Magnetic bead based immunoassay for enumeration of CD4 T lymphocytes on a microfluidic device

Human immunodeficiency virus (HIV) diagnostics are urgently needed in resource-scarce settings. Monitoring of HIV-infected patients requires accurate counting of CD4⁺ T lymphocytes. However, the current methods for enumeration of CD4⁺ T lymphocytes are of high cost, technically complex and time-consuming. In this paper, we developed a simple, rapid and inexpensive one-step immunomagnetic method for separating and counting CD4⁺ T lymphocytes on microfluidic devices with enlarged reaction chambers. CD4⁺ T lymphocytes were successfully separated and captured from the cell suspension obtained from mouse thymus. CD4 counts were determined under an optical microscope in a rapid and simple format. In order to acquire the maximum efficiency of cell capture, relative parameters were investigated, including section area of the reaction chamber and injection flow rate of the cell suspension. The enlarged reaction chamber with two symmetrical cone-shaped ends was helpful for cell capture, and the maximum capability of captured CD4⁺ T lymphocytes was about 700 cells μL⁻¹. Our investigations avoided the complex sample pre-treatment, and the entire analysis time was significantly reduced to 15 min. This CD4 counting microdevice had the potential to reduce the cost for HIV diagnosis in resource-limited settings.     

Design on head-to-tail directly linked homogeneous and heterogeneous cyclodextrin dimers and their evaluation of hydrophobic cavity

Covalent directly head-to-tail linked homogeneous and heterogeneous cyclodextrin (CD) dimers were synthesized, and that the reaction of 6-tosylated α-, β-, or γ-CD with a β-CD mono-oxyanion linked the second CD to the secondary hydroxyl side of β-CD was demonstrated. Moreover, deprotonation of α- and γ-CD using NaOH gave corresponding mono-oxyanions, which reacted with the 6-tosylated CDs to produce the CD dimers. The binding of the dimers to sodium 6-(4-tert-butylaniline)-2-naphthalenesulfonate (BNS) was investigated. The binding constant of the ⁶β–²β-CD dimer with BNS was estimated as 3.2 × 10⁶ M⁻¹, about 10² times larger than that of β-CD monomer.     

A rapid sandwich immunoassay for human fetuin A using agarose-3-aminopropyltriethoxysilane modified microtiter plate

A rapid sandwich immunoassay (IA) with enhanced signal response for human fetuin A (HFA) was developed by modifying the surface of a KOH-treated polystyrene microtiter plate (MTP) with agarose and 3-aminopropyltriethoxysilane (APTES). The agarose-APTES complex binds covalently to the hydroxyl moiety of the MTP plate to serve as a binding platform for bioconjugation of EDC-activated anti-HFA antibody (Ab) via carbodiimide coupling. The one-step kinetics-based sandwich enzyme-linked immunosorbent assay (ELISA) enabled the detection of HFA in 30 min with a limit of detection (LOD) and a linear range of 0.02 ng mL⁻¹ and 1–243 ng mL⁻¹, respectively. It detected HFA spiked in diluted human whole blood and serum, and HFA in ethylenediaminetetraacetic acid (EDTA)-plasma of patients with high precision similar to that of conventional ELISA. The anti-HFA Ab-bound agarose-functionalized MTPs retained their functional activity after 6 weeks of storage in 0.1 M PBS, pH 7.4 at 4 °C.     

Highly sensitive and reproducible cyclodextrin-modified gold electrodes for probing trace lead in blood

A highly sensitive and reproducible lead sensor based on a cyclodextrin-modified gold electrode was created. A self-assembled monolayer (SAM) of thiolated β-cyclodextrin (6-(2-mercapto-ethylamino)-6-deoxy-β-cyclodextrin (MEA-β-CD)) was prepared and modified on a gold electrode (MCGE) for specific Pb²⁺-sensing. Thus the mercury-free sensors for Pb²⁺ assay based on MCGE were established. A linear calibration response for Pb²⁺ was found in the range of 1.7 × 10⁻⁸ M to 9.3 × 10⁻⁷ M. The detection limit was 7.1 × 10⁻⁹ M (with S/N > 3), which was 10 times lower than other reported methods of detection Pb²⁺ with CD. The measurement results via this method for real blood samples were well agree with those obtained by ICP-AES, and thus presented a novel strategy in design of specific lead sensors with high sensitivity and stability for analysis of trace Pb²⁺ in real blood samples.     

Synthesis, characterization and antitumor activity of 1,2-disubstituted ferrocenes and cyclodextrin inclusion complexes

Seven different ferrocene derivatives have been tested in vitro against Ehrlich ascites tumor cells. Neither ferrocene nor the monosubstituted derivative N,N-dimethylaminomethylferrocene showed cytotoxic activity (IC₅₀ > 1000 μM for 3 h treatments). Better results were obtained with 1,2-disubstituted derivatives. The IC₅₀ values ranged from 376.6 μM for 1,2-diformylferrocene to 71.2 μM for racemic 2-(N,N-dimethylaminomethyl)ferrocenecarboxamide. The latter derivative was also encapsulated in native β-cyclodextrin (CD), heptakis-2,3,6-tri-O-methyl-β-CD (TRIMEB) and 2-hydroxypropyl-β-CD (HPβCD) to give 1:1 (host:guest) inclusion compounds. The existence of true inclusion complexes in the solid state was confirmed by a combination of powder X-ray diffraction, thermogravimetric analysis, FTIR and ¹³C CP MAS NMR spectroscopy. The IC₅₀ value for the β-CD inclusion compound was identical to that obtained for the nonincluded ferrocene derivative. By contrast, the inclusion compounds comprising TRIMEB and HPβCD yielded IC₅₀ values of 25.2 and 20.0 μM, respectively. No obvious relationship could be established between the redox behavior of the compounds determined by cyclic voltammetry and the biochemical data.     

Ab initio ground and the first excited adiabatic and quasidiabatic potential energy surfaces of H + CO system

Ab initio global adiabatic as well as quasidiabatic potential energy surfaces for the ground and the first excited electronic states of the H⁺ + CO system have been computed as a function of the Jacobi coordinates (R, r, γ) using Dunning’s cc-pVTZ basis set at the internally contracted multi-reference (single and double) configuration interaction level of accuracy. In addition, nonadiabatic coupling matrix elements arising from radial motion, mixing angle and coupling potential have been computed using the ab initio procedure [Simah et al. (1999) [66]] for the purpose of dynamics study. The geometrical properties corresponding to the minimum energy of the bound HCO⁺ and HOC⁺ isomers have been obtained and compared with those predicted by previous theoretical and experimental results. The HCO⁺ has been found to be more stable than the HOC⁺. The minimum energy pathway in the ground electronic state for the isomerization process, HCO⁺ ? HOC⁺ has also been obtained as a function of γ.     

Inclusion of molybdenocene dichloride (Cp2MoCl2) in 2-hydroxypropyl- and trimethyl-β-cyclodextrin: Structural and biological properties

Organometallic-cyclodextrin inclusion compounds were obtained by the treatment of molybdenocene dichloride (Cp₂MoCl₂) with the modified cyclodextrins (CDs) heptakis-2,3,6-tri-O-methyl-β-CD (TRIMEB) and 2-hydroxypropyl-β-CD (HPβCD) in aqueous solution. The products were isolated by liophilisation and characterised in the solid-state by powder XRD, thermogravimetric analysis, Raman and FTIR spectroscopy, and ¹³C CP MAS NMR spectroscopy. The results are consistent with inclusion of Cp₂MoCl₂, rather than hydrolysis products such as [Cp₂Mo(H₂O)X]⁺ (X = Cl, OH) or [Cp₂Mo(H₂O)₂]²⁺. The pure non-included metallocene Cp₂MoCl₂ and its inclusion compounds with unmodified β-CD, TRIMEB and HPβCD were screened for their potential antiproliferative and cytotoxic activity, in both human cancer and healthy cell lines. Inclusion in CD was found to enhance the cytotoxic effect of Cp₂MoCl₂, with the TRIMEB adduct displaying the highest anti-tumour activity, along with the lowest toxicity towards non-neoplastic cells.     

Development and validation of a sensitive enzyme immunoassay for surveillance of Cry1Ab toxin in bovine blood plasma of cows fed Bt-maize (MON810)

The increasing global adoption of genetically modified (GM) plant derivatives in animal feed has provoked a strong demand for an appropriate detection method to evaluate the existence of transgenic protein in animal tissues and animal by-products derived from GM plant fed animals. A highly specific and sensitive sandwich enzyme immunoassay for the surveillance of transgenic Cry1Ab protein from Bt-maize in the blood plasma of cows fed on Bt-maize was developed and validated according to the criteria of EU-Decision 2002/657/EC. The sandwich assay is based on immuno-affinity purified polyclonal antibody raised against Cry1Ab protein in rabbits. Native and biotinylated forms of this antibody served as capture antibody and detection antibody for the ELISA, respectively. Streptavidin-horseradish peroxidase conjugate and TMB substrate provided the means for enzymatic colour development.The immunoassay allowed Cry1Ab protein determination in bovine blood plasma in an analytical range of 0.4-100 ng mL⁻¹ with a decision limit (CCα) of 1.5 ng mL⁻¹ and detection capability (CCβ) of 2.3 ng mL⁻¹. Recoveries ranged from 89 to 106% (mean value of 98%) in spiked plasma.In total, 20 plasma samples from cows (n = 7) fed non-transgenic maize and 24 samples from cows (n = 8) fed transgenic maize (collected before and, after 1 and 2 months of feeding) were investigated for the presence of the Cry1Ab protein. There was no difference amongst both groups (all the samples were below 1.5 ng mL⁻¹; CCα). No plasma sample was positive for the presence of the Cry1Ab protein at CCα and CCβ of the assay.     

Determining the stoichiometry and binding constants of inclusion complexes formed between aromatic compounds and β-cyclodextrin by solid-phase microextraction coupled to high-performance liquid chromatography

The complexation of native β-cyclodextrin (CD) and seven aromatic compounds, namely, phenetole, toluene, m-xylene, naphthalene, biphenyl, fluorene and phenanthrene, has been studied for first time utilizing a solid-phase microextraction (SPME)–high-performance liquid chromatography (HPLC) method. The stoichiometries of the analyte:β-CD complexes were found to be either 1:1 or 1:2. The formation of 1:2 complexes was confirmed for naphthalene, biphenyl, fluorene, and phenanthrene only when utilizing relatively high concentrations of β-CD (up to 6.6 mM). The 1:2 stoichiometries were confirmed using the classical modified Benesi–Hildebrand (BH) method. The calculated binding constants for 1:1 stoichiometries (K₁) using the SPME method varied from 115.3 M⁻¹ for toluene to 3510 M⁻¹ for phenanthrene, whereas the corresponding values to the 1:2 stoichiometries (K₃) varied from 7.30 × 10⁵ M⁻² for biphenyl to 9.03 × 10⁶ M⁻² for naphthalene.     

Spectrofluorimetric study of the interaction of copper(II) and bis-heterocyclictriazene reagent in the presence of beta-cyclodextrin

A new reagent, 1,3-bis(2-benzothiazolyl-diazoamino)benzene (BBTAB), was first synthesized and characterized by elemental analysis, ¹H NMR and IR spectra. The inclusion complex of BBTAB with β-cyclodextrin (β-CD) was formed. BBTAB in the inclusion complex or alone reacts with copper(II) to form chelate complex in a slight basic medium, which results in drastic or slight fluorescence enhancement, respectively. The spectrofluorimetric method of trace amount of copper(II) based on the enhancement of inclusion complex by binding with copper(II) was established. The excitation and emission wavelengths of the BBTAB/β-CD/Cu system are 389 and 480 nm, respectively. Under optimal conditions, a linear response of BBTAB/β-CD to copper(II) is obtained in the range of 3.0 × 10⁻⁷ to 1.0 × 10⁻⁵ mol L⁻¹, and the detection limit is determined to be 1.2 × 10⁻⁸ mol L⁻¹. The method is selective, sensitive and simple, and has been used for the determination of trace copper(II) in water samples with satisfactory results. The possible response mechanism of BBTAB/β-CD or BBTAB to copper(II) and the role of β-CD in the drastic enhancement of fluorescence of BBTAB/β-CD/Cu system have been discussed.     

Electrochemical sensor for cinchonine based on a competitive host-guest complexation

An electrochemical sensor for cinchonine (CCN) using the β-cyclodextrin (β-CD) modified poly(N-acetylaniline) (PAA) electrode has been developed, in which 1,4-hydroquinone (HQ) was chosen as a probe. Complexation of HQ with β-CD modified on the glassy carbon electrode (GCE) was examined by cyclic voltammetry (CV). HQ was included in the cavity of β-CD and reversible voltammograms were observed. In the presence of CCN, a competitive inclusion equilibrium with β-CD was established between HQ and CCN, lowering the peak current of HQ. The decrease in the peak current of HQ is directly proportional to the amount of CCN. Linear calibration plot was obtained over the range from 4.0 × 10⁻⁶ to 8.0 × 10⁻⁵ M with a detection limit (S/N = 3) of 2.0 × 10⁻⁶ M. From the inhibitory effect of CCN on the inclusion of HQ by β-CD, the apparent formation constant of CCN with the immobilized β-CD was estimated. This electrochemical sensor showed excellent sensitivity, repeatability, stability and recovery for the determination of CCN. The response mechanism of the sensor was discussed in detail. The optimum steric configuration of inclusion complex was presented by molecular dynamics simulation.     

Characterization of Endogenous Protoporphyrin IX Induced by δ-Aminolevulinic Acid in Resting and Activated Peripheral Blood Lymphocytes by Four-Color Flow Cytometry

Lymphocytes treated with δ-aminolevulinic acid (ALA) can accumulate the photoactive, fluorescent heme precursor, protoporphyrin IX (PpIX). With visible light illumination, PpIX can be used in photodynamic therapy (ALA-PDT) to kill or functionally alter cells. The aim of this study was to characterize the effects of ALA and ALA-PDT on resting and activated human peripheral blood T lymphocytes. Accumulation of PpIX depends inversely on the rate of its iron-dependent conversion into heme. Activated, replicating lymphocytes have low intracellular iron levels, with corresponding increases in the transferrin receptor (CD71). Thus, we expected activated lymphocytes would preferentially accumulate PpIX. Using four-color flow cytometry, we examined ALA-induced PpIX levels in T-cell subsets of resting and activated human peripheral blood mononuclear cells and the relationship between CD71 and PpIX. Peripheral blood mononuclear cells stimulated by phytohemagglutinin (PHA) were simultaneously phenotyped for PpIX, CD71 and the T-cell markers CD3 and CD4 or CDS. In activated cells treated with 0-6mM ALA for 4 h, PpIX fluorescence was maximal at 1 mM ALA. On a single cell basis, there was a strong correlation between PpIX ac-cumulation and CD71 expression. The ALA-treated, PHA-stimulated, CD71⁺ lymphocytes had an eight-fold greater mean PpIX fluorescence than nonactivated, CD71- cells. Approximately 87% of the CD4* and 85% of the CD8⁺ T cells accumulated PpIX. The PpIX levels of CDS⁺ cells were about 5% greater than CD4⁺ cells. In addition, mixed lymphocyte reaction-stimulated cells treated with ALA accumulated more PpIX than controls. Thus, activated cells preferentially accumulate endogenous PpIX when exogenous ALA is administered. Cytotoxicity studies showed that the majority of the activated cells following ALA-PDT were killed but resting cells were spared. Also, in examining activation markers by flow cytometry the number of cells that were positive for activation markers CD38 or CD71 dramatically decreased after ALA and light treatment in activated populations. The data suggest a role for ALA-PDT as an immunomodulator or photocytotoxic agent targeting activated lymphocytes.     

Electroanalytical method of TCPP and its supramolecular system with cyclodextrins

In this paper, electroanalytical method of tetrakis (4-carboxylphenyl) porphyrin (TCPP) has been established. In a supporting electrolyte of KH₂PO₄-Na₂HPO₄ (pH 7.0), a sensitive second derivative reduction peak of TCPP was found by single-sweep oscillopolarography. The potential peak is −0.70 V (versus SCE).The relationship between peak height and the concentration of TCPP is linear from 1×10⁻⁷ to 2×10⁻⁵ mol l⁻¹, the relative standard deviation (R.S.D.) was 0.41% (n=8), and the recovery of TCPP varied from 95.8-105.4%.The interaction of cyclodextrins (CD) with TCPP in NH₃-NH₄Cl (pH 8.0) has been studied by polarography. The TCPP can form the 1:1 inclusion complex with β-CD, γ-CD, hydroxylpropyl-β-CD, sulfurbutylether-β-CD and trimethyl-β-CD. “Current method” has been used to determine the formation constants of TCPP with five CDs. The result shows that the inclusion ability of hydroxylpropyl-β-CD is very strong. Moreover, modified β-CD has stronger inclusion capacity than native β-CD. The formation constant of TCPP with γ-CD is much greater than that of TCPP with β-CD, because the γ-CD has a bigger cavity that can match with the size of the meso-phenyl of TCPP. The supramolecular data will provide useful information for further application of TCPP.     

Determination of aminoglutethimide enantiomers in pharmaceutical formulations by capillary electrophoresis using methylated-β-cyclodextrin as a chiral selector and computational calculation for their respective inclusion complexes

A capillary electrophoretic method for the separation of the aminoglutethimide (AGT) enantiomers using methylated-β-cyclodextrin (M-β-CD) as chiral selector is described. Several parameters affecting the separation were studied, including the type and concentration of chiral selector, buffer pH, voltage and temperature. Good chiral separation of the racemic mixture was achieved in less than 9 min with resolution factor Rs = 2.1, using a fused-silica capillary and a background electrolyte (BGE) of tris-phosphate buffer solution (50 mmol L⁻¹, pH 3.0) containing 30 mg mL⁻¹ of M-β-CD. The separation was carried out in normal polarity mode at 25 °C, 16 kV and using hydrostatic injection. Acceptable validation criteria for selectivity, linearity, precision, and accuracy/recovery were included. The proposed method was successfully applied to the assay of AGT enantiomers in pharmaceutical formulations. The computational calculations for the inclusion complexes of the R- and S-AGT-M-β-CD rationalized the reasons for the different migration times between the AGT enantiomers.     

Electrochemical determination of L-phenylalanine at polyaniline modified carbon electrode based on β-cyclodextrin incorporated carbon nanotube composite material and imprinted sol-gel film

A sensitive and selective electrochemical sensor based on a polyaniline modified carbon electrode for the determination of l-phenylalanine has been proposed by utilizing β-cyclodextrin (β-CD) incorporated multi-walled carbon nanotube (MWNT) and imprinted sol-gel film. The electrochemical behavior of the sensor towards l-phenylalanine was investigated by cyclic voltammetry (CV), differential pulse voltammetry (DPV), and amperometric i-t curve. The surface morphologies of layer-by-layer assembly electrodes were displayed by scanning electron microscope (SEM). The response mechanism of the imprinted sensor for l-phenylalanine was based on the inclusion interaction of β-CD and molecular recognition capacity of the imprinted film for l-phenylalanine. A linear calibration plot was obtained covering the concentration range from 5.0 × 10⁻⁷ to 1.0 × 10⁻⁴ mol L⁻¹ with a detection limit of 1.0 × 10⁻⁹ mol L⁻¹. With excellent sensitivity, selectivity, stability, reproducibility and recovery, the electrochemical imprinted sensor was used to detect l-phenylalanine in blood plasma samples successfully.     

Determination of mercury species by the diffusive gradient in thin film technique and liquid chromatography – atomic fluorescence spectrometry after microwave extraction

A diffusive gradient in thin films technique (DGT) was combined with liquid chromatography (LC) and cold vapor atomic fluorescence spectrometry (CV-AFS) for the simultaneous quantification of four mercury species (Hg²⁺, CH₃Hg⁺, C₂H₅Hg⁺, and C₆H₅Hg⁺). After diffusion through an agarose diffusive layer, the mercury species were accumulated in resin gels containing thiol-functionalized ion-exchange resins (Duolite GT73, and Ambersep GT74). A microwave-assisted extraction (MAE) in the presence of 6 M HCl and 5 M HCl (55 °C, 15 min) was used for isolation of mercury species from Ambersep and Duolite resin gels, respectively. The extraction efficiency was higher than 95.0% (RSD 3.5%). The mercury species were separated with a mobile phase containing 6.2% methanol + 0.05% 2-mercaptoethanol + 0.02 M ammonium acetate with a stepwise increase of methanol content up to 80% in the 16th min on a Zorbax C18 reverse phase column. The LODs of DGT–MAE–LC–CV-AFS method were 38 ng L⁻¹ for CH₃Hg⁺, 13 ng L⁻¹ for Hg²⁺, 34 ng L⁻¹ for C₂H₅Hg⁺ and 30 ng L⁻¹ for C₆H₅Hg⁺ for 24 h DGT accumulation at 25 °C.     

Development of a methodology for the simultaneous determination of inorganic and organolead compounds using supercritical fluid extraction followed by gas chromatography-mass spectrometry and its application to environmental matrices

A method for the extraction of triethyl lead (TEL⁺), trimethyl lead (TML⁺), and Pb²⁺ from sand was developed using supercritical modified CO₂-CH₃OH extraction and in situ complexation with sodium diethyldithiocarbamate (NaDDTC) using a 2⁵ factorial exploratory design is described. The screened variables were (i) pressure (69-193 bar), (ii) temperature (40-150 °C), (iii) ligand amount (0-100 mg), (iv) methanol volume (0.0-0.5 mL) and (v) static time (0-45 min). The optimum extraction conditions found were as follow: pressure, 193 bar; temperature, 40 °C; amount of NaDDTC, 100 mg; methanol volume, 0.5 mL; static time 45 min; and CO₂ flow rate, 1 mL min⁻¹. Under these conditions the following recoveries were obtained (TML⁺ 97 ± 2%, TEL⁺ 70 ± 5%, and Pb²⁺ 100 ± 4%). The presence of NaDDTC is not necessary for the extraction of TML⁺ and TEL⁺, but it is a very significative parameter for Pb²⁺. A second experimental design 2² + star for temperature and pressure was realized, but the results were not better than those of the first model. SFE extract derivatization was achieved with pentylmagnesium bromide, and target analyte determination was carried out by gas chromatography-mass spectrometry. Detection limits in the full-scan mode were 4, 10, and 39 pg as lead for TMPeL, TEPeL and PbPe₄, respectively. The method was validated with urban dust containing TML⁺ (CRM 605. Pb 7.9 ±1.2 μg kg⁻¹) and river sediment containing inorganic lead (GBW08301. Pb 79.0 ± 12.0 mg kg⁻¹) as reference materials. The proposed method was applied to lead analysis in sand collected from an oil-polluted beach in Chile.     

Assemblies of brilliant cresyl violet to DNA in the presence of γ-cyclodextrin

The interactions of brilliant cresyl violet (BCV) with herring sperm DNA in γ-cyclodextrin (γ-CD) supramolecular system were studied by UV-vis absorption spectroscopy and cyclic voltammetry (CV). Both UV-vis absorption and CV data show that the interaction of BCV with DNA depends on the concentration ratio of BCV to DNA (R), the initial concentration of BCV and γ-CD. The binding constants of BCV monomer, (BCV)₂ dimer and (BCV)₂-γ-CD inclusion complex with DNA are 1.64 × 10⁵, 2.56 × 10⁴ and 2.32 × 10³ M⁻¹, respectively. It was observed that γ-CD can affect the interactive mode of BCV with DNA. If R is larger than 0.5, the (BCV)₂-γ-CD inclusion complex will retain intact and bind to DNA via the electrostatic attraction forces. By contrast, when R is smaller than 0.5, the inclusion complex will be partially dissociated and the free BCV monomer is intercalated into the double-helix structure of DNA attributing to the more favorable microenvironment of DNA for the BCV monomer. Our work postulates the importance of the initial concentration of dye and host molecule on the interaction of dye with DNA in living bodies.     

Detecting interferon-gamma release from human CD4 T-cells using surface plasmon resonance

Cytokine secretion by leukocytes is an important indicator of immune response to pathogens and therefore has significant implications in disease diagnostics. Given heterogeneity of leukocyte subsets and the ability of multiple cell subsets to secrete the same cytokines, connecting cytokine production to a specific leukocyte subset is a distinct challenge. In the present paper we describe a strategy combining antibody (Ab)-based affinity cell separation and surface plasmon resonance (SPR) for capturing human CD4 T-cells and for label-free detection of cell-secreted interferon (IFN)-γ – an important inflammatory cytokine. Human blood was introduced into a flow chamber modified with anti-CD4 Abs resulting in capture of CD4⁺ T-cells. After mitogenic activation of cells inside the flow chamber, culture medium was routed onto an SPR chip modified with monoclonal IFN-γ Abs. SPR signal observed in this experiment correlated with cytokine production by T-cells. The strategy of combining SPR detection with cell purification may be used in the future for label-free, sensitive detection of multiple cytokines or proteins secreted by the desired cell subset.     

Biosensor system for continuous glucose monitoring in fish

A biosensor system was developed for continuous estimation of blood glucose in fish. Because it is difficult to measure blood components in real-time due to decreased sensor output resulting from blood coagulation and coalescing blood proteins at the sensor placement site, we used the eyeball scleral interstitial fluid (EISF) as the site of sensor implantation. Evaluation of the relationship between EISF and blood glucose concentrations revealed that the blood glucose concentration correlated closely with the EISF glucose concentration (y = 2.2996 + 0.9438x, R = 0.960, n = 112). To take advantage of the close correlation between blood and EISF glucose, we prepared a needle-type enzyme sensor for implantation in the fish sclera using a flexible wire electrode. The sensor provided a rapid response, good linearity, and reproducibility. Continuous glucose monitoring could be carried out by implanting this needle-type glucose sensor onto the eye. The findings indicated that the glucose concentration increased with sensor output current over time, and that changes in the blood glucose were continuously reflected in the EISF. The glucose concentration was estimated based on the one-point or two-point calibration methods. The two-point calibration method yielded the most accurate glucose monitoring (blood glucose range of 70-420 mg dL⁻¹) over 160 min. Sensor-estimated glucose and whole blood glucose values were highly correlated (y = 0.4401 + 0.8656x, R = 0.958).