ABSTRACT
Significant progress has been achieved in the field of solid-state biosensors over the past 50 years. Various sensing devices with high-density integration and flexible configuration, as well as new applications for clinical diagnosis and healthcare, have been developed using blood, serum, and other body fluids such as sweat, tears, and saliva. A high-density array of ion-sensitive field effect transistors was developed by exploiting the advantages of advanced semiconductor technologies and commercialized in combination with an enzymatic primer extension reaction as a DNA sequencer in 2011. Different types of materials such as inorganic materials, metals, polymers, and biomolecules are mixed together on the surface of the gate while maintaining their own functions; therefore, compatibility among different materials has to be optimized so that the best detection performance of solid-state biosensors, including stability and reliability, is achieved as designed. Solid-state biosensors are suitable for the rapid, cost-effective, and noninvasive identification of biomarkers at various timepoints over the course of a disease.
Subject(s)
Body Fluids , Reproducibility of Results , SemiconductorsABSTRACT
A novel strategy for epidermal growth factor receptor (EGFR) detection using a cell-based field-effect transistor (FET) with enzymatic chemical signal amplification is proposed. Four human breast cancer cell lines [BT474, MDA-MB-231 (MM231), MDA-MB-468 (MM468), and MDA-MB-453 (MM453)] were used to compare the expression levels of EGFR. The cells were non-specifically captured on the surface of the gate of the FET, irrespective of their surface antigens. With this configuration, the heterogeneity of the cells would be analyzed using secondary antibodies conjugated to different kinds of enzymes. Four breast cancer cell lines with different levels of EGFR expression were captured on the respective surfaces of the extracellular matrix (ECM) gel-coated gates of the FETs. Glucose oxidase (GOx) was conjugated to the secondary antibody, and the output signals of the cell-based FETs changed depending on the expression levels of EGFR upon addition of glucose. The order of the expression levels of EGFR among the four cell lines, determined with the cell-based FETs, was consistent with the results of fluorescence detection determined by fluorescence-activated cell sorting (FACS). The cell-based FETs are advantageous for miniaturization and in massive parallel analyses of target molecules expressed on the membranes of cells and EVs, and their small size and cost effectiveness for cancer testing could enable their realization in a future liquid biopsy.
Subject(s)
Breast Neoplasms , Cell Line, Tumor , ErbB Receptors/analysis , Female , Humans , MCF-7 CellsABSTRACT
Researchers widely apply enzyme inhibition to chemicals such as pesticides, nerve gases, and anti-Alzheimer's drugs. However, application of enzyme inhibition to odorant sensors is less common because the corresponding reaction mechanisms have not yet been clarified in detail. In this study, we propose a new strategy for highly selective detection of odorant molecules by using an inhibitor-specific enzyme. As an example, we analyzed the selective interactions between acetylcholinesterase (AChE) and limoneneâthe major odorant of citrus and an AChE inhibitorâusing molecular dynamics simulations. In these simulations, limonene was found to be captured at specific binding sites of AChE by modifying the binding site of acetylcholine (ACh), which induced inhibition of the catalytic activity of AChE toward ACh hydrolysis. We confirmed the simulation results by experiments using an ion-sensitive field-effect transistor, and the degree of inhibition of ACh hydrolysis depended on the limonene concentration. Accordingly, we quantitatively detected limonene at a detection limit of 5.7 µM. We furthermore distinguished the response signals to limonene from those to other odorants, such as pinene and perillic acid. Researchers will use our proposed odorant detection method for other odorant-enzyme combinations and applications of miniaturized odorant-sensing systems based on rapid testing.
Subject(s)
Acetylcholinesterase , Pesticides , Acetylcholine/chemistry , Acetylcholinesterase/metabolism , Cholinesterase Inhibitors/chemistry , Cholinesterase Inhibitors/pharmacology , Hydrolysis , LimoneneABSTRACT
A poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate) (PEDOT:PSS)-based conducting polymer, which has biorecognition capabilities, has promising biosensing applications. Previously, we developed a facile method for post-printing chemical modification of PEDOT:PSS thin films from commercial sources. Molecular recognition elements were directly introduced into the PSS side chain by a two-step chemical reaction: introduction of an ethylenediamine linker via an acid chloride reaction of the sulfonate moiety, and subsequent receptor attachment to the linker via amine coupling. In this study, the same method was used to introduce 6-carboxypyridine-3-boronic acid (carboxy-PyBA) into the linker for specifically detecting N-acetylneuraminic acid (sialic acid, SA), as a cancer biomarker. The surface-modified PEDOT:PSS films were characterized by X-ray photoelectron spectroscopy, attenuated total reflection Fourier-transform infrared spectroscopy, and static water contact angle and conductivity measurements. The specific interaction between PyBA and SA was detected by label-free reagent-free potentiometry. The SA-specific negative potential responses of modified PEDOT:PSS electrodes, which was ascribed to an SA carboxyl anion, were observed in a physiologically relevant SA range (1.6-2.9 mM) at pH 5, in a concentration-dependent manner even in the presence of 10% fetal bovine serum. The sensitivity was -2.9 mV/mM in 1-5 mM SA with a limit of detection of 0.7 mM. The sensing performances were almost equivalent to those of existing graphene-based electrical SA sensors. These results show that our chemical derivatization method for printing PEDOT:PSS thin films will have applications in SA clinical diagnostics.
ABSTRACT
Despite tremendous complexity in glycan structure, sialic acid (SA) provides an analytically accessible index for glycosylation, owing to its uniquely anionic nature and glycan-chain terminal occupation. Taking advantage of boronic acid (BA) based SA-recognition chemistry, we here demonstrate a label-free, no enzymatic, potentiometric determination of fetuin, a blood-circulating glycoprotein implicated in physiological and various pathological states. A phenylboronic acid (PBA) ω-end-functionalized poly(ethylene glycol) (PEG) with an α-tethering unit bearing pendent alkyne groups was "grafted-to" a gold electrode modified with 11-azide-undecathiol by a copper-catalyzed azide-alkyne cycloaddition reaction. Using the electrode, fetuin was potentiometrically detectable with a µM-order-sensitivity that is comparable to what is found in blood-collected specimen. Our finding may have implications for developing a remarkably economic hemodiagnostic technology with ease of downsizing and mass production.
Subject(s)
Boronic Acids/chemistry , Electrodes , Fetuins/metabolism , Glycoproteins/blood , Polyethylene Glycols/chemistry , Potentiometry/instrumentation , Limit of DetectionABSTRACT
Graphene's remarkable attributes make it suitable for application to biosensors for biomolecular recognition. Specific and precise target detection is realized by designing robust methods for immobilization of probe molecules, such as oligonucleotides, antibodies, receptors, and sugar chains, to a device surface. In this research, we developed a chemical modification method with a plasma treatment of amino groups on natural defects of graphene, which is compatible with a wafer-scalable semiconductor process, to prevent deterioration of the carrier mobility. The plasma treatment was optimized in terms of the efficiency of the amino radical generation, length of the mean free path, and reaction energy on graphene. The density of the modified amino groups on graphene was approximately 0.065 groups/nm2, and the change in the ΔId/ΔVg characteristic of the graphene field-effect transistor (FET) was negligible. DNA probes were then attached to the amino groups on the graphene FET. The target complementary DNA was detected at 1 nM after hybridization using the graphene FET devices. The plasma-assisted modification of the amino groups on the graphene surface was developed for immobilization of the DNA probes, and hybridization with the target DNA was demonstrated without deterioration of the carrier mobility.
ABSTRACT
RNA interference (RNAi) by small interfering RNAs (siRNAs) is a promising therapeutic approach. Because siRNA has limited intracellular access and is rapidly cleared in vivo, the success of RNAi depends on efficient delivery technologies. Particularly, polyion complexation between block catiomers and siRNA is a versatile approach for constructing effective carriers, such as unit polyion complexes (uPIC), core-shell polyion complex (PIC) micelles and vesicular siRNAsomes, by engineering the structure of block catiomers. In this regard, the flexibility of block catiomers could be an important parameter in the formation of PIC nanostructures with siRNA, though its effect remains unknown. Here, we studied the influence of block catiomer flexibility on the assembly of PIC structures with siRNA using a complementary polymeric system, i.e. poly(ethylene glycol)-poly(L-lysine) (PEG-PLL) and PEG-poly(glycidylbutylamine) (PEG-PGBA), which has a relatively more flexible polycation segment than PEG-PLL. Mixing PEG-PGBA with siRNA at molar ratios of primary amines in polymer to phosphates in the siRNA (N/P ratios) higher than 1.5 promoted the multimolecular association of uPICs, whereas PEG-PLL formed uPIC at all N/P ratios higher than 1. Moreover, uPICs from PEG-PGBA were more stable against counter polyanion exchange than uPICs from PEG-PLL, probably due to a favorable complexation process, as suggested by computational studies of siRNA/block catiomer binding. In in vitro experiments, PEG-PGBA uPICs promoted effective intracellular delivery of siRNA and efficient gene knockdown. Our results indicate the significance of polycation flexibility on assembling PIC structures with siRNA, and its potential for developing innovative delivery systems.
ABSTRACT
Tracing cell surface sialylation dynamics at a scale of the glycolipoprotein microdomain (lipid rafts) formations remains an intriguing challenge of cellular biology. Here, we demonstrate that this goal is accessible, taking advantage of a boronic acid (BA)-based reversible molecular recognition chemistry. A BA-end-functionalized poly(ethylene glycol) was decorated onto an atomic force microscopy (AFM) cantilever, which provided a dynamic and sialic acid (SA)-specific imaging mode. Using this technique, we were able to heat map the SA expression levels not only on protein-decorated substrates but also directly on the cell surfaces, with a submicrometer scale resolution that may be relevant to that of the lipid rafts formation. The SA specificity and the binding reversibility of the probe were confirmed from its pH-dependent characteristics and an inhibition assay using free state SA. This finding may provide a noninvasive means for assessing a variety of SA-involved glycosylation dynamics spanning from physiology to pathology.
Subject(s)
Boronic Acids/chemistry , Microscopy, Atomic Force/methods , N-Acetylneuraminic Acid/chemistry , Cell Membrane , HumansABSTRACT
Understanding the interactions of soft nanomatters with cell membranes is particularly important for research into nanocarrier-based drug delivery systems, cell engineering, and subcellular imaging. Most nanoparticles, vesicles, micelles, and polymeric aggregates are internalized into endosomes and, eventually, lysosomes in the cytosol because of energy-dependent endocytic processes. Endocytic uptake substantially limits the access to the cytoplasm where a cargo agent acts. Bypassing the endocytic pathways by direct penetration into plasma membrane barriers would enhance the efficacy of nanomedicines. Some zwitterionic polymer nanoaggregates have been shown to permeate into the cell interior in an energy-independent manner. We have elucidated this phenomenon by observing changes in the biomembrane barrier functions against protons as the smallest indicator and have used these results to further design and develop poly(betaines). In this work, we investigated the translocation mechanisms for a series of zwitterionic poly(methacrylamide) and poly(methacrylate) species bearing a pyridinium propane sulfonate moiety in the monomers. Minor differences in the monomer structures and functional groups were observed to have dramatic effects on the interaction with plasma membranes during translocation. The ability to cross the plasma membrane involves a balance among the betaine dipole-dipole interaction, NH-π interaction, π-π interaction, cation-π interaction, and amide hydrogen bonding. We found that the cell-penetrating polysulfobetaines had limited or no detrimental effect on cell proliferation. Our findings enhance the opportunity to design and synthesize soft nanomatters for cell manipulations by passing across biomembrane partitions.
Subject(s)
Betaine , Polymers , Betaine/analogs & derivatives , Cell Membrane , MicellesABSTRACT
Tight junctions (TJs) in the epithelial cell gap play primary roles in body defense and nutrient absorption in multicellular organisms. Standard in vitro assays lack sensitivity, selectivity, temporal resolution, and throughput for bioengineering applications. Prompted by the rigorous barrier functions of TJ, we developed a TJ assay that senses proton leaks in the cell gap using ion-sensitive field-effect transistors. Upon exposure of Madin-Darby canine kidney (MDCK) cells cultured on a gate dielectric to a calcium-chelator EGTA, ammonia-assisted pH perturbation was enhanced solely in TJ-forming cells. This was supported by simulations with increased ion permeability in the paracellular pathway. Following administration of Clostridium perfringens enterotoxin as a specific TJ inhibitor to the MDCK cells, our method detected TJ breakdown at a 13× lower concentration than a conventional trans-epithelial electrical resistance assay. Thus, the semiconductor-based active pH sensing could offer an alternative to current in vitro assays for TJs in pathological, toxicological, and pharmaceutical studies.
Subject(s)
Protons , Tight Junctions/metabolism , Animals , Bioengineering , Cells, Cultured , Clostridium perfringens/chemistry , Dogs , Enterotoxins/administration & dosage , Enterotoxins/pharmacology , Hydrogen-Ion Concentration , Madin Darby Canine Kidney Cells/drug effects , Madin Darby Canine Kidney Cells/metabolism , Semiconductors , Tight Junctions/drug effectsABSTRACT
Conferring antifouling properties can extend the use of conducting polymers in biosensors and bioelectronics under complex biological conditions. On the basis of the antifouling properties of a series of zwitterionic polymers, we synthesized new thiophene-based compounds bearing a phosphorylcholine, carboxybetaine, or sulfobetaine pendant group. The monomers were synthesized by a facile reaction of thiol-functionalized 3,4-ethylenedioxythiophene with zwitterionic methacrylates. Electrochemical copolymerization was performed to deposit zwitterionic poly(3,4-ethylenedioxythiophene) (PEDOT) films with tunable conducting and antifouling properties on a conducting substrate. Electrochemical impedance spectroscopy showed that the conductivity and capacitance decreased with increasing zwitterionic content in the films. Protein adsorption and cell adhesion studies showed the effects of the type and content of zwitterions on the antifouling characteristics. Optimization of the electrodeposition conditions enabled development of both conducting and antifouling polymer films. These antifouling conjugated functional polymers have promising applications in biological environments.
ABSTRACT
Resistive pulse sensing (RPS) is an analytical technique for detecting particles with nano- to micrometer diameters, such as proteins, viruses, and bacteria. RPS is a promising tool for diagnosis as it can analyze the characteristics of target particles individually from ion current blockades as pulse waveforms. However, it is difficult to discriminate analog targets because RPS merely provides physical information such as size, shape, concentration, and charge density of the analyte. Influenza A virus, which is 80-120 nm in diameter, has various subtypes, demonstrating the diversity of virus characteristics. For example, highly pathogenic avian influenza infections in humans are recognized as an emerging infectious disease with high mortality rates compared with human influenza viruses. Distinguishing human from avian influenza using their differing biological characteristics would be challenging using RPS. To develop a highly selective diagnostic system for infectious diseases, we combined RPS with molecular recognition. Gold nanoparticles (GNPs) that have human influenza A (H1N1 subtype) virus-specific sialic acid receptors on the surface were prepared as a virus label for RPS analysis. A sulfobetaine and sialic acid (ligand) hybrid surface was formed on the GNPs for the suppression of nonspecific interaction. The results show a size change of viruses derived from specific interactions with GNPs. In contrast, no size shift was observed when nonspecific sialic acid receptor-immobilized GNPs were used. Detection of viruses by individual particle counting could be a new facet of diagnosis.
Subject(s)
Influenza A Virus, H1N1 Subtype/isolation & purification , Metal Nanoparticles/chemistry , Sialic Acids/chemistry , Animals , Chickens/virology , Dogs , Electrochemical Techniques/methods , Gold/chemistry , Hemagglutinins/metabolism , Influenza A Virus, H1N1 Subtype/chemistry , Ligands , Madin Darby Canine Kidney Cells/virology , Microbiological Techniques/methods , Sialic Acids/metabolism , Viral Proteins/metabolismABSTRACT
Unlike the majority of nanomaterials designed for cellular uptake via endocytic pathways, some of the functional nanoparticles and nanospheres directly enter the cytoplasm without overt biomembrane injuries. Previously, we have shown that a water-soluble nanoaggregate composed of amphiphilic random copolymer of 2-methacryloyloxyethyl phosphorylcholine (MPC) and n-butyl methacrylate (BMA), poly(MPC- random-BMA) (PMB), passes live cell membranes in an endocytosis-free manner. Yet, details in its translocation mechanism remain elusive due to the lack of proper analytical methods. To understand this phenomenon experimentally, we elaborated the original pH perturbation assay that is extremely sensitive to the pore formation on cell membranes. The ultimate sensitivity originates from the detection of the smallest indicator H+ (H3O+) passed through the molecularly sized transmembrane pores upon challenge by exogenous reagents. We revealed that water-soluble PMB at the 30 mol % MPC unit (i.e., PMB30W) penetrated into the cytosol of model mammalian cells without any proton leaks, in contrast to conventional cell-penetrating peptides, TAT and R8 as well as the surfactant, Triton X-100. While exposure of PMB30W permeabilized cytoplasmic lactate dehydrogenase out of the cells, indicating the alteration of cell membrane polarity by partitioning of amphiphilic PMB30W into the lipid bilayers. Nevertheless, the biomembrane alterations by PMB30W did not exhibit cytotoxicity. In summary, elucidating translocation mechanisms by proton dynamics will guide the design of nanomaterials with controlled permeabilization to cell membranes for bioengineering applications.
Subject(s)
Nanoparticles/chemistry , Polymers/chemistry , Cell Proliferation/drug effects , Hep G2 Cells , Humans , Methacrylates/chemistry , Phosphorylcholine/analogs & derivatives , Phosphorylcholine/chemistry , Polymers/toxicity , Protons , SolubilityABSTRACT
A redox-sensitive inter-conversion between ascorbic acid (ASC) and its oxidized form dehydroascorbic acid (DHA) in the intracellular environment has been of exceptional interest to recent metabolomics and pharmaceutical research. We developed a chromatographic protocol to instantly determine these vitamers with each identity from cellular extracts, without any labeling and pretreatments. Owing to its simplicity, one can readily continue the assay for hours, an otherwise difficult to cover timescale at which the intracellular DHA-ASC conversion comes into play. The method was validated for the analysis of pancreatic cancer cells, to our knowledge the first-ever study on a nucleated cell type, to trace in detail their kinetics of glucose transporter-dependent DHA uptake and, simultaneously, that for the intracellular ASC conversion. The simplest of all the relevant techniques and yet with the unique ability to provide each vitamer identity on a high-throughput basis, this method should offer the most practical option for VC-involved physiological and pharmaceutical studies including high-dose VC cancer therapy.
Subject(s)
Ascorbic Acid/analysis , Ascorbic Acid/metabolism , Chromatography, High Pressure Liquid/methods , Dehydroascorbic Acid/analysis , Dehydroascorbic Acid/metabolism , Ascorbic Acid/chemistry , Cell Line, Tumor , Dehydroascorbic Acid/chemistry , Erythrocytes/metabolism , Glucose Transporter Type 1/metabolism , Humans , Oxidation-Reduction , Pancreas/cytology , Pancreas/metabolism , Phosphorous Acids/chemistryABSTRACT
A quantitative diagnostic method for dental caries would improve oral health, which directly affects the quality of life. Here we describe the preparation and application of Ir/IrOx pH sensors, which are used to measure the surface pH of dental caries. The pH level is used as an indicator to distinguish between active and arrested caries. After a dentist visually inspected and defined 18 extracted dentinal caries at various positions as active or arrested caries, the surface pH values of sound and caries areas were directly measured with an Ir/IrOx pH sensor with a diameter of 300 µm as a dental explorer. The average pH values of the sound root, the arrested caries, and active caries were 6.85, 6.07, and 5.30, respectively. The pH obtained with an Ir/IrOx sensor was highly correlated with the inspection results by the dentist, indicating that the types of caries were successfully categorized. This caries testing technique using a micro Ir/IrOx pH sensor provides an accurate quantitative caries evaluation and has potential in clinical diagnosis.
Subject(s)
Dental Caries/diagnostic imaging , Iridium/chemistry , Tooth/diagnostic imaging , Electrodes , Hydrogen-Ion Concentration , Sensitivity and Specificity , Surface PropertiesABSTRACT
Boronic acids are well known for their ability to reversibly interact with the diol groups, a common motif of biomolecules including sugars and ribose. Due to their ability to interact with carbohydrates, they can be regarded as synthetic mimics of lectins, termed 'borono-lectins'. The borono-lectins can be tailored to elicit a broad profile of binding strength and specificity. This special property has been translated into many creative biomedical applications in a way interactive with biology. This review provides a brief overview of recent efforts of polymeric materials-based engineering taking advantage of such virtue of 'borono-lectins' chemistry, related to the field of biomaterials and drug delivery applications.
ABSTRACT
An NH4Cl-superfused system for a cell-cultured pH-sensing transistor was developed for detecting ion leakage across the plasma membranes of model HepG2 cells. The screening of chemical species by the method developed and conventional membrane-leakage assays identified the types of membrane injuries: structural membrane disruption and pore formation. Apoptosis-mediated membrane disordering was detected by continuously monitoring the ion-barrier breakdown of the membranes using the transistor system for an extended period. Comparisons of the ISFET assay with conventional cytotoxicity assays distinguished the cell death by direct membrane injury from that by other organelle damage. Our cell-based transistor system is fast and sensitive to ion leakage of the plasma membrane due to the small hydrodynamic size of the proton and ammonium ions as the indicators. The combination of the ion leakage assay with the existing cytotoxicity assays is a new way of classifying membrane injury and cell death induced by external chemical stimuli.
Subject(s)
Cell Death , Cell Membrane/ultrastructure , Protons , Transistors, Electronic , Apoptosis , Hep G2 Cells , HumansABSTRACT
The extracellular ionic microenvironment has a close relationship to biological activities such as by cellular respiration, cancer development, and immune response. A system composed of ion-sensitive field-effect transistors (ISFET), cells, and program-controlled fluidics has enabled the acquisition of real-time information about the integrity of the cell membrane via pH measurement. Here we aimed to extend this system toward floating cells such as T lymphocytes for investigating complement activation and pharmacokinetics through alternations in the plasma membrane integrity. We functionalized the surface of tantalum oxide gate insulator of ISFET with oleyl-tethered phosphonic acid for interacting with the plasma membranes of floating cells without affecting the cell signaling. The surface modification was characterized by X-ray photoelectron spectroscopy and water contact angle measurements. The Nernst response of -37.8 mV/pH was obtained for the surface-modified ISFET at 37 °C. The oleyl group-functionalized gate insulator successfully captured Jurkat T cells in a fluidic condition without acute cytotoxicity. The system was able to record the time course of pH changes at the cells/ISFET interface during the process of instant addition and withdrawal of ammonium chloride. Further, the plasma membrane injury of floating cells after exposure by detergent Triton™ X-100 was successfully determined using the modified ISFET with enhanced sensitivity as compared with conventional hemolysis assays.
ABSTRACT
BACKGROUND: Field effect transistor (FET) based signal-transduction (Bio-FET) is an emerging technique for label-free and real-time basis biosensors for a wide range of targets. Glucose has constantly been of interest due to its clinical relevance. Use of glucose oxidase (GOD) and a lectin protein Concanavalin A are two common strategies to generate glucose-dependent electrochemical events. However, these protein-based materials are intolerant of long-term usage and storage due to their inevitable denaturing. METHODS: A phenylboronic acid (PBA) modified self-assembled monolayer (SAM) on a gold electrode with an optimized disassociation constant of PBA, that is, 3-fluoro-4-carbamoyl-PBA possessing its pKa of 7.1, was prepared and utilized as an extended gate electrode for Bio-FET. RESULTS: The prepared electrode showed a glucose-dependent change in the surface potential under physiological conditions, thus providing a remarkably simple rationale for the glyco-sensitive Bio-FET. Importantly, the PBA modified electrode showed tolerance to relatively severe heat and drying treatments; conditions under which protein based materials would surely be denatured. CONCLUSIONS: A PBA modified SAM with optimized disassociation constant (pKa) can exhibit a glucose-dependent change in the surface potential under physiological conditions, providing a remarkably simple but robust method for the glyco-sensing. GENERAL SIGNIFICANCE: This protein-free, totally synthetic glyco-sensing strategy may offer cheap, robust and easily accessible platform that may be useful in developing countries. This article is part of a Special Issue entitled Organic Bioelectronics-Novel Applications in Biomedicine.
Subject(s)
Biosensing Techniques/instrumentation , Biosensing Techniques/methods , Boronic Acids/chemistry , Glucose/analysis , Potentiometry/instrumentation , Potentiometry/methods , Electrodes , Glucose/chemistry , Glucose Oxidase/chemistry , Gold/chemistry , Halogenation , Transistors, ElectronicABSTRACT
Exactly 1/3 mol of AlCl3 is sufficient to cyclize 1 mol of 1,ω-dibenzoylbutane (or pentane) to a cyclopentenone (or hexenone) derivative in high yield at room temperature in 40 min to several hours. This condensation is driven by removing elements of water as HCl and Al(OH)3, and the product enones are exclusively unconjugated, unlike the base-catalyzed condensations providing thermodynamically more stable conjugated enones.