration of <i>Pf</i>GDH. The method could detect <i>Pf</i>GDH with an LOD of 2.85 nM in diluted serum sample. This novel simple, sensitive and specific protein induced fluorescence enhancement based detection of <i>Pf</i>GDH has a great potential to develop as a method for malaria detection

Silk-Hydrogel-Immobilized Gold-Nanocluster-Seeded Catalase Protein as a Pre-Plasmonic Probe for Colorimetric Peroxide Sensing

Protein-stabilized gold nanoclusters (AuNCs) are emerging as luminescent probes for various sensing applications. However, the technical difficulty of designing solid analytical platforms with these probes impedes their sensor applications. Herein, blue fluorescent (emission ∼ λ460nm) catalase protein (Cat)-AuNCs with peroxidase-mimicking activity were synthesized through an aurichlorohydric acid-led reaction process that concurrently released the heme prosthetic group from the protein matrix, turning off the catalase activity. A significant structural transition with a ∼1.9-fold increase in the β-sheets of the protein occurred during the process of seeding AuNCs of size ∼1.5 nm (dia) in protein molecules, as revealed from the respective circular dichroism and scanning electron microscopy studies. An atomic force microscopy study revealed the presence of AuNCs in the surface periphery of the catalase protein. Upon interaction with the substrate H2O2, these nanoclusters were destabilized and transformed into free plasmonic gold nanoparticles (AuNPs) with an average size of 10 nm through an internal aggregation process. The plasmonic signal (absorbance λ520nm) intensity of the released AuNPs was increased with the increasing concentration of H2O2, offering a linear detection range of 20–200 mM (R2 = 0.99) and a limit of detection of 1 mM for the peroxide. The phenomenon was utilized to develop a colorimetric sensor by immobilizing Cat-AuNCs in silk fibroin hydrogels, which offered stability to the clusters for up to 3 months. The plasmonic response signal from the sensor surface could be captured as a red pixel intensity and color change (yes/no format) for the respective quantitative and qualitative detections of H2O2 for diverse applications

Development of an Indicator Displacement Based Detection of Malaria Targeting HRP-II as Biomarker for Application in Point-of-Care Settings

A novel label free spectrophotometric detection of malarial biomarker HRP-II following an indicator displacement assay has been developed. The assay is based on competitive displacement of murexide dye from its complex with Ni²⁺ by HRP-II present in serum samples. The binding constant (<i>K</i>_d) discerned for the dye and HRP-II to Ni²⁺ were 1.4 × 10^–6 M^–1 and 6.8 × 10^–9 M^–1, respectively. The progress of the reaction could be monitored from the change of color from orange (∼λ_482 nm) to pink (∼λ_515 nm) with the concomitant increase in HRP-II concentration in the mixture. A linear response (<i>R</i>² = 0.995) curve was generated by plotting the ratio of absorbance (λ_515 nm/λ_482 nm) against the HRP-II concentrations. The method offers to detect HRP-II as low as 1 pM without any interference from some common salts and the major protein, HSA, present in the blood serum. The detection method was reproduced in a microfluidic paper based analytical device (μPAD), fabricated by printing hydrophobic alkyl ketene dimer on a chromatographic paper to create hydrophilic microchannels, test zone, and sample application zone. The device offers to use a maximum sample volume of 20 ± 0.06 μL and detects HRP-II within 5 min with LOD of 30 ± 9.6 nM in a dynamic range of 10 to 100 nM. The method has thus immense potential to develop as rapid, selective, simple, portable, and inexpensive malarial diagnostic device for point-of-care and low resource setting applications

Molecular Characterization and Expression of a Novel Alcohol Oxidase from <i>Aspergillus terreus</i> MTCC6324

<div><p>The alcohol oxidase (AOx) cDNA from <i>Aspergillus terreus</i> MTCC6324 with an open reading frame (ORF) of 2001 bp was constructed from <i>n</i>-hexadecane induced cells and expressed in <i>Escherichia coli</i> with a yield of ∼4.2 mg protein g⁻¹ wet cell. The deduced amino acid sequences of recombinant rAOx showed maximum structural homology with the chain B of aryl AOx from <i>Pleurotus eryngii</i>. A functionally active AOx was achieved by incubating the apo-AOx with flavin adenine dinucleotide (FAD) for ∼80 h at 16°C and pH 9.0. The isoelectric point and mass of the apo-AOx were found to be 6.5±0.1 and ∼74 kDa, respectively. Circular dichroism data of the rAOx confirmed its ordered structure. Docking studies with an <i>ab-initio</i> protein model demonstrated the presence of a conserved FAD binding domain with an active substrate binding site. The rAOx was specific for aryl alcohols and the order of its substrate preference was 4-methoxybenzyl alcohol >3-methoxybenzyl alcohol>3, 4-dimethoxybenzyl alcohol > benzyl alcohol. A significantly high aggregation to ∼1000 nm (diameter) and catalytic efficiency (<i>k_cat/K_m</i>) of 7829.5 min⁻¹ mM⁻¹ for 4-methoxybenzyl alcohol was also demonstrated for rAOx. The results infer the novelty of the AOx and its potential biocatalytic application.</p></div

Docking view of modeled rAOx (FAD docked) with its alcohol substrates.

<p>Docking view of aromatic alcohols (highlighted as thick stick CPK model) as substrates with FAD docked (highlighted as thick stick CPK model) apo-rAOx holoenzyme complex. Conserved amino acid residues hypothesized to take part in catalytic reaction in oxidizing its substrates are highlighted as thin stick Corey-Pauling-Koltun (CPK) model with its residues labelled. Panel (<b>A</b>), (<b>B</b>), (<b>C</b>) and (<b>D</b>) shows the close-up docking view generated by Molegro Virtual Docker version 4.0.2 (CLC bio-Qiagen company) of <i>ρ</i>-methoxybenzyl alcohol; <i>m</i>-methoxybenzyl alcohol; 3,4 dimethoxybenzyl alcohol and benzyl alcohol, respectively.</p

Steady-state kinetic parameters of <i>in-vitro</i> refolded recombinant alcohol oxidase from <i>E.coli</i>.

<p>Mean <i>K</i>_m, <i>k</i>_cat and <i>k</i>_cat/<i>K</i>_m values were determined and all assays were performed in replicates of 3 (n = 3).</p

Nucleotide and deduced amino acid sequence of AOx from <i>A.terreus</i> MTCC6324.

<p>Double stranded primer walking confirmed an ORF of 2001(−) denotes a stop codon. The N-terminal conserved amino acids taking part in Rossmann fold architecture (GXGXXG motif) are underlined in black with its residues in bold. The full length cDNA is submitted to NCBI GenBank with accession no: JX139751.</p