RESUMO
Highly sensitive and selective choline microbiosensors were constructed by microcontact printing (µCP) of choline oxidase (ChOx) in a crosslinked, polyamine-functionalized zwitterionic polymer matrix on microelectrode arrays (MEAs). µCP has emerged as a potential means to create implantable, multiplexed sensor microprobes, which requires the targeted deposition of different sensor materials to specific microelectrode sites on a MEA. However, the less than sufficient enzyme loading and inadequate spatial resolution achieved with current µCP approaches has limited adoption of the method for electroenzymatic microsensors. A novel polymer, poly(2-methacryloyloxyethyl phosphorylcholine)-g-poly(allylamine hydrochloride) (PMPC-g-PAH), has been developed to address this challenge. PMPC-g-PAH contributes to a higher viscosity "ink" that enables thicker immobilized ChOx deposits of high spatial resolution while also providing a hydrophilic, biocompatible microenvironment for the enzyme. Electroenzymatic choline microbiosensors with sensitivity of 639 ± 96 nA µM-1 cm-2 (pH 7.4; n = 4) were constructed that also are selective against both ascorbic acid and dopamine, which are potential electroactive interfering compounds in the mammalian brain. The high sensitivities achieved can lead to smaller MEA microprobes that minimize tissue damage and make possible the monitoring of multiple neurochemicals simultaneously in vivo with high spatial resolution.
Assuntos
Oxirredutases do Álcool , Polímeros , Animais , Colina , MamíferosRESUMO
A high performance, electroenzymatic microsensor for choline based on choline oxidase (ChOx) immobilized on Pt coated with permselective polymer layers has been created that exhibits sensitivity approaching the theoretical performance limit. Sensor construction was guided by simulations performed with a detailed mathematical model. Implantable microsensors with an array of electroenzymatic sensing sites provide a means to record concentration changes of choline, an effective surrogate for acetylcholine due to its very rapid turnover in the brain, and other neurochemicals in vivo. However, electroenzymatic sensors generally have insufficient sensitivity and response time to monitor neurotransmitter signaling on the millisecond timescale with cellular-level spatial resolution. Model simulations suggested that choline sensor performance can be improved significantly by optimizing immobilized ChOx layer thickness and minimizing the thicknesses of permselective polymer coatings as well. Electroenzymatic choline sensors constructed with a â¼5 µm-thick crosslinked ChOx layer atop 200 nm-thick permselective films (poly(m-phenylenediamine) and Nafion) exhibited unprecedented sensitivity and response time of 660 ± 40 nA µM-1 cm-2 at 37 °C and 0.36 ± 0.05 s, respectively, while maintaining excellent selectivity. Such performance characteristics provide greater flexibility in the design of microelectrode array (MEA) probes with near cellular-scale sensing sites arranged in more dense arrays. Also, faster response times enable better resolution of transient acetylcholine signals and better correlation of these events with electrophysiological recordings so as to advance study of brain function.
Assuntos
Técnicas Biossensoriais , Colina , Acetilcolina , Microeletrodos , PolímerosRESUMO
The sensitivity and response time of glutamate sensors based on glutamate oxidase immobilized on planar platinum microelectrodes have been improved to near the theoretical performance limits predicted by a detailed mathematical model. Microprobes with an array of electroenzymatic sensing sites have emerged as useful tools for the monitoring of glutamate and other neurotransmitters in vivo; and implemented as such, they can be used to study many complex neurological diseases and disorders including Parkinson's disease and drug addiction. However, less than optimal sensitivity and response time has limited the spatiotemporal resolution of these promising research tools. A mathematical model has guided systematic improvement of an electroenzymatic glutamate microsensor constructed with a 1-2 µm-thick crosslinked glutamate oxidase layer and underlying permselective coating of polyphenylenediamine and Nafion reduced to less than 200 nm thick. These design modifications led to a nearly 6-fold improvement in sensitivity to 320 ± 20 nA µM-1 cm-2 at 37 °C and a â¼10-fold reduction in response time to 80 ± 10 ms. Importantly, the sensitivity and response times were attained while maintaining a low detection limit and excellent selectivity. Direct measurement of the transport properties of the enzyme and polymer layers used to create the biosensors enabled improvement of the mathematical model as well. Subsequent model simulations indicated that the performance characteristics achieved with the optimized biosensors approach the theoretical limits predicted for devices of this construction. Such high-performance glutamate biosensors will be more effective in vivo at a size closer to cellular dimension and will enable better correlation of glutamate signaling events with electrical recordings.
Assuntos
Aminoácido Oxirredutases/metabolismo , Técnicas Eletroquímicas/métodos , Ácido Glutâmico/análise , Aminoácido Oxirredutases/química , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Polímeros de Fluorcarboneto/química , Ácido Glutâmico/metabolismo , Peróxido de Hidrogênio/química , Sistemas Microeletromecânicos , Microeletrodos , Oxirredução , Polímeros/químicaRESUMO
High performance microprobes for combined sensing of glucose and choline were fabricated using microcontact printing (µCP) to transfer choline oxidase (ChOx) and glucose oxidase (GOx) onto targeted sites on microelectrode arrays (MEAs). Most electroenzymatic sensing sites on MEAs for neuroscience applications are created by manual enzyme deposition, which becomes problematic when the array feature size is less than or equal to â¼100 µm. The µCP process used here relies on use of soft lithography to create features on a polydimethylsiloxane (PDMS) microstamp that correspond to the dimensions and array locations of targeted, microscale sites on a MEA. Precise alignment of the stamp with the MEA is also required to transfer enzyme only onto the specified microelectrode(s). The dual sensor fabrication process began with polyphenylenediamine (PPD) electrodeposition on all Pt microelectrodes to block common interferents (e.g., ascorbic acid and dopamine) found in brain extracellular fluid. Next, a chitosan film was electrodeposited to serve as an adhesive layer. The two enzymes, ChOx and GOx, were transferred onto different microelectrodes of 2 × 2 arrays using two different PDMS stamps and a microscope for stamp alignment. Using constant potential amperometry, the combined sensing microprobe was confirmed to have high sensitivity for choline and glucose (286 and 117 µA mM cm-2, respectively) accompanied by low detection limits (1 and 3 µM, respectively) and rapid response times (≤2 s). This work demonstrates the use of µCP for facile creation of multianalyte sensing microprobes by targeted deposition of enzymes onto preselected sites of a microelectrode array.
Assuntos
Técnicas Biossensoriais/métodos , Colina/análise , Dimetilpolisiloxanos/química , Glucose/análise , Oxirredutases do Álcool/química , Técnicas Eletroquímicas/métodos , Enzimas Imobilizadas/química , Glucose Oxidase/química , Limite de Detecção , Microeletrodos , Sensibilidade e EspecificidadeRESUMO
Target DNA fragments at 10 fM concentration (approximately 6 × 10(5) molecules) were detected against a DNA background simulating the noncomplementary genomic DNA present in real samples using a simple, PCR-free, optics-free approach based on electromechanical signal transduction. The development of a rapid, sensitive, and cost-effective nucleic acid detection platform is highly desired for a range of diverse applications. We previously described a potentially low-cost device for sequence-specific nucleic acid detection based on conductance change measurement of a pore blocked by electrophoretically mobilized bead-(peptide nucleic acid probe) conjugates upon hybridization with target nucleic acid. Here, we demonstrate the operation of our device with longer DNA targets, and we describe the resulting improvement in the limit of detection (LOD). We investigated the detection of DNA oligomers of 110, 235, 419, and 1613 nucleotides at 1 pM to 1 fM and found that the LOD decreased as DNA length increased, with 419 and 1613 nucleotide oligomers detectable down to 10 fM. In addition, no false positive responses were obtained with noncomplementary, control DNA fragments of similar length. The 1613-base DNA oligomer is similar in size to 16S rRNA, which suggests that our device may be useful for detection of pathogenic bacteria at clinically relevant concentrations based on recognition of species-specific 16S rRNA sequences.
Assuntos
DNA/química , Transdução de Sinais , Limite de DetecçãoRESUMO
The ability to make rapid, informed decisions about whether or not to engage in a sequence of actions to earn reward is essential for survival. Modeling in rodents has demonstrated a critical role for the basolateral amygdala (BLA) in such reward-seeking actions, but the precise neurochemical underpinnings are not well understood. Taking advantage of recent advancements in biosensor technologies, we made spatially discrete near-real-time extracellular recordings of the major excitatory transmitter, glutamate, in the BLA of rats performing a self-paced lever-pressing sequence task for sucrose reward. This allowed us to detect rapid transient fluctuations in extracellular BLA glutamate time-locked to action performance. These glutamate transients tended to precede lever-pressing actions and were markedly increased in frequency when rats were engaged in such reward-seeking actions. Based on muscimol and tetrodotoxin microinfusions, these glutamate transients appeared to originate from the terminals of neurons with cell bodies in the orbital frontal cortex. Importantly, glutamate transient amplitude and frequency fluctuated with the value of the earned reward and positively predicted lever-pressing rate. Such novel rapid glutamate recordings during instrumental performance identify a role for glutamatergic signaling within the BLA in instrumental reward-seeking actions.
Assuntos
Tonsila do Cerebelo/metabolismo , Condicionamento Operante/fisiologia , Glutamatos/metabolismo , Recompensa , Potenciais de Ação/efeitos dos fármacos , Tonsila do Cerebelo/efeitos dos fármacos , Análise de Variância , Animais , Comportamento Animal , Condicionamento Operante/efeitos dos fármacos , Vias Eferentes/efeitos dos fármacos , Vias Eferentes/fisiologia , Eletrodos Implantados , Lobo Frontal/citologia , Lobo Frontal/efeitos dos fármacos , Lobo Frontal/fisiologia , Lateralidade Funcional/efeitos dos fármacos , Agonistas de Receptores de GABA-A/farmacologia , Masculino , Muscimol/farmacologia , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Ratos , Ratos Sprague-Dawley , Tempo de Reação/efeitos dos fármacos , Bloqueadores dos Canais de Sódio/farmacologia , Sacarose/administração & dosagem , Edulcorantes/administração & dosagem , Tetrodotoxina/farmacologiaRESUMO
[This corrects the article DOI: 10.1039/D2SD00128D.].
RESUMO
An amplification-free, nanopore-based nucleic acid detection platform has been demonstrated for rapid, 16S rRNA sequence-specific detection of Neisseria gonorrhoeae at 10-100 CFU mL-1 in human urine against background bacterial flora at 1000 CFU mL-1. Gonorrhea is a very common notifiable communicable disease, antibiotic resistant strains have emerged, and the rate of reported gonococcal infections continues to increase. Since rapid clinical identification of bacterial pathogens in clinical samples is needed to guide proper antibiotic treatment and to control disease spread, it is important to engineer rapid, sensitive, selective, and inexpensive point-of-care (POC) diagnostic devices for pathogens such as N. gonorrhoeae. Our detector technology is based on straightforward conductometric detection of sustained blockage of a glass nanopore. Charge neutral, complementary peptide nucleic acid probes are conjugated to polystyrene beads to capture N. gonorrhoeae 16S rRNA selectively. In the presence of an electric field applied externally through a glass nanopore, the PNA-microbead conjugates that acquire substantial negative charge upon target hybridization are driven to the smaller diameter nanopore. At least partial blockage of the nanopore results in a sustained drop in ionic current that can be measured easily with simple electronics. The ability to detect N. gonorrhoeae over the range of 10 to 100 CFU mL-1 spiked in human urine was demonstrated successfully with estimated sensitivity and specificity of â¼98% and â¼100%, respectively. No false positives were observed for the control group of representative background flora (E. coli, K. pneumoniae, and E. faecalis) at 1000 CFU mL-1. Also, N. gonorrhoeae at 50 CFU mL-1 was successfully detected against 1000 CFU mL-1 of background flora in urine. These results suggest that this amplification-free technology may serve as the basis for rapid, inexpensive, low-power detection of pathogens in clinical samples at the POC.
RESUMO
We describe a platform for sequence-specific nucleic acid (NA) detection utilizing a micropipet tapered to a 2 µm diameter pore and 3 µm diameter polystyrene beads to which uncharged peptide nucleic acid (PNA) probe molecules have been conjugated. As the target NAs hybridize to the complementary PNA-beads, the beads acquire negative charge and become electrophoretically mobile. An applied electric field guides these NA-PNA-beads toward the pipet tip, which they obstruct, leading to an indefinite, electrically detectable, partial blockade of the pore. In the presence of noncomplementary NA, even to the level of single base mismatch, permanent pore blockade is not seen. We show application of this platform to detection of the anthrax lethal factor sequence.
Assuntos
Ácidos Nucleicos Peptídicos/análise , Sequência de Bases , DNA de Cadeia Simples , Ensaio de Desvio de Mobilidade Eletroforética , Hibridização de Ácido NucleicoRESUMO
Selective generation of an amine-terminated self-assembled monolayer bound to silicon wafers via a silicon-carbon linkage was realized by photocatalytically reducing the corresponding azide-terminated, self-assembled monolayers (Az-SAMs). The Az-SAM was obtained by thermal deposition of 11-chloroundecene onto a hydrogen-terminated silicon wafer followed by nucleophilic substitution of the chloride with the azide ion in warm N,N'-dimethylformamide (DMF). The presence of the terminal azide group on the SAM was confirmed by reflection absorption infrared spectroscopy (RAIRS), by X-ray photoelectron spectroscopy (XPS), and by detecting the formation of a triazole upon reaction of the azide with an activated alkyne. The desired terminal amine groups were generated by photocatalytic reduction of the Az-SAM with cadmium selenide quantum dots (CdSe Qdots) using λ > 400 nm. Analysis of the reduced SAM by XPS gave results that were consistent with those obtained with an amine-terminated surface obtained by reducing the Az-SAM with triphenylphosphine. To demonstrate the feasibility of using the Az-SAM for surface patterning, a sample was coated with adsorbed CdSe Qdots and exposed to the output of a diode laser at λ = 407 nm through a micropatterned mask. Using a SEM, the pattern formed in this manner was revealed after removing the CdSe Qdots and subsequently adsorbing 10 nm gold nanoparticles (AuNPs) to the positively charged terminal-amine groups. The formation of the pattern by CdSe-photocatalyzed reduction of the azide demonstrates a novel route to create features by selective modification of organic monolayers on silicon wafers.
RESUMO
Monitoring neurochemical signaling across time scales is critical to understanding how brains encode and store information. Flexible (vs stiff) devices have been shown to improve in vivo monitoring, particularly over longer times, by reducing tissue damage and immunological responses. Here, we report our initial steps toward developing flexible and implantable neuroprobes with aptamer-field-effect transistor (FET) biosensors for neurotransmitter monitoring. A high-throughput process was developed to fabricate thin, flexible polyimide probes using microelectromechanical-system (MEMS) technologies, where 150 flexible probes were fabricated on each 4 in. Si wafer. Probes were 150 µm wide and 7 µm thick with two FETs per tip. The bending stiffness was 1.2 × 10-11 N·m2. Semiconductor thin films (3 nm In2O3) were functionalized with DNA aptamers for target recognition, which produces aptamer conformational rearrangements detected via changes in FET conductance. Flexible aptamer-FET neuroprobes detected serotonin at femtomolar concentrations in high-ionic strength artificial cerebrospinal fluid. A straightforward implantation process was developed, where microfabricated Si carrier devices assisted with implantation such that flexible neuroprobes detected physiological relevant serotonin in a tissue-hydrogel brain mimic.
Assuntos
Aptâmeros de Nucleotídeos , Técnicas Biossensoriais , Serotonina , Semicondutores , Aptâmeros de Nucleotídeos/químicaRESUMO
A key characteristic of the analyte-reporter enzyme conjugate used in the enzyme-multiplied immunoassay technique (EMIT) is the inhibition of the conjugate enzyme upon anti-analyte antibody binding. To improve our understanding of the antibody-induced inhibition mechanism, we characterized morphine-glucose-6-phosphate dehydrogenase (G6PDH) conjugates as model EMIT analyte-reporter enzyme conjugates. Morphine-G6PDH conjugates were prepared by acylating predominantly the primary amines on G6PDH with morphine 3-glucuronide NHS ester molecules. In this study, morphine-G6PDH conjugates were characterized using a combination of methods, including tryptic digestion, immunoprecipitation, matrix-assisted laser desorption ionization mass spectrometry, and electrospray ionization tandem mass spectrometry. Twenty-six conjugation sites were identified. The identified sites all were found to be primary amines. The degree of conjugation was determined to be less than the number of conjugation sites, suggesting heterogeneity within the morphine-G6PDH conjugate population. Two catalytically important residues in the active site (K22 and K183) were among the identified conjugation sites, explaining at least partially the cause of loss of activity due to the coupling reaction.
Assuntos
Técnica de Imunoensaio Enzimático de Multiplicação , Glucosefosfato Desidrogenase , Espectrometria de Massas/métodos , Morfina , Aminas/química , Anticorpos , Sítios de Ligação , Domínio Catalítico , Inibidores EnzimáticosRESUMO
The stability of Langmuir monolayers of CdSe Qdots capped with dodecan-ethiol (DDT), with dithiocarbamates having one, two, or three long alkyl chains (DTC-1, DTC-2 and DTC-3) or with tri-n-octylphosphine oxide (TOPO), was investigated and linked to the transport of Qdots into the subphase via a dissolution and diffusion mechanism. Langmuir films of Qdots were created by depositing droplets of purified Qdots in chloroform at the air-water interface. While holding the Qdot films at 13 mN/m for 1 h in a Langmuir trough, the average monolayer areas decreased by roughly 9% for TOPO-capped Qdots, â¼15-18% for the three DTC-capped Qdot preparations, and â¼21% for DDT-capped Qdots. Using the model of Ter Minassian-Saraga, the relative stabilities of the Qdot films studied were related to differences in equilibrium partitioning into the subphase and to apparent Qdot diffusivities within the subphase. An analysis of the Qdot preparations by Fourier-transform infrared spectroscopy (FTIR) revealed that the aliphatic tails of capping ligands were assembled on Qdot surfaces with similar packing densities for all ligand chemistries. A combined analysis of the film-area contraction and FTIR data suggested that, for the chemistries examined in this study, both the capping-ligand headgroup and the aliphatic tail groups impact Qdot Langmuir film stability through their joint influence on nanoparticle wettability and the tendency to aggregate upon partitioning into the subphase.
RESUMO
A convenient homogeneous enzyme immunoassay for estradiol is described. Unlike heterogeneous immunoassays, which require time-consuming separation steps or expensive automated systems, homogeneous immunoassays, wherein all reagents are freely suspended in bulk solution, can be simple and fast without costly instrumentation. The key component of this assay system, an estradiol-reporter enzyme conjugate, was prepared by covalently binding ß-estradiol-6-(O-carboxymethyl)oxime to glucose-6-phosphate dehydrogenase (G6PDH) by an N-hydroxysuccinimide-enhanced, carbodiimide-mediated coupling reaction. The estradiol-G6PDH activity can be repressed up to 46% upon anti-estradiol antibody binding. The lower detection limit of the assay is 1 nM estradiol in aqueous solution, and the standard curve is linear on logit-log scale-up to 6.7 µM estradiol. A detection limit of 11.5 nM in estradiol-spiked human serum samples suggests the feasibility of applying this assay to monitor estradiol levels for the prediction and prevention of ovarian hyperstimulation syndrome.
Assuntos
Estradiol/análise , Técnicas Imunoenzimáticas/métodos , Síndrome de Hiperestimulação Ovariana/diagnóstico , Estradiol/análogos & derivados , Estradiol/sangue , Estradiol/metabolismo , Feminino , Glucosefosfato Desidrogenase/metabolismo , Humanos , Limite de Detecção , Síndrome de Hiperestimulação Ovariana/metabolismo , Síndrome de Hiperestimulação Ovariana/patologia , Succinimidas/metabolismoRESUMO
A practical approach for constructing enzyme-multiplied immunoassay technique (EMIT)-based protein/peptide assays is described. Normally used in small-molecule drug testing, EMIT is a homogeneous assay method that is attractive for its simplicity, sensitivity, and rapidity. The EMIT-based peptide/protein assay was developed by conjugating a cysteine-modified HA peptide (from influenza hemagglutinin A) to the reporter enzyme, glucose-6-phosphate dehydrogenase. The 13-min assay gave a free HA limit of detection of 10 nM and proved effective for detection of a high-molecular-weight model protein tagged with HA. Similar EMIT-based assay approaches may be developed for applications in biotoxin and infectious disease detection.
Assuntos
Técnica de Imunoensaio Enzimático de Multiplicação , Glicoproteínas de Hemaglutininação de Vírus da Influenza/análise , Calibragem , Glucosefosfato Desidrogenase/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/químicaRESUMO
Detailed simulations show that the relationship between electroenzymatic glutamate (Glut) sensor performance in vitro and that modeled in vivo is complicated by the influence of both resistances to mass transfer and clearance rates of Glut and H2O2 in the brain extracellular space (ECS). Mathematical modeling provides a powerful means to illustrate how these devices are expected to respond to a variety of conditions in vivo in ways that cannot be accomplished readily using existing experimental techniques. Through the use of transient model simulations in one spatial dimension, it is shown that the sensor response in vivo may exhibit much greater dependence on H2O2 mass transfer and clearance in the surrounding tissue than previously thought. This dependence may lead to sensor signals more than double the expected values (based on prior sensor calibration in vitro) for Glut release events within a few microns of the sensor surface. The sensor response in general is greatly affected by the distance between the device and location of Glut release, and apparent concentrations reported by simulated sensors consistently are well below the actual Glut levels for events occurring at distances greater than a few microns. Simulations of transient Glut concentrations, including a physiologically relevant bolus release, indicate that detection of Glut signaling likely is limited to events within 30 µm of the sensor surface based on representative sensor detection limits. It follows that important limitations also exist with respect to interpretation of decays in sensor signals, including relation of such data to actual Glut concentration declines in vivo. Thus, the use of sensor signal data to determine quantitatively the rates of Glut uptake from the brain ECS likely is problematic. The model is designed to represent a broad range of relevant physiological conditions, and although limited to one dimension, provides much needed guidance regarding the interpretation in general of electroenzymatic sensor data gathered in vivo.
Assuntos
Técnicas Biossensoriais , Ácido Glutâmico , Calibragem , Peróxido de Hidrogênio , Modelos TeóricosRESUMO
The broad spectrum of approaches for nucleic acid amplification-free detection of DNA and RNA at single-digit attomolar (10-18 M) concentration and lower is reviewed. These low concentrations correspond roughly to the most clinically desirable detection range for pathogen-specific nucleic acid as well as the detection limits of commercially available, nucleic acid amplification tests based primarily on polymerase chain reaction (PCR). The need for more rapid and inexpensive, yet still highly accurate tests, has become evident during the pandemic. It is expected that publication of reports describing improved tests will accelerate soon, and this review covers the wide variety of detection methods based on both optical and electrical measurements that have been conceived over recent years, enabled generally by the advent of nanotechnology.
Assuntos
Ácidos Nucleicos , RNA , DNA/genética , Técnicas de Amplificação de Ácido Nucleico , Reação em Cadeia da Polimerase , RNA/genéticaRESUMO
While tools for monitoring in vivo electrophysiology have been extensively developed, neurochemical recording technologies remain limited. Nevertheless, chemical communication via neurotransmitters plays central roles in brain information processing. We developed implantable aptamerfield-effect transistor (FET) neuroprobes for monitoring neurotransmitters. Neuroprobes were fabricated using high-throughput microelectromechanical system (MEMS) technologies, where 150 probes with shanks of either 150- or 50-µm widths and thicknesses were fabricated on 4-inch Si wafers. Nanoscale FETs with ultrathin (~3 to 4 nm) In2O3 semiconductor films were prepared using sol-gel processing. The In2O3 surfaces were coupled with synthetic oligonucleotide receptors (aptamers) to recognize and to detect the neurotransmitter serotonin. Aptamer-FET neuroprobes enabled femtomolar serotonin detection limits in brain tissue with minimal biofouling. Stimulated serotonin release was detected in vivo. This study opens opportunities for integrated neural activity recordings at high spatiotemporal resolution by combining these aptamer-FET sensors with other types of Si-based implantable probes to advance our understanding of brain function.
RESUMO
A multifunctional chemical neural probe fabrication process exploiting PDMS thin-film transfer to incorporate a microfluidic channel onto a silicon-based microelectrode array (MEA) platform, and enzyme microstamping to provide multi-analyte detection is described. The Si/PDMS hybrid chemtrode, modified with a nano-based on-probe IrOx reference electrode, was validated in brain phantoms and in rat brain.
Assuntos
Microfluídica , Próteses e Implantes , Animais , Microeletrodos , RatosRESUMO
The membrane lipids of archaea are characterized by unique isoprenoid biochemistry, which typically is based on two core lipid structures, sn-2,3-diphytanylglycerol diether (archaeol) and sn-2,3-dibiphytanyldiglycerol tetraether (caldarchaeol). The biosynthetic pathway for the tetraether lipid entails unprecedented head-to-head coupling of isoprenoid intermediates by an unknown mechanism involving unidentified enzymes. To investigate the isoprenoid ether lipid biosynthesis pathway of the hyperthermophilic archaeon, Archaeoglobus fulgidus, its lipid synthesis machinery was reconstructed in an engineered Escherichia coli strain in an effort to demonstrate, for the first time, efficient isoprenoid ether lipid biosynthesis for the production of the intermediate, digeranylgeranylglyceryl phosphate (DGGGP). The biosynthesis of DGGGP was verified using an LC/MS/MS technique and was accomplished by cloning and expressing the native E. coli gene for isopentenyl diphosphate (IPP) isomerase (idi), along with the A. fulgidus genes for G1P dehydrogenase (egsA) and GGPP synthase (gps), under the control of the lac promoter. The A. fulgidus genes for GGGP synthase (GGGPS) and DGGGP synthase (DGGGPS), under the control of the araBAD promoter, were then introduced and expressed to enable DGGGP biosynthesis in vivo. This investigation established roles for four A. fulgidus genes in the isoprenoid ether lipid pathway for DGGGP biosynthesis and provides a platform useful for identification of subsequent, currently unknown, steps in tetraether lipid biosynthesis proceeding from DGGGP, which is the presumed substrate for the head-to-head coupling reaction yielding unsaturated caldarchaeol.