A thermostat chip of indium tin oxide glass substrate for static polymerase chain reaction and in situ real time fluorescence monitoring

A thermostat chip of indium-tin oxide glass substrate for static chip polymerase chain reaction (PCR) is, for the first time, introduced in this paper. The transparent conductive layer was used as an electro-heating element. Pulse width modulation and fuzzy proportional integration-differentiation algorithm were adopted in the temperature programming of the chip. The temperature distribution was investigated, and a dynamic control precision within ±2 °C was achieved. The highest ramping rates were 37 °C s⁻¹ for heating and 8 °C s⁻¹ for cooling with an electric fan. The PCR reaction vials were constructed with polyethylene tubes or poly(dimethylsiloxane) directly on the thermostat chip; the chip had a typical size of 25 mm × 25 mm and a thickness of 1.1 mm. Static chip PCR was successfully demonstrated either in a single vial or in an up to 8-parallel array vials. In situ real time fluorescence monitoring during PCR of a λ DNA fragments (236 bp) with SYBR Green I was demonstrated using a blue light emission diode as a light source and a photomultiplier as a detector. The method proposed here is characterized by open access, easy fabrication and low cost. This work could be the basis for developing a portable real time PCR system with disposable chips for point of care tests.     

The development of miniSTRs as a method for high-speed direct PCR

There are situations in which it would be very valuable to have a DNA profile within a short time; for example, in mass disasters or airport security. In previous work, we have promoted reduced size STR amplicons for the analysis of degraded DNA. We also noticed that shorter amplicons are more robust during amplification, making them inhibition resistant, and potentially applicable to high-speed direct PCR. Here, we describe a set of miniSTRs capable of rapid direct PCR amplification. The selected markers are a subset of the Combined DNA Index System (CODIS) loci modified to permit high-speed amplification. Using the proposed protocol, the amplification of eight loci plus amelogenin directly from a saliva sample can be completed in 7 min and 38 s using a two-step PCR with 30 cycles of 98°C for 2 s and 62°C for 7 s on a Streck Philisa thermocycler. Selection of DNA polymerase, optimization of the two-step PCR cycling conditions, the primer concentrations, and the dilution of saliva is described. This method shows great potential as a quick screening method to obtain a presumptive DNA profile when time is limited, particularly when combined with high-speed separation and detection methods.     

Application of adaptive pressure-driven microfluidic chip in thyroid function measurement

The improvement in accuracy of in vitro diagnosis has always been the focus of early screening of thyroid dysfunction. We constructed a microfluidic chip based on a polystyrene polymer substrate. Total triiodothyronine (TT3), total thyroxine (TT4), free triiodothyronine (FT3), free thyroxine (FT4), and thyrotropin (TSH) in human whole blood samples were analysed by fluorescence immunoassay to evaluate thyroid function. The results indicate that the microfluidic chip shows a good linear relationship in the detection of TT3, TT4, FT3, FT4, and TSH standards, and the correlation coefficient (r) is not less than 0.9900. In addition, the chip also has strong anti-interference (RSD% ≤ 5%) and good repeatability (CV ≤ 8%), and its inter-batch differences are small (CV ≤ 15%). The results of practical application in clinical thyroid function measurement indicated its high accuracy (r ≥ 0.9900). It provides a new method for the determination of thyroid function and lays a foundation for subsequent clinical application.     

Determination of parathion in biological fluids by means of direct solid-phase microextraction

A new and simple procedure for the determination of parathion in human whole blood and urine using direct immersion (DI) solid-phase microextraction (SPME) and gas chromatography/mass spectrometry (GC/MS) is presented. This technique was developed using only 100 μL of sample, and ethion was used as internal standard (IS). A 65-μm Carbowax/divinylbenzene (CW/DVB) SPME fibre was selected for sampling, and the main parameters affecting the SPME process such as extraction temperature, adsorption and desorption time, salt addition, agitation and pH effect were optimized to enhance the sensitivity of the method. This optimization was also performed to allow the qualitative determination of parathion’s main metabolite, paraoxon, in blood. The limits of detection and quantitation for parathion were 3 and 10 ng/mL for urine and 25 and 50 ng/mL for blood, respectively. For paraoxon, the limit of detection was 50 ng/mL in blood. The method showed linearity between the LOQ and 50 μg/mL for both matrices, with correlation coefficients ranging from 0.9954 to 0.9999. Precision and accuracy were in conformity with the criteria normally accepted in bioanalytical method validation. The mean absolute recoveries were 35.1% for urine and 6.7% for blood. Other parameters such as dilution of sample and stability were also validated. Its simplicity and the fact that only 100 μL of sample is required to accomplish the analysis make this method useful in forensic toxicology laboratories to determine this compound in intoxications, and it can be considered an alternative to other methods normally used for the determination of this compound in biological media.     

Simultaneous determination of Fe, P, Ca, Mg, Zn, and Cu in whole blood by two-jet plasma atomic emission spectrometry

Availability of many biological samples in ample quantity for biomedical investigations sometimes is very restricted. Therefore, there is the need for the simple techniques allowing the analysis of small amount samples. In the present work the two-jet plasma atomic emission spectrometry techniques for the determination of Fe, P, Ca, Mg, Zn, and Cu in whole blood are proposed. The first technique is developed for direct analysis of freeze-dried blood. The sample preparation consisted in a dilution of blood powder (particle size 20 μm or less) with a spectroscopic buffer (graphite powder containing 15 wt.% NaCl). For the analysis of liquid whole blood, previous carbonization (not ashing) of blood evaporated on graphite powder was applied. Calibration samples based on graphite powder containing 15 wt.% NaCl were used. The validation of the techniques was confirmed by the use of different sample preparation procedures (wet acid digestion and dry carbonization), the analysis of IAEA A-13 reference material (freeze-dried bovine whole blood), and the comparison of the results obtained by the proposed technique with the results of the stripping voltammetry technique. Just 20-50 μL of whole blood is quite enough for all determinations. The proposed techniques were successfully applied for the simultaneous determination of Fe, P, Ca, Mg, Zn, and Cu in whole blood of living experimental rats and mice and human blood.     

Determination of total and oxidized glutathione in human whole blood with a sequential injection analysis system

This work reports the development of a simple, robust, automated sequential injection analysis (SIA) system for the enzymatic determination of total (tGSH) and oxidized (GSSG) glutathione in human whole blood. The reduced (GSH) glutathione concentration is then obtained as the difference between the tGSH and GSSG concentrations. The determination was based on the DTNB–GSSG reductase recycling assay, which couples the specificity of the GSSG reductase (GR) with an amplification of the response to glutathione, followed by spectrophotometric detection of the 2-nitro-5-thiobenzoic acid (TNB) formed (λ = 412 nm). The implementation of this reaction in a SIA flow system with an in-line dilution strategy permitted the necessary distinct application ranges for tGSH and for GSSG. It also guaranteed the exact timing of fluidic manipulations and precise control of the reaction conditions.

The influence of parameters such as reagents concentration, temperature, pH, flow rate of the carrier buffer solution, as well as reaction coil length, etc., on the sensitivity and performance of the SIA system were studied and the optimum reaction conditions subsequently selected. Linear calibration plots were obtained for GSH and GSSG concentrations up to 3.00 and 1.50 μM, with detection limits of 0.031 and 0.014 μM, respectively. The developed methodology showed good precision, with a relative standard deviation (R.S.D.) < 5.0% (n = 10) for determination of both glutathione forms. Statistical evaluation showed good compliance, for a 95% confidence level, between the results obtained with the SIA system and those furnished by the comparison batch procedure.     

流动型微流控PCR扩增芯片的研究

组装了由注射泵进样系统、微流控芯片和三温区加热器组成的流动型PCR扩增系统,该系统具有扩增速度快、交叉污染小、芯片可重复使用和操作方便等特点.优化了芯片厚度、隔热材料和流速等影响PCR扩增的因素.在4.9min内经24个循环成功地扩增了浓度为1ng/100μL的λ-DNA(500bp).     

Successful direct amplification of nuclear markers from single dog hairs using DogFiler multiplex

We report on successful amplification of canine STR DNA profiles from single dog hairs. Dog hairs are commonly found on clothing or items of interest in forensic casework and may be crucial associative evidence if linked to an individual dog. We used direct amplification from these hairs to increase the DNA yield of the sample, as well as greatly reducing analysis time. Hairs from different somatic regions were used from several different dog breeds to amplify a selection of eight loci from the validated DogFiler multiplex. Naturally shed canine hairs were processed, with a mix of coarse topcoat (guard) hairs and thinner soft undercoat hairs. Multiple sections of single hairs were amplified in 5 mm segments to determine the viability of DNA recovery from the shaft of the hair. Single guard hairs were cut into 5 mm sections and added directly into a PCR tube. Undercoat hairs, which are very fine, were amplified together in a single tube (approximately ten small hairs). Coarse hairs were found to be the most successful in producing full DNA profiles at all eight loci, matching the corresponding reference profile for that dog.     

Mixed‐mode SPE for a multi‐residue analysis of benzodiazepines in whole blood using rapid GC with negative‐ion chemical ionization MS

A sensitive and selective method for simultaneous quantitation of 15 benzodiazepines in human whole blood using rapid GC with negative‐ion chemical ionization MS is proposed. A mixed‐mode cation‐exchange polymeric sorbent was used for SPE. Different extraction solvents or mixtures of solvents of different compositions for elution of the adsorbed analytes, and washing steps for eliminating interferences in the column were tested. Analytes were eluted from the column using 5% v/v NH₄OH in methanol. A derivatization step using different silylation reagents, time, and temperature was tested. Extracts from SPE were silylated by a mixture of N‐(tert‐butyldimethylsilyl)‐N‐methyltrifluoroacetamide, acetonitrile, ethyl acetate, and subjected to gas chromatographic analysis. The LODs of 15 benzodiazepines in whole blood samples ranged from 0.24–0.62 ng mL^?1. The RSDs of samples used for three different quality control concentration levels were lower than 7.0%, and the accuracy ranged from 89.5 to 110.5%. The results show that the developed method is accurate, sensitive, selective, and very fast. Finally, the applicability of this method for determination of trace concentrations of several benzodiazepines in real blood samples has been demonstrated.     

Electrochemical detection of glucose from whole blood using paper-based microfluidic devices

Electrochemical paper-based analytical devices (ePADs) with integrated plasma isolation for determination of glucose from whole blood samples have been developed. A dumbbell shaped ePAD containing two blood separation zones (VF2 membranes) with a middle detection zone was fabricated using the wax dipping method. The dumbbell shaped device was designed to separate plasma while generating homogeneous flow to the middle detection zone of the ePAD. The proposed ePADs work with whole blood samples with 24–60% hematocrit without dilution, and the plasma was completely separated within 4 min. Glucose in isolated plasma separated was detected using glucose oxidase immobilized on the middle of the paper device. The hydrogen peroxide generated from the reaction between glucose and the enzyme pass through to a Prussian blue modified screen printed electrode (PB-SPEs). The currents measured using chronoamperometry at the optimal detection potential for H₂O₂ (−0.1 V versus Ag/AgCl reference electrode) were proportional to glucose concentrations in the whole blood. The linear range for glucose assay was in the range 0–33.1 mM (r² = 0.987). The coefficients of variation (CVs) of currents were 6.5%, 9.0% and 8.0% when assay whole blood sample containing glucose concentration at 3.4, 6.3, and 15.6 mM, respectively. Because each sample displayed intra-individual variation of electrochemical signal, glucose assay in whole blood samples were measured using the standard addition method. Results demonstrate that the ePAD glucose assay was not significantly different from the spectrophotometric method (p = 0.376, paired sample t-test, n = 10).     

An electrophoretic method for the detection of chymotrypsin and trypsin activity directly in whole blood

In biomedical research and clinical diagnostics, it is a major challenge to measure disease‐related degradative enzyme activity directly in whole blood. Present techniques for assaying degradative enzyme activity require sample preparation, which makes the assays time‐consuming and costly. This study now describes a simple and rapid electrophoretic method that allows detection of degradative enzyme activity directly in whole blood using charge‐changing fluorescent peptide substrates. Charge‐changing substrates eliminate the need for sample preparation by producing positively charged cleavage fragments that can be readily separated from the oppositely charged fluorescent substrate and blood components by electrophoresis. Two peptide substrates have been developed for pancreatic α‐chymotrypsin and trypsin. For the first substrate, a detection limit of 3 ng for both α‐chymotrypsin and trypsin was achieved in whole rat blood using a 4% agarose gel. This substrate had minimal cross‐reactivity with the trypsin‐like proteases thrombin, plasmin, and kallikrein. For the second substrate (trypsin‐specific), a detection limit of about 10–20 pg was achieved using thinner higher resolution 20 and 25% polyacrylamide gels. Thus, the new charge changing peptide substrates enable a simple electrophoretic assay format for the measurement of degradative enzyme activity, which is an important step toward the development of novel point‐of‐care diagnostics.     

Investigation of whole blood of SJL/J mice using neutron activation analysis

The Br (0.0022 ± 0.0006 gL⁻¹), Ca (0.113 ± 0.012 gL⁻¹), Cl (3.07 ± 0.36 gL⁻¹), K (2.63 ± 0.14 gL⁻¹), Mg (0.045 ± 0.002 gL⁻¹) and Na (2.09 ± 0.10 gL⁻¹) concentrations were determined in whole blood of SJL/J mice using the Neutron Activation Analysis (NAA) technique. Eleven whole blood samples were analyzed in the IEA-R1 nuclear reactor at IPEN (S?o Paulo, Brazil). These data contribute for applications in veterinary medicine related to biochemistry analyses using whole blood. Moreover, the correlation with human blood estimation allows to checking the similarities for studying muscular dystrophy using this model animal.     

Fabrication of glucose biosensor for whole blood based on Au/hyperbranched polyester nanoparticles multilayers by antibiofouling and self-assembly technique

Acknowledging the benefits of hyperbranched polymers and their nanoparticles, herein we report the design and synthesis of sulfonic acid group functionalized hydroxyl-terminated hyperbranched polyester (H30-SO₃H) nanoparticles and their biomedical application. The H30-SO₃H nanoparticles were characterized by transmission electron microscopy (TEM), Fourier transform infrared (FTIR) spectroscopy and proton nuclear magnetic resonance spectroscopy (¹H NMR). The good hemocompatibility of H30-SO₃H nanoparticles was also investigated by coagulation tests, complement activation and platelet activation. The novel glucose biosensor was fabricated by immobilizing the positively charged Au nanoparticles, H30-SO₃H nanoparticles and glucose oxidase (GOx) onto the surface of glassy carbon electrode (GCE). It can be applied in whole blood directly, which was based on the good hemocompatibility and antibiofouling property of H30-SO₃H nanoparticles. The biosensor had good electrocatalytic activity toward glucose with a wide linear range (0.2–20 mM), a low detection limit 1.2 × 10⁻⁵ M in whole blood and good anti-interference property. The development of materials science will offer a novel platform for application to substance detection in whole blood.     

Element assessment in whole blood, serum and urine of three Italian healthy sub-populations by INAA

This research, that is a framework of a big project regarding the knowledge of human metabolism in patients affected by renal disease, is aimed to assess the levels of some selected essential (Co, Fe, K, Na, Rb, Se and Zn) and non-essential elements (Br Cr, Cs, Hg, Ni and Sb) in different biological matrices such as whole blood, serum and urine, of three Italian sub-populations.The analytical methodology involved is the Instrumental Neutron Activation Analysis (INAA), a primary analytical technique: the samples (totally 144) were irradiated in the Triga nuclear reactor at the R.C.-Casaccia ENEA for 12 h at a neutron flux of 2.6 × 10¹² n × cm^{− 2} × s^{− 1}.The results show a similar distribution for essential elements whereas quite different for non-essential elements. The statistical treatment has evidenced no differences among the samples grouping the subpopulations into unique one. In this way, the levels found can be considered “reference values” in the investigated matrices.     

Rapid amplification of genetically modified organisms using a circular ferrofluid-driven PCR microchip

The use of genetically modified organisms (GMOs) as food and in food products is becoming more and more widespread. Polymerase chain reaction (PCR) technology is extensively used for the detection of GMOs in food products in order to verify compliance with labeling requirements. In this paper, we present a novel close-loop ferrofluid-driven PCR microchip for rapid amplification of GMOs. The microchip was fabricated in polymethyl methacrylate by CO₂ laser ablation and was integrated with three temperature zones. PCR solution was contained in a circular closed microchannel and was driven by magnetic force generated by an external magnet through a small oil-based ferrofluid plug. Successful amplification of genetically modified soya and maize were achieved in less than 13 min. This PCR microchip combines advantages of cycling flexibility and quick temperature transitions associated with two existing microchip PCR techniques, and it provides a cost saving and less time-consuming way to conduct preliminary screening of GMOs. Figure Schematic of the circular ferrofluid-driven PCR microchip     

Hollow‐fiber liquid‐phase microextraction and gas chromatography‐mass spectrometry of barbiturates in whole blood samples

Here, we present a method for measuring barbiturates (butalbital, secobarbital, pentobarbital, and phenobarbital) in whole blood samples. To accomplish these measurements, analytes were extracted by means of hollow‐fiber liquid‐phase microextraction in the three‐phase mode. Hollow‐fiber pores were filled with decanol, and a solution of sodium hydroxide (pH 13) was introduced into the lumen of the fiber (acceptor phase). The fiber was submersed in the acidified blood sample, and the system was subjected to an ultrasonic bath. After a 5 min extraction, the acceptor phase was withdrawn from the fiber and dried under a nitrogen stream. The residue was reconstituted with ethyl acetate and trimethylanilinium hydroxide. An aliquot of 1.0 μL of this solution was injected into the gas chromatograph/mass spectrometer, with the derivatization reaction occurring in the hot injector port (flash methylation). The method proved to be simple and rapid, and only a small amount of organic solvent (decanol) was needed for extraction. The detection limit was 0.5 μg/mL for all the analyzed barbiturates. The calibration curves were linear over the specified range (1.0 to 10.0 μg/mL). This method was successfully applied to postmortem samples (heart blood and femoral blood) collected from three deceased persons previously exposed to barbiturates.     

Hydrogen peroxide in basic media for whole blood sample dissolution for determination of its lead content by electrothermal atomization atomic absorption spectrometry

Hydrogen peroxide in basic media is proposed as a means for dissolving whole blood samples to be analyzed by electrothermal atomization atomic absorption spectrometry, ET AAS. Approximately 2 g of the whole blood sample were directly weighed in a 150 mL volumetric flask; 3 mL of a NaOH 0.2 mol L⁻¹ solution, two drops of 1-octanol, as an antifoaming agent, and 1 mL of 30% volume hydrogen peroxide were added to the flask to promote oxidation. The solution was then manually shaken and after approximately three minutes of shaking, a clear solution, with no apparent suspended solids or greasy layers, was obtained. Distilled-deionized water was used to complete the volume. Ten μL of the resulting solution along with 10 μL of a solution containing 5000 mg L⁻¹ of NH₄H₂PO₄ and 300 mg L⁻¹ of Mg(NO₃)₂ as a modifier, were injected into transversely heated graphite tubes for lead determination. Both aqueous standards and standard addition calibration curves produced results not significantly different at a 95% confidence limit level. Accuracy of the measurements was assessed by analysis of the IAEA A-13 (concentration of trace and minor elements in freeze dried animal blood) standard reference material containing 0.18 mg L⁻¹ lead on a dry basis and by means of recovery tests. Analysis of the IAEA A-13 standard produced 0.17 ± 0.02 mg L⁻¹ lead on a dry basis; recovery tests afforded values from 95 to 105%. Ten consecutive measurements of a 5 ppb lead solution gave a characteristic mass of 47.2 pg and a (3S) detection limit of 1.77 μg L⁻¹ Pb. Results obtained from analysis of whole blood samples of volunteer donors covered a lead concentration range between 8 and 21 μg L⁻¹ with a mean value of 11.9 ± 4.7 μg L⁻¹.     

An analytical method for the determination of perfluorinated compounds in whole blood using acetonitrile and solid phase extraction methods

A method for the analysis of perfluorinated compounds (perfluoroalkyl sulfonates: C4, C6, C8, C10; perfluoroalkyl sulfinates: C6, C8, C10; perfluorooctanesulfonamide, N-ethyl perfluorooctanesulfonamide, N-ethyl perfluorooctanesulfonamidoacetate, perfluorocarboxylates: C4–C14; fluorotelomer carboxylate (7:3, 8:2) in whole blood using acetonitrile and OASIS WAX^® solid phase extraction (SPE) cartridge was developed. Separation of target compounds in two HPLC columns (ion exchange JJ50-2D and C18 Betasil columns) was examined. Matrix recoveries of the developed methods ranged from 70% to 120%. Separation of possible inferences such as taurodeoxycholic acid (TDC) was accomplished using an ion exchange JJ50-2D column, and this separation was validated using whole blood of different animals.     

Interference by complex structures of target DNA with specific PCR amplification

It has been considered that target DNA is a forgiving component for PCR amplification. Herein we present evidence to demonstrate that secondary structure located at the end of a template may interfere with the specificity of amplification. Experiments indicate that nonspecific amplification results from a long stretch of stem and loop structures at the 3′ end of prochymosin cDNA. Based on the sequence of mRNA coding for prochymosin, it is argued that the sequence responsible for the formation of the complex structure described here is most likely generated during synthesis of the second cDNA strand.     

Injection by hydrostatic pressure in conjunction with electrokinetic force on a microfluidic chip

A simple method was developed for injecting a sample on a cross-form microfluidic chip by means of hydrostatic pressure combined with electrokinetic forces. The hydrostatic pressure was generated simply by adjusting the liquid level in different reservoirs without any additional driven equipment such as a pump. Two dispensing strategies using a floating injection and a gated injection, coupled with hydrostatic pressure loading, were tested. The fluorescence observation verified the feasibility of hydrostatic pressure loading in the separation of a mixture of fluorescein sodium salt and fluorescein isothiocyanate. This method was proved to be effective in leading cells to a separation channel for single cell analysis.