HtrA, fatty acids, and membrane protein interplay in Chlamydia trachomatis to impact stress response and trigger early cellular exit.

Chlamydia trachomatis is an intracellular bacterial pathogen that undergoes a biphasic developmental cycle, consisting of intracellular reticulate bodies and extracellular infectious elementary bodies. A conserved bacterial protease, HtrA, was shown previously to be essential for Chlamydia during the reticulate body phase, using a novel inhibitor (JO146). In this study, isolates selected for the survival of JO146 treatment were found to have polymorphisms in the acyl-acyl carrier protein synthetase gene (aasC). AasC encodes the enzyme responsible for activating fatty acids from the host cell or synthesis to be incorporated into lipid bilayers. The isolates had distinct lipidomes with varied fatty acid compositions. A reduction in the lipid compositions that HtrA prefers to bind to was detected, yet HtrA and MOMP (a key outer membrane protein) were present at higher levels in the variants. Reduced progeny production and an earlier cellular exit were observed. Transcriptome analysis identified that multiple genes were downregulated in the variants especially stress and DNA processing factors. Here, we have shown that the fatty acid composition of chlamydial lipids, HtrA, and membrane proteins interplay and, when disrupted, impact chlamydial stress response that could trigger early cellular exit. IMPORTANCE: Chlamydia trachomatis is an important obligate intracellular pathogen that has a unique biphasic developmental cycle. HtrA is an essential stress or virulence protease in many bacteria, with many different functions. Previously, we demonstrated that HtrA is critical for Chlamydia using a novel inhibitor. In the present study, we characterized genetic variants of Chlamydia trachomatis with reduced susceptibility to the HtrA inhibitor. The variants were changed in membrane fatty acid composition, outer membrane proteins, and transcription of stress genes. Earlier and more synchronous cellular exit was observed. Combined, this links stress response to fatty acids, membrane proteins, and HtrA interplay with the outcome of disrupted timing of chlamydial cellular exit.

Dimeric peptoids as antibacterial agents.

Building upon our previous study on peptoid-based antibacterials which showed good activity against Gram-positive bacteria only, herein we report the synthesis of 34 dimeric peptoid compounds and the investigation of their activity against Gram-positive and Gram-negative pathogens. The newly designed peptoids feature a di-hydrophobic moiety incorporating phenyl, bromo-phenyl, and naphthyl groups, combined with variable lengths of cationic units such as amino and guanidine groups. The study also underscores the pivotal interplay between hydrophobicity and cationicity in optimizing efficacy against specific bacteria. The bromophenyl dimeric guanidinium peptoid compound 10j showed excellent activity against S. aureus 38 and E. coli K12 with MIC of 0.8 µg mL-1 and 6.2 µg mL-1, respectively. Further investigation into the mechanism of action revealed that the antibacterial effect might be attributed to the disruption of bacterial cell membranes, as suggested by tethered bilayer lipid membranes (tBLMs) and cytoplasmic membrane permeability studies. Notably, these promising antibacterial agents exhibited negligible toxicity against mammalian red blood cells. Additionally, the study explored the potential of 12 active compounds to disrupt established biofilms of S. aureus 38. The most effective biofilm disruptors were ethyl and octyl-naphthyl guanidinium peptoids (10c and 10 k). These compounds 10c and 10 k disrupted the established biofilms of S. aureus 38 with 51 % at 4x MIC (MIC = 17.6 µg mL-1 and 11.2 µg mL-1) and 56 %-58 % at 8x MIC (MIC = 35.2 µg mL-1 and 22.4 µg mL-1) respectively. Overall, this research contributes insights into the design principles of cationic dimeric peptoids and their antibacterial activity, with implications for the development of new antibacterial compounds.

E-Cigarette Aerosol Condensate Leads to Impaired Coronary Endothelial Cell Health and Restricted Angiogenesis.

Cardiovascular disease (CVD) is a leading cause of mortality worldwide, with cigarette smoking being a major preventable risk factor. Smoking cessation can be difficult due to the addictive nature of nicotine and the withdrawal symptoms following cessation. Electronic cigarettes (e-Cigs) have emerged as an alternative smoking cessation device, which has been increasingly used by non-smokers; however, the cardiovascular effects surrounding the use of e-Cigs remains unclear. This study aimed to investigate the effects of e-Cig aerosol condensate (EAC) (0 mg and 18 mg nicotine) in vitro on human coronary artery endothelial cells (HCAEC) and in vivo on the cardiovascular system using a mouse model of 'e-vaping'. In vitro results show a decrease in cell viability of HCAEC when exposed to EAC either directly or after exposure to conditioned lung cell media (p < 0.05 vs. control). Reactive oxygen species were increased in HCAEC when exposed to EAC directly or after exposure to conditioned lung cell media (p < 0.0001 vs. control). ICAM-1 protein expression levels were increased after exposure to conditioned lung cell media (18 mg vs. control, p < 0.01). Ex vivo results show an increase in the mRNA levels of anti-angiogenic marker, FKBPL (p < 0.05 vs. sham), and endothelial cell adhesion molecule involved in barrier function, ICAM-1 (p < 0.05 vs. sham) in murine hearts following exposure to electronic cigarette aerosol treatment containing a higher amount of nicotine. Immunohistochemistry also revealed an upregulation of FKBPL and ICAM-1 protein expression levels. This study showed that despite e-Cigs being widely used for tobacco smoking cessation, these can negatively impact endothelial cell health with a potential to lead to the development of cardiovascular disease.

Bioinformatic Analysis of Na+, K+-ATPase Regulation through Phosphorylation of the Alpha-Subunit N-Terminus.

The Na+, K+-ATPase is an integral membrane protein which uses the energy of ATP hydrolysis to pump Na+ and K+ ions across the plasma membrane of all animal cells. It plays crucial roles in numerous physiological processes, such as cell volume regulation, nutrient reabsorption in the kidneys, nerve impulse transmission, and muscle contraction. Recent data suggest that it is regulated via an electrostatic switch mechanism involving the interaction of its lysine-rich N-terminus with the cytoplasmic surface of its surrounding lipid membrane, which can be modulated through the regulatory phosphorylation of the conserved serine and tyrosine residues on the protein's N-terminal tail. Prior data indicate that the kinases responsible for phosphorylation belong to the protein kinase C (PKC) and Src kinase families. To provide indications of which particular enzyme of these families might be responsible, we analysed them for evidence of coevolution via the mirror tree method, utilising coevolution as a marker for a functional interaction. The results obtained showed that the most likely kinase isoforms to interact with the Na+, K+-ATPase were the θ and η isoforms of PKC and the Src kinase itself. These theoretical results will guide the direction of future experimental studies.

Cholic Acid-Based Antimicrobial Peptide Mimics as Antibacterial Agents.

There is a significant and urgent need for the development of novel antibacterial agents to tackle the increasing incidence of antibiotic resistance. Cholic acid-based small molecular antimicrobial peptide mimics are reported as potential new leads to treat bacterial infection. Here, we describe the design, synthesis and biological evaluation of cholic acid-based small molecular antimicrobial peptide mimics. The synthesis of cholic acid analogues involves the attachment of a hydrophobic moiety at the carboxyl terminal of the cholic acid scaffold, followed by the installation of one to three amino acid residues on the hydroxyl groups present on the cholic acid scaffold. Structure-activity relationship studies suggest that the tryptophan moiety is important for high antibacterial activity. Moreover, a minimum of +2 charge is also important for antimicrobial activity. In particular, analogues containing lysine-like residues showed the highest antibacterial potency against Gram-positive S. aureus. All di-substituted analogues possess high antimicrobial activity against both Gram-positive S. aureus as well as Gram-negative E. coli and P. aeruginosa. Analogues 17c and 17d with a combination of these features were found to be the most potent in this study. These compounds were able to depolarise the bacterial membrane, suggesting that they are potential antimicrobial pore forming agents.

Calcium Ion Binding at the Lipid-Water Interface Alters the Ion Permeability of Phospholipid Bilayers.

Calcium ions (Ca2+) play a fundamental role in membrane-associated physiological processes. Ca2+ can also significantly modulate the physicochemical properties of phospholipid bilayers, but whether this occurs at physiologically relevant concentrations is difficult to determine because of the uncertainty in the reported affinity of Ca2+ for phospholipid bilayers. In this article, we determine the apparent affinity of Ca2+ for zwitterionic phospholipid bilayers using tethered bilayer lipid membranes (tBLMs) used in conjunction with swept-frequency electrical impedance spectroscopy (EIS). We report that Ca2+ binds to phospholipid bilayers at physiologically relevant concentrations and modulates membrane permeability. We present direct experimental evidence that this effect is governed by specific interactions with select lipid headgroup moieties, which is supported by data from molecular dynamics (MD) simulations. This is the first reported use of tBLM/EIS to estimate cation-membrane affinity. Combined with MD simulations, this technique provides a novel methodology to elucidate the molecular details of cation-membrane interactions at the water-phospholipid interface.

Effect of polar amino acid incorporation on Fmoc-diphenylalanine-based tetrapeptides.

Peptide hydrogels show great promise as extracellular matrix mimics due to their tuneable, fibrous nature. Through incorporation of polar cationic, polar anionic or polar neutral amino acids into the Fmoc-diphenylalanine motif, we show that electrostatic charge plays a key role in the properties of the subsequent gelators. Specifically, we show that an inverse relationship exists for biocompatibility in the solution state versus the gel state for cationic and anionic peptides. Finally, we use tethered bilayer lipid membrane (tBLM) experiments to suggest a likely mode of cytotoxicity for tetrapeptides which exhibit cytotoxicity in the solution state.

The Role of Structure and Biophysical Properties in the Pleiotropic Effects of Statins.

Statins are a class of drugs used to lower low-density lipoprotein cholesterol and are amongst the most prescribed medications worldwide. Most statins work as a competitive inhibitor of 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGR), but statin intolerance from pleiotropic effects have been proposed to arise from non-specific binding due to poor enzyme-ligand sensitivity. Yet, research into the physicochemical properties of statins, and their interactions with off-target sites, has not progressed much over the past few decades. Here, we present a concise perspective on the role of statins in lowering serum cholesterol levels, and how their reported interactions with phospholipid membranes offer a crucial insight into the mechanism of some of the more commonly observed pleiotropic effects of statin administration. Lipophilicity, which governs hepatoselectivity, is directly related to the molecular structure of statins, which dictates interaction with and transport through membranes. The structure of statins is therefore a clinically important consideration in the treatment of hypercholesterolaemia. This review integrates the recent biophysical studies of statins with the literature on the physiological effects and provides new insights into the mechanistic cause of statin pleiotropy, and prospective means of understanding the cholesterol-independent effects of statins.

Maternal E-Cigarette Exposure Results in Cognitive and Epigenetic Alterations in Offspring in a Mouse Model.

Electronic cigarette (e-cigarette) use is on the rise worldwide and is particularly attractive to young people and as a smoking substitute by pregnant woman. There is a perception in pregnant women and women of child-bearing age that the use of e-cigarettes (vaping) is safer than smoking tobacco cigarettes during pregnancy. However, there is little evidence to support this perception. Here, we examined the offspring from mouse dams that had been exposed during and after pregnancy to ambient air (sham) ( n = 8), e-cigarette aerosols with nicotine ( n = 8), or e-cigarette aerosols without nicotine ( n = 8). Offspring underwent cognitive testing at 12 weeks of age and epigenetic testing of brain tissues at 1 day, 20 days, and 13 weeks after birth. The findings showed deficits in short-term memory, reduced anxiety, and hyperactivity in offspring following maternal e-cigarette exposure using the novel object recognition and elevated plus maze tests. In addition, global DNA methylation was increased in the brains of offspring soon after birth. Using a quantitative-PCR array specific to chromatin modification enzymes on genomic DNA and histones,13 key genes were identified to be significantly altered in the offspring brains from the e-cigarette groups compared to the nonexposed groups. The changes to genes Aurka, Aurkb, Aurkc, Kdm5c, Kdm6b, Dnmt3a, Dnmt3b, and Atf2, all associated with modulating neurological activity, were validated using RT-qPCR. In conclusion, in a mouse model, maternal exposure to e-cigarette aerosols resulted in both cognitive and epigenetic changes in offspring. This suggests that the use of e-cigarettes during pregnancy may have hitherto undetected neurological consequences on newborns.

Lipid Membrane Interactions of the Cationic Antimicrobial Peptide Chimeras Melimine and Cys-Melimine.

Melimine and its derivatives are synthetic chimeric antimicrobial agents based on protamine and melittin. The binding of solubilized melimine and its derivative, with a cysteine on N-terminus, (cys-melimine) on tethered bilayer lipid membranes (tBLMs) was examined using ac electrical impedance spectroscopy. The addition of melimine and cys-melimine initially increased membrane conduction, which subsequently falls over time. The results were obtained for tBLMs comprising zwitterionic phosphatidylcholine, anionic phosphatidylglycerol, or tBLMs made using purified lipids from Escherichia coli. The effect on conduction is more marked with the cysteine variant than the noncysteine variant. The variation in membrane conduction most probably arises from individual melimines inducing increased ionic permeability, which is then reduced as the melimines aggregate and phase-separate within the membrane. The actions of these antimicrobials are modeled in terms of altering the critical packing parameter (CPP) of the membranes. The variations in the peptide length of cys-melimine were compared with a truncated version of the peptide, cys-mel4. The results suggest that the smaller molecule impacts the membrane by a mechanism that increases the average CPP, reducing membrane conduction. Alternatively, an uncharged alanine-replacement version of melimine still produced an increase in membrane conduction, further supporting the CPP model of geometry-induced toroidal pore alterations. All the data were then compared to their antimicrobial effectiveness for the Gram-positive and Gram-negative strains of bacteria, and their fusogenic properties were examined using dynamic light scattering in 1-oleoyl-2-hydroxy- sn-glycero-3-phosphocholine lipid spheroids. We conclude that a degree of correlation exists between the antimicrobial effectiveness of the peptides studied here and their modulation of membrane conductivity.

Kalata B1 and Kalata B2 Have a Surfactant-Like Activity in Phosphatidylethanolomine-Containing Lipid Membranes.

Cyclotides are cyclic disulfide-rich peptides that are chemically and thermally stable and possess pharmaceutical and insecticidal properties. The activities reported for cyclotides correlate with their ability to target phosphatidylethanolamine (PE)-phospholipids and disrupt cell membranes. However, the mechanism by which this disruption occurs remains unclear. In the current study we examine the effect of the prototypic cyclotides, kalata B1 (kB1) and kalata B2 (kB2), on tethered lipid bilayer membranes (tBLMs) using swept frequency electrical impedance spectroscopy. We confirmed that kB1 and kB2 bind to bilayers only if they contain PE-phospholipids. We hypothesize that the increase in membrane conduction and capacitance observed upon addition of kB1 or kB2 is unlikely to result from ion channel like pores but is consistent with the formation of lipidic toroidal pores. This hypothesis is supported by the concentration dependence of effects of kB1 and kB2 being suggestive of a critical micelle concentration event rather than a progressive increase in conduction arising from increased channel insertion. Additionally, conduction behavior is readily reversible when the peptide is rinsed from the bilayer. Our results support a mechanism by which kB1 and kB2 bind to and disrupt PE-containing membranes by decreasing the overall membrane critical packing parameter, as would a surfactant, which then opens or increases the size of existing membrane defects. The cyclotides need not participate directly in the conductive pore but might exert their effect indirectly through altering membrane packing constraints and inducing purely lipidic conductive pores.

Amphipathic guanidine-embedded glyoxamide-based peptidomimetics as novel antibacterial agents and biofilm disruptors.

Antimicrobial resistance in bacteria is becoming increasingly prevalent, posing a critical challenge to global health. Bacterial biofilm formation is a common resistance mechanism that reduces the effectiveness of antibiotics. Thus, the development of compounds that can disrupt bacterial biofilms is a potential strategy to combat antimicrobial resistance. We report herein the synthesis of amphipathic guanidine-embedded glyoxamide-based peptidomimetics via ring-opening reactions of N-naphthoylisatins with amines and amino acids. These compounds were investigated for their antibacterial activity by the determination of minimum inhibitory concentration (MIC) against S. aureus and E. coli. Compounds 35, 36, and 66 exhibited MIC values of 6, 8 and 10 µg mL-1 against S. aureus, respectively, while compounds 55 and 56 showed MIC values of 17 and 19 µg mL-1 against E. coli, respectively. Biofilm disruption and inhibition activities were also evaluated against various Gram-positive and Gram-negative bacteria. The most active compound 65 exhibited the greatest disruption of established biofilms by 65% in S. aureus, 61% in P. aeruginosa, and 60% in S. marcescens respectively, at 250 µM concentration, while compound 52 inhibited the formation of biofilms by 72% in S. marcescens at 250 µM. We also report here the in vitro toxicity against MRC-5 human lung fibroblast cells. Finally, the pore forming capability of the three most potent compounds were tested using tethered bilayer lipid membrane (tBLM) technology.

The effect of hydronium ions on the structure of phospholipid membranes.

This work seeks to identify the mechanisms by which hydronium ions (H3O+) modulate the structure of phospholipid bilayers by studying the interactions of H3O+ with phospholipids at the molecular level. For this, we carried out multiple microsecond-long unrestrained molecular dynamics (MD) simulations of a POPC bilayer at different H3O+ concentrations. The results show that H3O+ accumulates at the membrane surface where it displaces water and forms strong and long-lived hydrogen bonds with the phosphate and carbonyl oxygens in phospholipids. This results in a concentration-dependent reduction of the area per lipid and an increase in bilayer thickness. This study provides an important molecular-level insight into the mechanism of how H3O+ modulates the structure of biological membranes and is a critical step towards a better understanding of the effect of low pH on mammalian and bacterial membranes.

Evidence of the Key Role of H3O+ in Phospholipid Membrane Morphology.

This study explains the importance of the phosphate moiety and H3O+ in controlling the ionic flux through phospholipid membranes. We show that despite an increase in the H3O+ concentration when the pH is decreased, the level of ionic conduction through phospholipid bilayers is reduced. By modifying the lipid structure, we show the dominant determinant of membrane conduction is the hydrogen bonding between the phosphate oxygens on adjacent phospholipids. The modulation of conduction with pH is proposed to arise from the varying H3O+ concentrations altering the molecular area per lipid and modifying the geometry of conductive defects already present in the membrane. Given the geometrical constraints that control the lipid phase structure of membranes, these area changes predict that organisms evolving in environments with different pHs will select for different phospholipid chain lengths, as is found for organisms near highly acidic volcanic vents (short chains) or in highly alkaline salt lakes (long chains). The stabilizing effect of the hydration shells around phosphate groups also accounts for the prevalence of phospholipids across biology. Measurement of ion permeation through lipid bilayers was made tractable using sparsely tethered bilayer lipid membranes with swept frequency electrical impedance spectroscopy and ramped dc amperometry. Additional evidence of the effect of a change in pH on lipid packing density is obtained from neutron reflectometry data of tethered membranes containing perdeuterated lipids.

Synthesis and biological evaluation of N-naphthoyl-phenylglyoxamide-based small molecular antimicrobial peptide mimics as novel antimicrobial agents and biofilm inhibitors.

Antimicrobial peptides (AMPs) are a key component of the human immune system. Synthetic AMP mimics represent a novel strategy to counteract the increasing incidence of antimicrobial resistance. Here, we describe the synthesis of novel glyoxamide derivatives via ring-opening reactions of N-hexanoyl, N-benzoyl and N-naphthoylisatins with N,N-dimethylethane-1,2-diamine and N,N-dimethylpropane-1,3-diamine. These were converted to both the hydrochloric acid (HCl) or quaternary ammonium iodide (MeI) salts and their antibacterial activity against Staphylococcus aureus was investigated by their zone-of-inhibition and minimum inhibitory concentration (MIC). The HCl salt 22b exhibited the lowest MIC of 16 µg mL(-1), whereas the corresponding MeI salt 22c had a MIC of 39 µg mL(-1). We also investigated the in vitro toxicity of active compounds against the MRC-5 normal human lung fibroblasts and their activity against established biofilm in S. aureus.

Nanoscale ion sequestration to determine the polarity selectivity of ion conductance in carriers and channels.

The nanoscale spacing between a tethered lipid bilayer membrane (tBLM) and its supporting gold electrode can be utilized to determine the polarity selectivity of the conduction of ion channels and ion carriers embedded in a membrane. The technique relies upon a bias voltage sequestering or eliminating ions, of a particular polarity, into or out of the aqueous electrolyte region between the gold electrode and the tethered membrane. A demonstration is given, using ac swept frequency impedance spectrometry, of the bias polarity dependence of the ionophore conductance of gramicidin A, a cationic selective channel, and valinomycin, a potassium ion selective carrier. We further use pulsed amperometry to show that the intrinsic voltage dependence of the ion conduction is actually selective of the polarity of the transported ion and not simply of the direction of the ionic current flow.

Differential effects of lipids and lyso-lipids on the mechanosensitivity of the mechanosensitive channels MscL and MscS.

Mechanosensitive (MS) channels of small (MscS) and large (MscL) conductance are the major players in the protection of bacterial cells against hypoosmotic shock. Although a great deal is known about structure and function of these channels, much less is known about how membrane lipids may influence their mechanosensitivity and function. In this study, we use liposome coreconstitution to examine the effects of different types of lipids on MscS and MscL mechanosensitivity simultaneously using the patch-clamp technique and confocal microscopy. Fluorescence lifetime imaging (FLIM)-FRET microscopy demonstrated that coreconstitution of MscS and MscL led to clustering of these channels causing a significant increase in the MscS activation threshold. Furthermore, the MscL/MscS threshold ratio dramatically decreased in thinner compared with thicker bilayers and upon addition of cholesterol, known to affect the bilayer thickness, stiffness and pressure profile. In contrast, application of micromolar concentrations of lysophosphatidylcholine (LPC) led to an increase of the MscL/MscS threshold ratio. These data suggest that differences in hydrophobic mismatch and bilayer stiffness, change in transbilayer pressure profile, and close proximity of MscL and MscS affect the structural dynamics of both channels to a different extent. Our findings may have far-reaching implications for other types of ion channels and membrane proteins that, like MscL and MscS, may coexist in multiple molecular complexes and, consequently, have their activation characteristics significantly affected by changes in the lipid environment and their proximity to each other.

An engineered membrane to measure electroporation: effect of tethers and bioelectronic interface.

This article reports on the construction and predictive models for a platform comprised of an engineered tethered membrane. The platform provides a controllable and physiologically relevant environment for the study of the electroporation process. The mixed self-assembled membrane is formed via a rapid solvent exchange technique. The membrane is tethered to the gold electrode and includes an ionic reservoir separating the membrane and gold surface. Above the membrane, there is an electrolyte solution, and a gold counterelectrode. A voltage is applied between the gold electrodes and the current measured. The current is dependent on the energy required to form aqueous pores and the conductance of each pore. A two-level predictive model, consisting of a macroscopic and a continuum model, is developed to relate the pore dynamics to the measured current. The macroscopic model consists of an equivalent circuit model of the tethered membrane, and asymptotic approximations to the Smoluchowski-Einstein equation of electroporation that is dependent on the pore conductance and the energy required to form aqueous pores. The continuum model is a generalized Poisson-Nernst-Planck (GPNP) system where an activity coefficient to account for steric effects of ions is added to the standard PNP system. The GPNP is used to evaluate the conductance of aqueous pores, and the electrical energy required to form the pores. As an outcome of the setup of the device and the two-level model, biologically important variables can be estimated from experimental measurements. To validate the accuracy of the two-level model, the predicted current is compared with experimentally measured current for different tethering densities.

Transient potential gradients and impedance measures of tethered bilayer lipid membranes: pore-forming peptide insertion and the effect of electroporation.

In this work, we present experimental data, supported by a quantitative model, on the generation and effect of potential gradients across a tethered bilayer lipid membrane (tBLM) with, to the best of our knowledge, novel architecture. A challenge to generating potential gradients across tBLMs arises from the tethering coordination chemistry requiring an inert metal such as gold, resulting in any externally applied voltage source being capacitively coupled to the tBLM. This in turn causes any potential across the tBLM assembly to decay to zero in milliseconds to seconds, depending on the level of membrane conductance. Transient voltages applied to tBLMs by pulsed or ramped direct-current amperometry can, however, provide current-voltage (I/V) data that may be used to measure the voltage dependency of the membrane conductance. We show that potential gradients >~150 mV induce membrane defects that permit the insertion of pore-forming peptides. Further, we report here the novel (to our knowledge) use of real-time modeling of conventional low-voltage alternating-current impedance spectroscopy to identify whether the conduction arising from the insertion of a polypeptide is uniform or heterogeneous on scales of nanometers to micrometers across the membrane. The utility of this tBLM architecture and these techniques is demonstrated by characterizing the resulting conduction properties of the antimicrobial peptide PGLa.

Triglyceride-Tethered Membrane Lipase Sensor.

Sensors that can quickly measure the lipase activity from biological samples are useful in enzyme production and medical diagnostics. However, current lipase sensors have limitations such as requiring fluorescent labels, pH control of buffer vehicles, or lengthy assay preparation. We introduce a sparsely tethered triglyceride substrate anchored off of a gold electrode for the impedance sensing of real-time lipase activity. The tethered substrate is self-assembled using a rapid solvent exchange technique and can form an anchored bilayer 1 nm off the gold electrode. This allows for an aqueous reservoir region, providing access to ions transported through membrane defects caused by triglyceride enzymatic hydrolysis. Electrical impedance spectroscopy techniques can readily detect the decrease in resistance caused by enzymatically induced defects. This rapid and reliable lipase detection method can have potential applications in disease studies, monitoring of lipase production, and as point-of-care diagnostic devices.