Undiluted Blood Serum Research Articles

Highly efficient detection of single fluorophores in blood serum samples with high autofluorescence.

Single molecule detection (SMD) is usually performed on surface-immobilized molecules with diffraction-limited observation volumes, typically with confocal optics to suppress background from the sample and instrument. In this paper we consider the more difficult task of detecting single fluorophores in the presence of a substantial fluorescence background. We determined that for a readily accessible macroscopic observation volume of 1 pL that the background from undiluted blood serum was approximately equal to 2700 Cy5 molecules, and the background from whole blood equal to about 14 000 Cy5 molecules in whole blood. These high backgrounds appear to preclude the possibility of SMD of Cy5 molecules. However, we show that the signal-to-noise ratio (SNR) in high background samples can be increased dramatically by reduction of the observed volume. We were able to detect single surface-bound Cy5-labeled DNA (Cy5-DNA) oligomers in diluted blood serum with an SNR near 40. We also examined freely diffusing Cy5-DNA in blood serum. The data showed that single Cy5-DNA molecules could be detected even in the undiluted serum. We further investigated the SNR on silver island films. We found that the fluorescence signal was greatly enhanced in the presence of metallic nanostructures showing a larger SNR in the application tested. These results suggest the possibility of clinical assays based on SMD in blood serum and possibly whole blood. Increased SNR near metallic nanostructure could probably overcome the need for diffraction-limited volumes and enhance our ability to do in situ SMD.

Continuous, Real-Time Monitoring of Cocaine in Undiluted Blood Serum via a Microfluidic, Electrochemical Aptamer-Based Sensor

The development of a biosensor system capable of continuous, real-time measurement of small-molecule analytes directly in complex, unprocessed aqueous samples has been a significant challenge, and successful implementation has been achieved for only a limited number of targets. Toward a general solution to this problem, we report here the Microfluidic Electrochemical Aptamer-based Sensor (MECAS) chip wherein we integrate target-specific DNA aptamers that fold, and thus generate an electrochemical signal, in response to the analyte with a microfluidic detection system. As a model, we demonstrate the continuous, real-time (approximately 1 min time resolution) detection of the small-molecule drug cocaine at near physiological, low micromolar concentrations directly in undiluted, otherwise unmodified blood serum. We believe our approach of integrating folding-based electrochemical sensors with miniaturized detection systems may lay the groundwork for the real-time, point-of-care detection of a wide variety of molecular targets.

Combination of aptamer with gold nanoparticles for electrochemical signal amplification: Application to sensitive detection of platelet-derived growth factor

In this paper, we report a novel electrochemical detection approach for platelet-derived growth factor (PDGF) via “sandwich” structure and gold nanoparticles (Au-NPs) mediated amplification technique. The “sandwich” structure is fabricated based on the fact that PDGF has two aptamer-binding sites, which makes it possible for one PDGF molecule to connect with two aptamers simultaneously. It is found that this electrochemical system with “sandwich” structure and Au-NPs can significantly amplify the signal of electrochemical probe [Ru(NH 3) 5Cl] 2+ for PDGF detection, and thus increase the detection sensitivity significantly. As a result, this PDGF detection approach obtains an extraordinarily low detection limit of 1 × 10 −14 M for purified samples, 1 × 10 −12 M for contaminated-ridden samples or undiluted blood serum. This detection approach can also exhibit good stability and excellent specificity.

Determination of selenium in blood serum by ICP-OES including an on-line wet digestion and Se-hydride formation procedure

An analytical procedure for the determination of Se, as well as of other trace elements of clinical interest, in undiluted blood serum samples is presented. This procedure allows the on-line wet ashing of the sample, the hydride generation and the determination of the SeH2 formed in one step. For this purpose, carrier solution and the injected sample (300 μl) were merged with an acid stream (80% H2SO4, 12% HClO4, 8% HNO3) and passed through a reaction coil heated up to 240°C. To increase the dispersion, the wet digestion was carried out in an ultrasonic field. After trapping of the gas bubbles, 5 mol/l HCl and 0.053 mol/l NaBH4 solutions were added via T-junctions for Se-hydride generation. The nebulizing system of the ICP-OES was used as gas/liquid separator. The hydride was swept from the spray chamber into the plasma by an argon gas flow with droplets containing other sample constituents. This allows the simultaneous determination of other trace elements of clinical interest, for example Fe, Cu and Zn. The relative detection limit for Se in blood serum was 5 μg/l. The quality of the developed procedure was verified in two ways: by measuring SRM Seronorm 116 and by comparing the results for different serum samples with the results obtained with ETAAS. Our results were in good agreement in both cases.

Performance of a highly lipophilic Li+ lonophore in solvent polymeric membranes

Ionophores based on non-cyclic triamides exhibiting Li+ selectivity in solvent polymeric membranes are described. Their lipophilicity (water/1-n-octanol partition coefficient) is up to 1013.8 so that they can be used in ion-selective electrodes with a lifetime of up to 40 000 h even in permanent contact with whole blood or undiluted blood serum.

Neutral ionophore-based selective electrode for assaying the activity of magnesium in undiluted blood serum

The concentration of free, ionized magnesium in undiluted blood serum can be determined potentiometrically with a new magnesium-selective carrier in polymeric membrane electrodes (by weight 66% plasticizer, approximately 33% polyvinyl chloride and approximately 1% ionophore). However, discrimination of calcium by the new ionophore-added membrane is not sufficient to keep calcium interference to less than 1%. To correct for the interference by calcium, we determine the calcium activity of the serum sample with a calcium-selective electrode and calibrate the magnesium-selective electrode with standard solutions containing calcium in the concentrations found. In this way we determined the free ionized magnesium in five plasma samples from five different persons and found it to be within the range expected for magnesium in undiluted serum.

Very lipophilic sodium-selective ionophore for chemical sensors of high lifetime

Ionophores based on 1,2-phenylenedioxydiacetamides with water/1-octanol partition coefficients up to 10 17 were synthesized. They have sufficient lipophilicity to be used in solvent-polymeric membranes for optrodes, ISFETs and ion-selective electrodes with a lifetime from 10 × 10 4 hours even in permanent contact with whole blood or undiluted blood serum. Selectivities of Na + over K +, Ca 2+ and Mg 2+ of 10 1.5–10 2 and 10 4, respectively, were obtained.

Determination of creatinine in undiluted blood serum enzymatic flow injection analysis with optosensing

A specific enzymatic assay for creatinine in undiluted serum samples is described, exploiting the generation of ammonia from creatinine by immobilized creatinine iminohydrolase. The ammonia produced is separated from the sample matrix by gas diffusion into an acceptor stream containing a pH-sensitive indicator. The creatinine content is quantified by monitoring the resulting colour change of the indicator by means of reflectance measurement via optical fibers, the hydrophobic gas-permeable membrane serving as a diffuse reflector. Two approaches are used to overcome the interference caused by endogenous ammonia. The first is based on enzymatic abatement of endogenous ammonia by immobilized glutamate dehydrogenase. In the second, preferable, approach, endogenous ammonia, itself a parameter of clinical interest, is measured separately prior to the enzymatic degradation by creatinine iminohydrolase. Each assay requires only 30 μl of sample solution, and the sampling frequency is 60 h −1. The relative standard deviation is approximately 3%.

Determination of potassium ion concentration in serum using a highly selective liquid-membrane electrode

A simple method for the direct determination of the K + concentration in undiluted blood serum is described. The reproducibility of single determinations using the readings after 60-sec contact of a valinomycin liquid-membrane electrode with the sample is 0.07 mequiv/1 K + (standard deviation). No corrections are needed for the presence of other ions in the sample solutions.

Relationship of Pedersen's X-protein to the protein complex in undiluted blood serum

The present investigation is an electrophoretic study of X-protein obtained by separation from blood serum by ultracentrifugation. It has been found that the X-protein of Pedersen, as well as the complexes of undiluted serum are both dissociated by dialysis and dilution. It is suggested that these latter act on the binding material which is responsible for holding the proteins in complex association.

EFFECTS OF HETEROLOGOUS SERA ON FERTILIZED RABBIT OVA

Undiluted blood serum of various species was used to culture two-celled rabbit ova for 24 hours. It was found that there is an ovocidal factor present in the serum of man, sheep, cattle, goat, and fowl. The factor is lethal rather than inhibitory; exposure to it for 10 minutes will cause the death of the ova. This factor is unstable, thermolabile (destroyed at 55 degrees C. in 30 minutes), and of large molecular size. The strength or concentration of this factor varies according to the origin of the serum, increasing in the order man, sheep, cattle, goat, fowl. The blood serum of rabbit, horse, dog, guinea pig, rat, and pig contains no ovocidal factor against rabbit ova. The ovocidal factor differs from the spermicidal factor, which is present in all the sera of the different species studied with rabbit spermatozoa. Immunization of the guinea pig against rabbit ova is possible. Normal development of young rabbits was obtained by transplantation of ova cultured in the heated or normal serum of other species after 24 hours.