Development of an immunopredictor for the evaluation of the risk of cardiovascular diseases based on the level of soluble P-selectin.

Due to its physiologic role in modulating adhesive interactions between blood cells and the endothelium during inflammatory processes or at injury sites, the adhesion molecule P-selectin is of great interest. The level of soluble P-selectin in plasma or serum can be detected and used as a clinical predictor for adverse cardiovascular events, leading to the presumption that it is secreted, shed or cleaved from the cell membrane during the process of diseases. Increased levels of soluble P-selectin in the plasma have been shown to be associated with a range of cardiovascular disorders, including coronary artery disease, hypertension and atrial fibrillation. Therefore, it is of huge significance to develop simple, rapid and sensitive methods for the detection of such pathological predictors, not only for facilitating the surveillance of cardiovascular mortality/sudden cardiac death, but also for effectively monitoring the drug potency on platelets based on measurement of P-selectin performed on fixed blood samples following platelet stimulation in whole blood in a remote setting. We herein developed a simple, yet novel and sensitive electrochemical sandwich immunosensor for the detection of P-selectin; it operates through covalent linkage of anti-P-selectin antibody on CNT@GNB nanocomposites-modified disposable screen-printed electrode as the detection platform, with the potassium ferrocyanide-encapsulated, anti-P-selectin-tagged liposomal biolabels as the electrochemical signal probes. The immunorecognition of the sample P-selectin by the liposomal biolabels occurred on the surface of the electrodes; the release of potassium ferrocyanide from the bound liposomal biolabels extensively contributed to the increase in electrochemical signal, which was acquired in HCl solution at +0.32V in square wave voltammetry mode. The resulting sigmoidally shaped dose-response curves possessed a linear dynamic working range from 1×10(-13) to 1×10(-5)g/mL. This liposome-based electrochemical immunoassay provides an amplification approach for detecting P-selectin at trace levels, leading to a detection limit as low as 4.3fg (equivalent to 5µL of 0.85pg/mL solution). A commercially available ELISA kit was used as a reference method to validate the newly-developed assay through the analysis of mouse serum samples. A strong correlation was observed between the two data sets as the R-squared value of 0.997 from the linear regression line. This electrochemical immunosensor will be useful for the detection of P-selectin in biological fluids and tissue extracts.

Liposome-based artificial cells: From gene expression to reconstitution of cellular functions and phenotypes.

Bottom-up approaches in creating artificial cells that can mimic natural cells have significant implications for both basic research and translational application. Among various artificial cell models, liposome is one of the most sophisticated systems. By encapsulating proteins and associated biomolecules, they can functionally reconstitute foundational features of biological cells, such as the ability to divide, communicate, and undergo shape deformation. Yet constructing liposome artificial cells from the genetic level, which is central to generate self-sustained systems remains highly challenging. Indeed, many studies have successfully established the expression of gene-coded proteins inside liposomes. Further, recent endeavors to build a direct integration of gene-expressed proteins for reconstituting molecular functions and phenotypes in liposomes have also significantly increased. Thus, this review presents the development of liposome-based artificial cells to demonstrate the process of gene-expressed proteins and their reconstitution to perform desired molecular and cell-like functions. The molecular and cellular phenotypes discussed here include the self-production of membrane phospholipids, division, shape deformation, self-DNA/RNA replication, fusion, and intercellular communication. Together, this review gives a comprehensive overview of gene-expressing liposomes that can stimulate further research of this technology and achieve artificial cells with superior properties in the future.

Diagnostic detection of human lung cancer-associated antigen using a gold nanoparticle-based electrochemical immunosensor.

The development of rapid and sensitive methods for the detection of immunogenic tumor-associated antigen is important not only for understanding their roles in cancer immunology but also for the development of clinical diagnostics. Alpha-enolase (ENO1), a p48 molecule, is widely distributed in a variety of tissues, whereas gamma-enolase (ENO2) and beta-enolase (ENO3) are found exclusively in neuron/neuroendocrine and muscle tissues, respectively. Because ENO1 has been correlated with small cell lung cancer, nonsmall cell lung cancer, and head and neck cancer, it can be used as a potential diagnostic marker for lung cancer. In this study, we developed a simple, yet novel and sensitive, electrochemical sandwich immunosensor for the detection of ENO1; it operates through physisorption of anti-ENO1 monoclonal antibody on polyethylene glycol-modified disposable screen-printed electrode as the detection platform, with polyclonal secondary anti-ENO1-tagged, gold nanoparticle (AuNP) congregates as electrochemical signal probes. The immunorecognition of the sample ENO1 by the congregated AuNP@antibody occurred on the surface of the electrodes; the electrochemical signal from the bound AuNP congregates was obtained after oxidizing them in 0.1 M HCl at 1.2 V for 120 s, followed by the reduction of AuCl(4-) in square wave voltammetry (SWV) mode. The resulting sigmoidally shaped dose-response curves possessed a linear dynamic working range from 10(-8) to 10(-12) g/mL. This AuNP congregate-based assay provides an amplification approach for detecting ENO1 at trace levels, leading to a detection limit as low as 11.9 fg (equivalent to 5 microL of a 2.38 pg/mL solution).

Simple and Cost-effective Enzymatic Detection of Cholesterol Using Flow Injection Analysis.

A flow-injection analytical (FIA) system was developed for the determination of cholesterol concentrations based on enzymatic reactions that occurred in a cholesterol oxidase (CHOx)-immobilized, fused-silica capillary followed by electrochemical detection. The production of hydrogen peroxide from cholesterol in an enzymatic reaction catalyzed by CHOx was subsequently oxidized electrochemically at an electrode. Our FlA system demonstrated its cost-effectiveness and utility at an applied potential of 0.6 V (vs. Ag/AgCl), a flow rate of 100 µL/min and, under optimal conditions, the resulting signal demonstrated a linear dynamic range from 50 µM to 1.0 mM with a limit of detection (LOD) of 12.4 µM, limit of quantification (LOQ) of 44.9 µM, and the coefficient of variation of 5.17%. In addition, validation of our proposed system using a reference HDL-cholesterol kit used for clinical diagnosis suggested our FIA system was comparable to commercial kits for the determination of the cholesterol incorporation amount in various aqueous liposomal suspensions. These good analytical features achieved by FIA could make the implementation of this methodology possible for on-line monitoring of cholesterol in various types of samples.

Attomole DNA electrochemical sensor for the detection of Escherichia coli O157.

Enterohemorrhagic Escherichia coli O157, a verocytotoxin (VT1/2)-producing pathogen, can be deadly because it can induce acute or chronic renal failure. To speed up the clinical diagnosis of related syndromes caused by E. coli O157, there is an urgent need for rapid, simple, and reliable analytical tools for its quantitation. In this study, we developed a novel electrochemical competitive genosensor, featuring gold-electrodeposited screen-printed electrodes (nanoAu/SPE) modified with a self-assembled monolayer of thiol-capped single-stranded DNA (capture probe), for the detection of the rfbE gene, which is specific to E. coli O157. This assay functions based on competition between the target gene (complementary to the capture probe DNA) and reporter DNA-tagged, hexaammineruthenium(III) chloride-encapsulated liposomes. The current signal of the released liposomal Ru(NH(3))(6)(3+) was measured using square wave voltammetry, yielding a sigmoidally shaped dose-response curve whose linear portion was over the range from 1 to 10(6) fmol. This liposomal competitive assay provides an amplification route for the detection of the rfbE gene at ultratrace levels; indeed, we could detect as little as 0.75 amol of the target rfbE DNA (equivalent to the amount present in 5 microL of a 0.15 pM solution).

Folic acid-anchored PEGgylated phospholipid bioconjugate and its application in a liposomal immunodiagnostic assay for folic acid.

A folic acid-anchored, poly(ethylene glycol)-linked (PEGgylated) phospholipid and an immunoaffinity chromatographic column were prepared and employed to develop a liposomal immunodiagnostic assay for the direct determination of folic acid (FA) in this study. Distearoylphosphatidylethanolamine-poly(ethylene glycol)2000-folic acid (DSPE-PEG2000-FA) was synthesized through carbodiimide-mediated coupling of FA and DSPE-PEG2000-amine and characterized using thin layer chromatography, 1H nuclear magnetic resonance spectroscopy, and electrospray ionization-mass spectrometry. Liposomal biolabels were constructed using the synthesized DSPE-PEG2000-FA in conjunction with other phospholipids. A stationary phase having affinity for FA was prepared by covalently linking purified anti-FA monoclonal antibodies onto N-hydroxysuccinimide-activated Sepharose beads, which were subsequently packed into a 1.9 cm diameter polypropylene column. The calibration curve for FA had a linear range from 10(-8) to 10(-4) M. The limit of detection was 6.8 ng (equivalent to 500 microL of 3.1 x 10(-8) M FA). The elution buffer (35% methanol in Tris buffered saline containing 0.1% Tween 20) also served as the regeneration buffer, which allowed the same column to be used for up to 50 times without any observable loss of reactivity. The immunoaffinity chromatographic column was reusable and capable of concentrating analytes from sample solution; in conjunction with folic acid-sensitized liposomal biolabels, however, they hold great potential as sensitive immunoaffinity assays for the determination for FA. To confirm the feasibility of using this system in the analysis of real samples, the folic acid contents of three over-the-counter vitamin supplements were tested. The recoveries of folic acid of 90-112% for these three samples were obtained, suggesting contents that were consistent with the information obtained from their nutritional facts panels.

Carbon nanoparticle-enhanced immunoelectrochemical detection for protein tumor marker with cadmium sulfide biotracers.

We have developed a sensitive electrochemical immunoassay system for the detection of a protein tumor marker, carcinoembryonic antigen (CEA), that is based on a carbon nanoparticle (CNP)/poly(ethylene imine) (PEI)-modified screen-printed graphite electrode (CNP-PEI/SPGE) covered with anti-CEA antibodies. The signal amplification strategy--using CdS nanocrystals as biotracers and CNPs to enhance electron transfer--improves the sensitivity and detection limit for CEA, suggesting that this system holds promise for development into a point-of-care or disposable home-care self-diagnostic tool. This biosensor is based on a sandwich complex immunoassay, which we assembled from sequential layers of the anti-CEA antibody (alphaCEA) on CNP-PEI/SPGE, the CEA sample, and the CdS nanocrystal quantum dots (QDs) sensitized with alphaCEA (alphaCEA-CdS QD). We used square wave anodic stripping voltammetry (SWASV) to amplify the signal current response obtained from the dissolved alphaCEA-CdS QDs. The calibration curve for CEA concentration was linear in the range of 0.032-10 ng/mL; the detection limit (estimated as the mean of the blank sample plus three times the standard deviation obtained on the blank sample) was 32 pg/mL (equivalent to 160 fg in a 5 microL sample). This method is suitably precise and sensitive to function as a means of determining urinary CEA, which is a better marker than serum CEA for the early detection of urothelial carcinoma.

Gold-nanostructured immunosensor for the electrochemical sensing of biotin based on liposomal competitive assay.

This work describes the development of a cost-effective, easy-to-use, portable immunoanalytical platform technology with sufficient sensitivity for use in the detection of physiologically important targets. Biotin, also known as vitamin H, was selected as the model analyte. The detecting system employs biotin-tagged, potassium ferrocyanide-encapsulated liposomes as the signal amplifier and PAH (poly allylamine hydrochloride)-modified, nanosized-Au particles assembled screen-printed electrode (nanoAu-SPE) as the working electrode. The diagnostic procedures are based on selective immunoanalytical recognitions and sensitive electrochemical detection. The model analyte biotin was determined based on a "competitive-type" immunoassay in which competition occurs between the analyte biotin and potassium ferrocyanide-encapsulated, biotin-tagged liposomes for a limited number of anti-biotin antibody binding sites, which were immobilized on the PAH/nanoAu/SPE surface. The nanostructured Au SPE surface was covalently bonded to the PAH layer, which subsequently interacted with anti-biotin antibodies. The ferrocyanide released from ruptured bound-liposomes was finally measured using square-wave voltammetry. The calibration curve for biotin had a linear range of 10(-11)-10(-2) M, covering nine orders of magnitude. The detection limit of this immunodetecting system was as low as 9.1 pg of biotin (equivalent to 4.5/microL of 8.3 x 10(-9) M).

Using liposomal fluorescent biolabels to develop an immunoaffinity chromatographic biosensing system for biotin.

Although biotin is an important vitamin for cellular function and growth, there are no rapid, simple, and reliable analytical tools available for its quantitation in bodily fluids or foodstuffs. In this study, we developed an immunoaffinity chromatographic biosensing system for the direct determination of biotin. A stationary phase having affinity for biotin was synthesized by covalently bonding antibiotin monoclonal antibodies onto 90-microm, NHS-activated sepharose beads. The beads were then packed into 1.9-cm-diameter plastic tubes to form a column having a volume of 3.0 mL. The function of the proposed immunoaffinity chromatographic assay was based on competition between biotin and carboxyfluorescein (CF)-encapsulated, biotin-tagged liposomes (liposomal biolabels) for the limited number of antibiotin antibody binding sites. Buffers containing biotin standards at concentrations ranging from 10(-12) to 10(-3) M were passed through the column to trap and concentrate the biotin on the solid support. The unbound binding sites of the antibody were then occupied through subsequent addition of the liposomal biolabels. The addition of 35% methanol released the CF molecules from the lyzed bound liposomes; the fluorescence intensity of the released markers was then measured using a fluorometer. The calibration curve for biotin was linear over 8 orders of magnitude, from 10(-12) to 10(-4) M. The limit of detection of this immunoaffinity chromatographic biosensing system reached as low as 5.0 pg of biotin (equivalent to 500 microL of 4.10 x 10(-11) M biotin).

Liposome-based immunostrip for the rapid detection of Salmonella.

Salmonellae are ubiquitous human pathogens, which pose a danger to the elderly and children. Due to the increased number of outbreaks of human illness associated with the consumption of contaminated products in the USA and many other countries, there is an urgent need to develop rapid assays to detect common food-borne pathogens. This study demonstrates the feasibility of using a detectable label comprising methyl blue (MB), a visible dye, entrapped inside liposomes. Immunoliposomes tagged with anti-Salmonella common structural antigens (CSA) antibody encapsulating MB dye were prepared and used as the signal amplifier for the development of a field-portable colorimetric immunoassay to detect Salmonellae. Tapping mode atomic force microscopy (TMAFM), a scanning probe technique, was utilized to demonstrate the presence of anti-Salmonella antibody at the thus-prepared liposome. A plastic-backed nitrocellulose strip with two immobilized zones formed the basis of a sandwich assay. The first zone was the antigen capture zone (AC zone), used in a sandwich (noncompetitive) assay format; the other was the biotin capture zone (BC zone), used as a quality control index for the strip assay. During the capillary migration of the wicking reagent containing 80 µL of immunoliposomes and 40 µL of the test sample (heat-killed S. typhimurium), sample pathogens with surface-bound immunoliposomes were captured at the AC zone, while the unbound immunoliposomes continued to migrate and bind to the anti-biotin antibodies coated on the BC zone. The color density of the AC zone was directly proportional to the number of Salmonella typhimurium in the test sample. The detection limit of the current assay with heat-killed Salmonella typhimurium was 1,680 cells. The cross-reactivity of the proposed immunoassay was also investigated, and pathogens including E. coli O157:H7 and Listeria genus specific caused no interference with the detection of Salmonella typhimurium.

Tyramine detection using PEDOT:PSS/AuNPs/1-methyl-4-mercaptopyridine modified screen-printed carbon electrode with molecularly imprinted polymer solid phase extraction.

Tyramine (4-hydroxyphenethylamine), which is a monoamine metabolized by monoamine oxidase (MAO), exists widely in plants, animals, fermented foods, and salted foods. The incidence of hypertension, or "cheese effect", which is associated with a large dietary intake of tyramine while taking MAO inhibitors has been reported; therefore, the measurement of tyramine is an urgent concern. Herein, an efficient approach that integrates a molecular imprinting polymer for solid phase extraction (MISPE) technique with a sensitive electrochemical sensing platform (SPCE/PEDOT: PSS/AuNP/1-m-4-MP) for the quantification of tyramine is presented. Enhanced electrode conductivity was achieved sequentially by constructing a conductive polymer (PEDOT: PSS) on a screen-printed carbon electrode (SPCE), followed by electrodeposition with gold nanoparticles (AuNPs) and, finally, by modification with positively charged 1-methyl-4-mercaptopyridine (1-m-4-MP) using an Au-S bond. Tyramine was isolated selectively and pre-concentrated by the MISPE technique; electroanalysis that used differential pulse voltammetry (DPV) in NaOH (0.1M, pH 13) was conducted successively. Experimental parameters (such as modes of electrode modification, ratio of PEDOT: PSS, pH of electrolyte, time required for AuNP deposition, and 1-m-4-MP concentrations) that were associated with optimal detection conditions were evaluated also. We obtained a linear concentration range (5-100nM, R2=0.9939) with LOD and sensitivity at 2.31nM, and 3.11µAnM-1cm-2, respectively. The applicability of our technique was demonstrated by analyzing tyramine in spiked serum and milk. The feature of our newly developed analytical methods that coupled sample pre-treatment (sample clean-up and pre-concentration) with sensitive detection makes it a promising tool for quantifying of tyramine.

Resuscitation Using Liposomal Vasopressin in an Animal Model of Uncontrolled Hemorrhagic Shock.

BACKGROUND: Current research suggests that administration of vasopressin to patients with uncontrolled hemorrhagic shock (UHS) can avoid the detrimental effects associated with aggressive fluid resuscitation. However, vasopressin has a short half-life of 10~35 minutes in in vivo use and precludes its use in the pre-hospital setting. To increase the half-life of vasopressin, we proposed to synthesize liposome-encapsulated vasopressin and test it in a rat model of UHS. METHODS: The film hydration method was used to prepare liposomal vasopressin consisting of: Dipalmitoylphosphatidylcholine, cholesterol, and dipalmitoyl phosphatidylethanolamine (20:20:1 mole ratio). 42 rats were subjected to UHS and randomly received 5 different treatments (vasopressin, liposomal vasopressin, lactate ringer (LR), liposome only and sham). Outcome of UHS were measured using 4 common prognostic tests: mean arterial pressure (MAP), serum lactate level, inflammatory profile and pulmonary edema. RESULTS: The dynamic light scattering results confirmed that we had prepared a successful liposomal vasopressin complex. Comparing the serum vasopressin concentration of liposomal vasopressin and vasopressin treated animals by ELISA, we found that the concentration of vasopressin for the liposomal vasopressin treated group is higher at 60 minutes. However, there was no significant difference between the MAP profile of rats treated with vasopressin and liposomal vasopressin in UHS. We also observed that animals treated with liposomal vasopressin performed indifferently to vasopressin treated rats in serum lactate level, inflammatory profile and edema profile. For most of our assays, the liposome only control behaves similarly to LR resuscitation in UHS rats. CONCLUSION: We have synthesized a liposomal vasopressin complex that can prolong the serum concentration of vasopressin in a rat model of UHS. Although UHS rats treated with either liposomal vasopressin or vasopressin showed no statistical differences, it would be worthwhile to repeat the experiments with different liposomal compositions.

Monitoring the subcellular localization of doxorubicin in CHO-K1 using MEKC-LIF: liposomal carrier for enhanced drug delivery.

Doxorubicin (DOX) is an extensively used anthracycline that has proven to be effective against a variety of human malignant tumors, such as ovarian or breast cancer. While DOX was administered into cultured cancer cell targets (such as CHO-K1) in either free drug form or in drug carrier-associated form (i.e., DOX encapsulated in the drug delivery carrier), various action of mechanisms for DOX were initiated, among which, it has been long believed that DOX enters the nucleus, interacts with DNA in numerous ways, and finally halts cell proliferation. Aside from its therapeutic effect, regrettably DOX treatment may be accompanied by the occurrence of cardiac and liver toxicity and drug resistance that are attributed from cellular processes involving the parent drug or its metabolites. Liposomal formulation of DOX, known to be capable of attenuating direct uptake of reticuloendothelial system (RES) and prolonging the circulation time in blood, demonstrated reduced toxic side-effects. We herein report the development of a modified MEKC-LIF (Micellar electrokinetic chromatography-Laser-induced fluorescence) method suitable for analyzing DOX in biological samples. The MEKC migration buffer, consisting of 10 mM borate, 100 mM sodium dodecyl sulfate (SDS) (pH 9.3), was found to provide an efficient and stable electrophoretic separation and analysis for DOX. Responses were linear in the range of 11.3-725 ng/mL; the limit of quantitation (LOQ) was found to be 43.1 ng/mL (S/N=10) (equivalent to 74.3 nM) and limit of detection (LOD) was calculated as 6.36 ng/mL (S/N=3) (equivalent to 11.0 nM). This approach was subsequently employed to compare the intracellular accumulation in three subcellular fractions of DOX-treated CHO-K1 cells. These fractions form a pellet at <1400 g, 1400-14000 g, and >14000 g and are enriched in nuclei, organelles (mitochondria and lysosomes), and cytosole components, respectively, resulting from treatment of CHO-K1 cells with 25 µM (equivalent to 14.5 µg/mL) of two DOX formats (in free drug form or liposomal form synthesized in current study) for different periods of time. Our results indicated that the most abundant DOX was found in the nuclear-enriched fraction of cells treated for 12 h and 6 h with free and liposomal DOX, respectively, providing direct evidence to confirm the enhanced efficiency of liposomal carriers in delivering DOX into the nucleus. The observations presented herein suggest that subcellular fractionation followed by liquid-liquid extraction and MEKC-LIF could be a powerful diagnostic tool for monitoring intracellular DOX distribution, which is highly relevant to cytotoxicity studies of anthracycline-type anticancer drugs.

Biofunctionalized phospholipid-capped mesoporous silica nanoshuttles for targeted drug delivery: improved water suspensibility and decreased nonspecific protein binding.

A main challenge in nanobiomedicine is the engineering of nanostructures or nanomaterials that can efficiently encapsulate drugs at high load, cross cell membranes, and controllably release their cargo at target sites. Although mesoporous silica nanoparticles (MSNs) are safe, versatile, and promising carrier materials for targeted drug delivery, their aggregation phenomena under physiological conditions (or salt-containing environments) and their nonspecific binding in protein-containing solutions (or serum) limit their applications in biological science and biomedicine. To address this challenge, we have developed a novel delivery system, termed a nanoshuttle, comprising a nanoscale PEGylated-phospholipid coating and 13-(chlorodimethylsilylmethyl)heptacosane-derivatized MSNs, in which therapeutic or imaging agents may be trapped and ligand-assisted targeted delivery may be achieved through surface functionalization of the phospholipids. As a proof of concept in this study, we selected fluorescein isothiocyanate and folate as the imaging tracer and targeted ligand, respectively. Relative to the bare MSNs, the lipid-capped MSNs exhibited superior suspensibility in phosphate-buffered saline and much lower nonspecific binding in vitro. Furthermore, enhanced specific cellular uptake by Hela cells occurred after administering the folate-sensitized phospholipid-capped MSNs. Our results suggest that these highly versatile multifunctional MSNs are promising vectors for nanomedicine applications.

Ultrasensitive electrochemical detection of biotin using electrically addressable site-oriented antibody immobilization approach via aminophenyl boronic acid.

A common approach towards developing immunoassays is to attach antibodies onto the surfaces of assay devices via a solid support. When directly adsorbed onto surfaces, however, antibodies generally adopt random orientations and therefore, often fail to exhibit their immunoaffinity. To preserve the antigen-binding activity of antibodies, there is an urgent need to develop specific and novel linking chemistries for attaching the antibodies to the solid surfaces in an oriented manner. In this paper, we report 2 alternative immobilization methods to enhance the orientation of antibodies onto screen-printed graphite electrodes (SPGEs). The first approach involves the deposition of gold nanoparticles (AuNPs) onto the SPGE and subsequent adsorption of monovalent half-antibody (monoAb) fragments of the anti-biotin antibody via Au-thiol bonds. For the second technique, we exploited the affinity of boronic acid towards sugar moieties by preparing a boronic acid-presenting SPGE surface to interact with the carbohydrate unit of this anti-biotin antibody. Using such approaches, we prepared an ultrasensitive electrochemical immunosensor, possessing a maximized epitope density, for the detection of biotin at concentrations as low as 0.19pg.

Disposable electrochemical immunosensor for carcinoembryonic antigen using ferrocene liposomes and MWCNT screen-printed electrode.

Disposable electrochemical immunosensor for the detection of carcinoembryonic antigen (CEA) in saliva and serum was developed. Monoclonal anti-CEA antibodies (alphaCEA) were covalently immobilized on polyethyleneimine wrapped multiwalled carbon nanotubes screen-printed electrode. A sandwich immunoassay was performed with CEA and alphaCEA tagged ferrocene carboxylic acid encapsulated liposomes (alphaCEA-FCL). The square wave voltammetry (SWV) was employed to analyze faradic redox responses of the released ferrocene carboxylic acid from the immunoconjugated liposomes on the electrode surface. The magnitude of the SWV peak current was directly related to the concentration of CEA. The calibration curve for CEA concentration was in the range of 5 x 10(-12) to 5 x 10(-7)gmL(-1) with a detection limit of 1 x 10(-12)gmL(-1) (S/N=3). This method provides a high precise and sensitive determination of CEA in human blood serum and saliva samples.

Application of ganglioside-sensitized liposomes in a flow injection immunoanalytical system for the determination of cholera toxin.

Cholera, an acute infectious disease associated with water and seafood contamination, is caused by the bacterium Vibrio cholerae, which lives and colonizes in the small intestine and secretes cholera toxin (CT), a causative agent for diarrhea in humans. Based on earlier lateral flow assays, a flow injection liposome immunoanalysis (FILIA) system with excellent sensitivity was developed in this study for the determination of CT at zeptomole levels. Ganglioside (GM1), found to have specific affinity toward CT, was inserted into the phospholipid bilayer during the liposome synthesis. These GM1-sensitized, sulforhodamine B (SRB) dye-entrapping liposomes were used as probes in the FILIA system. Anti-CT antibodies were immobilized in its microcapillary. CT was detected by the formation of a sandwich complex between the immobilized antibody and GM1 liposomes. During the assay, the sample was introduced first into the column, and then liposomes were injected to bind to all CT captured by the antibody in the microcapillary. Subsequently, the SRB dye molecules were released from the bound liposomes via the addition of the detergent octyl glucopyranoside. The released dye molecules were transported to a flow-through fluorescence detector for quantification. The FILIA system was optimized with respect to flow rate, antibody concentration, liposome concentration, and injected sample volume. The calibration curve for CT had a linear range of 10-16 to 10-14 g mL-1. The detection limit of this immunosensor was 6.6 x 10(-17) g mL-1 in 200-microL samples (equivalent to 13 ag or 1.1 zmol).

Electrochemical immunosensor for cholera toxin using liposomes and poly(3,4-ethylenedioxythiophene)-coated carbon nanotubes.

A sensitive method for the detection of cholera toxin (CT) using an electrochemical immunosensor with liposomic magnification followed by adsorptive square-wave stripping voltammetry is described. Potassium ferrocyanide-encapsulated and ganglioside (GM1)-functionalized liposomes act as highly specific recognition labels for the amplified detection of cholera toxin. The sensing interface consists of monoclonal antibody against the B subunit of CT that is linked to poly(3,4-ethylenedioxythiophene) coated on Nafion-supported multiwalled carbon nanotube caste film on a glassy carbon electrode. The CT is detected by a "sandwich-type" assay on the electronic transducers, where the toxin is first bound to the anti-CT antibody and then to the GM1-functionalized liposome. The potassium ferrocyanide molecules are released from the bounded liposomes on the electrode by lyses with methanolic solution of Triton X-100. The released electroactive marker is measured by adsorptive square-wave stripping voltammetry. The sandwich assay provides the amplification route for the detection of the CT present in ultratrace levels. The calibration curve for CT had a linear range of 10(-14)-10(-7)g mL(-1). The detection limit of this immunosensor was 10(-16) g of cholera toxin (equivalent to 100 microL of 10(-15) g mL(-1)).

Application of a liposomal bioluminescent label in the development of a flow injection immunoanalytical system.

A flow injection liposome immunoanalytical system was developed using biotin as the model analyte and liposomal aequorin as the label. Aequorin is a photoprotein isolated from luminescent jellyfish (notably Aequorea victoria) and other marine organisms that emits visible light in the presence of a trace of Ca2+. Because of this characteristic, the aequorin complex has been used as an intracellular Ca2+ indicator. In this study, a bioluminescent label was designed by encapsulating aequorin inside the cavity of the liposome, whose outer surface was sensitized with the analyte of interest. The analyte-tagged liposomal aequorin was employed in the development of a heterogeneous bioluminescence immunoassay for the model analyte biotin. The proposed immunoassay was based on the competition between the model biotin and aequorin-encapsulating, biotin-tagged liposomes for a limited number of anti-biotin antibody-binding sites. The anti-biotin antibodies were immobilized via protein A in a capillary immunoreactor column, and 30% MeOH was used for the regeneration of antibody-binding sites after each measurement, which allowed the immunoreactor to be used for up to 50 sequential sample injections without any loss of reactivity. The calibration curve for biotin in Tris-buffered saline solution had a linear range of 1 x 10(-11)-1 x 10(-3) M. The detection limit of the assay was 50 pg (equivalent to 200-microL injection of 1 x 10(-9) M). This study demonstrates the procedures for the encapsulation of the photoprotein aequorin into the liposome, which can be used as a sensitive label in bioluminescence immunoassays for biotin or in other applications.