Development of a quantum dot-based fluorescent immunoassay for progesterone determination in bovine milk

The use of semiconductor quantum dots (QDs) as fluorescent labels to develop a competitive immunoassay for sensitive detection and quantification of progesterone in cow's milk is described. Colloidal water-soluble CdSe/ZnS QDs are conjugated to an antigen derivative (progesterone-BSA conjugate) and a simple methodology is optimised to determine the antigen concentration in the final bioconjugate. The obtained QD-linked antigens were then employed together with unlabelled anti-progesterone monoclonal antibodies, as the biological recognition elements, in the development of the quantitative QDs-based fluorescent immunoassay for progesterone in bovine milk. After optimization, the developed immunoassay proved to cover a progesterone concentration range from 0.3 to 14.5 ng/mL in cow milk. Milk samples were just diluted 10-fold with deionised water and directly analysed with the proposed immunoassay, without additional sample pre-treatment or analyte extraction. The minimum detectable level (IC(10)) of the developed immunoassay turned out to be 0.1 ng/mL of progesterone in bovine milk. The sensitivity (IC(50)) achieved was 2.2 ng/mL with a reproducibility of 3.5% RSD as obtained from the results of the analysis of the triplicate of same samples but in three different days. Applicability of the proposed methodology was evaluated by analyzing cow's milk samples enriched with known concentrations of progesterone and recoveries better than 90% were achieved.     

Direct enzymeimmunoassay of progesterone in bovine milk

A sensitive enzymeimmunoassay has been developed for measuring progesterone in unextracted bovine milk. An N-hydroxysuccinimide ester of 11 alpha -hydroxyprogesterone 11-hemisuccinate has been synthesised and used to form conjugates with beta-galactosidase in buffer at pH 7.0. The degree of incorporation of progesterone into the enzyme was demonstrated using (14C)-labelled steroid and by radioimmunoassay binding inhibition. Standard curves of comparable range and sensitivity to radioimmunoassay were obtained in the presence of whole milk taken from a cow at oestrus. These advances have allowed the development of a simple micro-titre plate enzymeimmunoassay of progesterone in whole milk and will be of particular value in determination of pregnancy, prediction of the day of oestrus and diagnosis of reproductive disorders.     

Development and validation of an enzyme immunoassay for progesterone in bovine milk or blood plasma

We have developed and validated a sensitive, simple and direct (i.e. without extraction) enzyme immunoassay (EIA) system for the measurement of progesterone in bovine milk and blood plasma. Progesterone (P) has been analysed by a microtiterplate EIA, employing polyclonal antibodies against P-7α-carboxyethylthioether-BSA as the antigen. The enzyme used as a label is horseradish peroxidase (HRP) and the chromogen is tetramethylbenzidine (TMB). Sensitivity of the EIA has been greatly improved by introduction of a heterologous tracer, in which progesterone is coupled to HRP at the 6β position. Compared to the radioimmunoassay (RIA) in which the same antiserum has been used, the sensitivity is 20 times greater. The detection limit of the assay is 0.4 pg per well. The working range of the standard curve is 0–20 pg per well (i.e. 0–40 ng per ml), and 50% reduction of the initial binding is obtained with 2.5-5.0 pg. Results can be obtained either by spectrophotometric measurement at 450 nm, or by naked eye. Total time needed for the assay of 40 replicate samples is approximately 3 h. Comparison of the EIA system with a previously validated RIA system gave a regression line EIA = 0.85 RIA + 2.11 (r = 0.93, n = 400 milk samples). Application of the milk-progesterone EIA to pregnancy testing (n = 66) gave an accuracy of 79.6% for positive diagnoses and 100% for negative diagnoses.     

Quantitative analysis of the response of an electrochemical biosensor for progesterone in milk

An electrochemical biosensor for progesterone in cow's milk was developed and used in a competitive immunoassay by Hart et al. (1977, Studies towards a disposable screenprinted amperometric biosensor for progesterone, Biosens. Bioelectron. 12, 1113-1121). The sensor was fabricated by depositing anti-progesterone monoclonal antibody (mAb) onto screen-printed carbon electrodes (SPCEs) which were coated with rabbit anti-sheep IgG (rIgG). This sensor was operated following the steps of competitive binding between sample and conjugate (alkaline-phosphatase-labelled progesterone) for the immobilised mAb sites and measurements of an amperometric signal in the presence of p-nitrophenylphosphate using either colorimetric assays or cyclic voltammetry. The hook effect of the progesterone biosensor was found in the concentration range of milk progesterone between 0 and 5 ng/ml when the sensor was fabricated using a loading of 25 ng rIgG per electrode of a diameter of 3 mm and a 1/50 dilution of mAb. A computer model has been developed in this study to simulate the operation of this progesterone biosensor with consideration of the fabrication processes. This paper presents the results of validating the computer model and the model has predicted the hook effect as observed in tests. The model thus reveals that the hook effect is determined by the total number of binding sites available and the rates of labelled and unlabelled progesterone diffusing towards the sensor surface and the binding rates.     

A sensitive photosystem II-based biosensor for detection of a class of herbicides

We have developed a biosensor for the detection of residual triazine-, urea- and phenolic-type herbicides, using isolated photosystem II (PSII) particles from the thermophilic cyanobacterium, Synechococcus elongatus, as biosensing elements. The herbicide detection was based on the fact that, in the presence of artificial electron acceptors, the light-induced electron transfer through isolated PSII particles is accompanied by the release of oxygen, which is inhibited by the herbicide in a concentration-dependent manner. The PSII particles were immobilized between dialysis membrane and the Teflon membrane of the Clark oxygen electrode mounted in a flow cell that was illuminated. Inclusion of the antibiotic chloramphenicol in the reaction mixtures prolonged, by 50%, the lifetime of the biosensor. The use of highly active PSII particles in combination with the flow system resulted in a reusable herbicide biosensor with good stability (50% of initial activity was still remaining after 35-h use at 25 degrees C) and high sensitivity (detection limit for diuron was 5 x 10(-10) M).     

Development of a sensitive enzymeimmunoassay (EIA) for progesterone determination in unextracted bovine plasma using the second antibody technique

A simple direct enzymeimmunoassay (EIA) on microtiter plates for plasma progesterone using the second antibody coating technique and horseradish peroxidase (HRP) as the enzyme label (EIA-HRP) is described and compared with an identical EIA procedure which employed alkaline phosphatase (AP) as the enzyme label (EIA-AP). The assays used antiserum raised against progesterone-7-carboxyethlthioether-BSA in rabbits. Both systems were further compared with the conventional direct progesterone radioimmunoassay (RIA) in regular use. The enzymes HRP and AP were coupled to progesterone-6 beta-hydroxy-hemisuccinate by a mixed anhydride method. While the precision of EIA-HRP was comparable to RIA, the sensitivity in terms of the lowest detection limit obtained in EIA-HRP was about 10 times better than that seen in RIA. Progesterone estimates from plasma samples in EIA-HRP showed good correlation (r = 0.94) with the RIA values and the levels measured in the two systems were identical. Progesterone estimates from plasma samples in EIA-AP were at least three times higher than those obtained by either EIA-HRP or RIA. Thus, only the EIA-HRP but not the EIA-AP was suitable for the reliable direct measurement of progesterone in plasma.     

核酸适体生物传感器快速检测牛奶中抗生素

为了现场快速检测牛奶溶液中四环素类抗生素的含量,对电极进行阳极氧化、氨基化、活化等一系列处理,将四环素核酸适体作为识别分子,共价固定于玻碳电极表面,构建出新型核酸适体生物传感器。传感器可以特异性结合标本中四环素类抗生素,并产生电化学信号,强度与抗生素浓度相关。其最低检测量是1μg/L,检测时间为5 m in。     

Immobilization of bovine serum albumin as a sensitive biosensor for the detection of trace lead ion in solution by piezoelectric quartz crystal impedance

A biosensor based on bovine serum albumin (BSA) for the detection of lead (Pb(2+)) ion was developed and characterized. BSA was immobilized onto a colloidal Au-modified piezoelectric quartz crystal (PQC) as a biosensor for the detection of Pb(2+) ion by piezoelectric quartz crystal impedance (PQCI). Calibration curves for the quantification of Pb(2+) ion showed excellent linearity throughout the concentration range from 1.0 x 10(-7) to 3.0 x 10(-9)mol/L. The interaction between the Pb(2+) ions and the sensor chip is influenced significantly by the pH of the reaction buffer, and the optimal pH for the experiment was 5.4. Under the optimal conditions, the detection limit of 1.0 x 10(-9)mol/L for Pb(2+) was obtained. Kinetic parameters of the Pb(2+)-BSA interactions were also determined by using this chip. The sensor chip could be regenerated for use by dipping in the ethylenediaminetetraacetic acid (EDTA) solution for approximately 2h, and the chip was used to detect Pb(2+) ion for eight times without obvious signal attenuation.     

Development of a sensitive enzymeimmunoassay for oxytocin determination in bovine plasma

A highly sensitive and specific second antibody enzymeimmunoassay (EIA) on microtiterplates for oxytocin determination in bovine plasma using the biotin–streptavidin amplification system was developed. Biotin was coupled to oxytocin and used to bridge between streptavidin-peroxidase and the immobilized oxytocin antiserum in the competitive assay. The assay was carried out directly in 200 μl of bovine plasma. Oxytocin standards prepared in hormone-free plasma were used. The sensitivity of the assay was 0.25 pg/well which corresponded to 1.25 pg/ml plasma; the 50% relative binding was seen at 2.8 pg/well. Plasma volumes for the assay ranging from 50 to 200 μl did not influence the shape of the oxytocin standard curve; however a distinct drop in the OD₄₅₀ was observed with higher plasma volumes. The oxytocin antiserum used in the assay showed no significant cross-reaction with other octapeptides tested. The assay was compared with a radioimmunoassay (RIA) procedure employing prior solvent extraction of plasma samples. The oxytocin concentrations assayed by EIA and RIA in plasma samples obtained from four cows before, during and after milking were highly correlated and very similar (r=0.97). Hence the assay developed offers an attractive alternative to the RIA since no prior laborious plasma extraction is needed. Further, the assay has the distinct advantage of being non-radioactive in nature.     

纳米多孔硅阻抗生物传感器的研究

构建了1种基于多孔硅材料,无需标记的纳米生物传感器,用于对牛血清白蛋白分子进行检测。通过对多孔硅进行表面处理,形成氧化膜,将抗体固定到多孔硅氧化层表面。在磷酸盐缓冲液中,通过电化学检测系统检测加入抗原后,传感器的阻抗值的变化。磷酸缓冲液（PBS）/抗体-氧化层/硅,构成电解液/绝缘层/半导体（electro-lyte-insulator-semiconductor,EIS）结构。传感器的线性检测范围为0.01～0.27mg/mL,检测限为0.01mg/mL。     

Rapid and sensitive galactose oxidase-peroxidase biosensor for galactose detection with prolonged stability

Galactose oxidase was co-immobilised with peroxidase by drop-coating on the surface of a graphite electrode with adsorbed ferrocene. This system offers low detection limit – 0.51 mg galactose l^–1 and fast response: 44 s in phosphate buffer or 25 s in borate buffer. Optimal working potential for galactose detection was 150 mV vs. SCE (saturated calomel electrode) with optimal pH of 7.85. The storage stability was highly improved, more than 12 times in comparison to control without stabilisers, by addition of DEAE-dextran and inositol. During repeated assays for 5.25 h, signal dropped only to 95% of original one. The response was linear in phosphate buffer in the range 1–110 mg l^–1, while in borate buffer linear range was extended to 3–210 mg l^–1 because of chelating effect of borate.     

Estrogen and progesterone concentrations in bovine milk during the estrous cycle

Estrogen and progesterone concentrations in milk during the estrous cycle were estimated in 18 normally cycling Holstein dairy cows. The estrogen and progesterone concentrations in milk during the estrous cycle followed the pattern described for them in blood in the corresponding period. During most of the estrous cycle, estrogen concentration remained at approximately 200 pg/ml and reached a proestrous peak of 360 +/- 127 pg/ml on day 19. The progesterone concentration in milk during the estrous cycle increased to a peak on day 13 (45.5 +/- 6.6 ng/ml) and thereafter declined towards estrus. Estrus detection/prediction based on milk progesterone concentrations appears feasible in view of the significant differences in milk progesterone concentrations between the early luteal (post-ovulatory), luteal and rapid follicular growth periods of the estrous cycle.     

Highly sensitive gold nanoparticles biosensor chips modified with a self-assembled bilayer for detection of Con A

In this paper, an improved method for detection of Concanavalin A (Con A) with label-free optical biosensors is reported. 1-Dodecanethiol (DDT) was self-assembled onto gold nanoparticles which were deposited on glass slides, and then glycolipid molecules were inserted into dodecanethiol by physical interactions only. The recognition between Con A and carbohydrate was observed by UV-vis spectrophotometry. The absorption spectrum shifted when Con A was bound to the sugar residues of glycolipids immobilized onto nanogold slides, while almost no spectrum change was observed when another nonspecific protein molecule met the nanogold slides. The self-assembled bilayer on nanogold substrates had very high sensitivity for Con A, the minimum detection concentration of Con A can be down to 0.1 nM. In addition to the ultra sensitivity for investigating carbohydrate-lectin interaction, the self-assembled bilayer structure, is expected to replace many receptors which require time-consuming organic syntheses for the fixation to the transducer. The simplicity and sensitivity of this biosensor architecture once again show the prospect of nanogold application in biosensor.     

Effects of sample handling temperatures on bovine skim milk progesterone concentrations

The effects of incubation of whole milk at various temperatures and times on the concentration of progesterone in the skim milk fraction was determined. For the study, milk samples were collected from 10 pregnant Holstein cows. The milk from each cow was transferred to culture tubes to provide 32 replicates of 3 ml volume. To begin the incubation study, all samples were placed in a 37 degrees C water bath for 4 h. The end of this incubation was designated as time 0 and a sample from each cow was centrifuged to harvest skim milk. At time 0, samples from each cow were divided among incubation temperatures of 0 degrees, 4 degrees, 20 degrees and 37 degrees C. Samples were removed from each incubation group at 30, 60, 90 and 120 min. After 120 min, all remaining samples were returned to the 37 degrees C incubation and skim milk was collected at 30, 60 and 90 min. Progesterone was measured in skim milk by radioimmunoassay. The mean +/- SE concentration of progesterone in skim milk at time 0 was 10.9 +/- 1.1 nmol/L. The mean concentration of progesterone in skim milk was higher (P < 0.05) in all samples incubated at 0 degrees and 4 degrees C, with incremental increases ranging from 34% to 67% above time 0. Progesterone in skim milk returned to time 0 concentrations in milk samples transferred from 0 degrees or 4 degrees C to 37 degrees C. There was no change in skim milk progesterone in whole milk samples incubated at 20 degrees or 37 degrees C. From this study, it can be concluded that the concentration of progesterone in skim milk is temperature dependent. Inconsistency in handling whole milk samples can have a profound effect in the concentration of progesterone on skim milk. The temperature-dependent effect was reversible and may be related to solubility of progesterone in milk fat.     

Rapid and sensitive biosensor for Salmonella

The rapid and sensitive detection of Salmonella typhymurium based on the use of a polyclonal antibody immobilized by the Langmuir-Blodgett method on the surface of a quartz crystal acoustic wave device was demonstrated. The binding of bacteria to the surface changed the crystal resonance parameters; these were quantified by the output voltage of the sensor instrumentation. The sensor had a lower detection limit of a few hundred cells/ml, and a response time of < 100 s over the range of 10(2)-10(10) cells/ml. The sensor response was linear between bacterial concentrations of 10(2)-10(7) cells/ml, with a sensitivity of 18 mV/decade. The binding of bacteria was specific with two binding sites needed to bind a single cell. The sensors preserve approximately 75% of their sensitivity over a period of 32 days.     

Highly sensitive single polyaniline nanowire biosensor for the detection of immunoglobulin G and myoglobin

A single polyaniline (PANI) nanowire-based biosensor was established to detect immunoglobulin G (IgG) and myoglobin (Myo), which is one of the cardiac biomarkers. The single PANI nanowires were fabricated via an electrochemical growth method, in which single nanowires were formed between a pair of patterned electrodes. The single PANI nanowires were functionalized with monoclonal antibodies (mAbs) of IgG or Myo via a surface immobilization method, using 1-ethyl-3-(3-dimethyaminopropyl) carbodiimide (EDC), and N-hydroxysuccinimde (NHS). The functionalization was then verified by Raman spectroscopy and fluorescence microscopy. The target proteins of IgG and Myo were detected by measuring the conductance change of functionalized single PANI nanowires owing to the capturing of target proteins by mAbs. The detection limit was found to be 3 ng/mL for IgG and 1.4 ng/mL for Myo. No response was observed when single nanowires were exposed to a non-specific protein, demonstrating excellent specificity to expected target detection. Together with the fast response time (a few seconds), high sensitivity, and good specificity, this single PANI nanowire-based biosensor shows great promise in the detection of cardiac markers and other proteins.     

Direct radioimmunoassay of progesterone in milk using a radioiodinated progesterone derivative

A heterologous radioimmunoassay technique which allows the measurement of progesterone in whole milk, without extraction is described. An antiserum against progesterone-12α-succinyl-BSA and progesterone-11α-(2′-methyl)succinyl-¹²⁵I-thyramine was used. When applied in pregnancy detection on day 20–22, progesterone levels of up to 6 ng/ml or above 8 ng/ml were indicative of an unsuccessful or a successful insemination, respectively.The sensitivity of the assay was 0.8 ng/ml, and the values found in cow milk ranged from 0.8 to 50 ng/ml.