滚环扩增信号放大技术在生物检测中应用的研究进展

滚环扩增(Rolling circle amplification,RCA)是一种快速、灵敏且恒温的单链DNA(Single-stranded DNA,ssDNA)扩增技术,与染色或探针联用可实现检测信号的放大,在生物检测等方面得到广泛的应用。文中对RCA的构建方法进行了简介,综述了近几年其在致病菌、核酸肿瘤标记物、蛋白质、生物小分子和病毒等检测中的研究进展,并对其未来的发展趋势进行了展望。     

Recovering circulating extracellular or cell-free RNA from bodily fluids

The presence of extracellular circulating or cell-free RNA in biological fluids is becoming a promising diagnostic tool for non invasive and cost effective cancer detection. Extracellular RNA or miRNA as biological marker could be used either for the early detection and diagnosis of the disease or as a marker of recurrence patterns and surveillance. In this review article, we refer to the origin of the circulating extracellular RNA, we summarise the data on the biological fluids (serum/plasma, saliva, urine, cerebrospinal fluid and bronchial lavage fluid) of patients suffering from various types of malignancies reported to contain a substantial amount of circulating extracellular (or cell-free) RNAs and we discuss the appropriate reagents and methodologies needed to be employed in order to obtain RNA material of high quality and integrity for the majority of the experimental methods used in RNA expression analysis. Furthermore, we discuss the advantages and disadvantages of the RT-PCR or microarray methodology which are the methods more often employed in procedures of extracellular RNA analysis.     

呼肠孤病毒Ⅲ型（Re03）RT—PCR检测方法的建立

目的建立呼肠孤病毒Ⅲ型（Re03）RT—PCR检测方法,应用于实验动物及人用动物源性材料及生物制品外源Re03的检测。方法根据已发表的呼肠孤病毒Ⅲ型（Re03）M1基因序列,设计合成引物。提取Re03细胞毒RNA,以其为模板,进行PCR扩增。优化反应条件,进行特异性、敏感性、重复性试验。结果建立的Re03RT—PCR检测方法特异、敏感、稳定。以Re03RNA逆转录产物为模板,所能检测RNA最小模板浓度为0．42pg／μL,可检测病毒最小滴度为10^-9／mL。结论建立的呼肠孤病毒Ⅲ型（Re03）RT—PCR检测方法可用于实验动物及人用动物源性材料及生物制品外源Re03的检测。     

Multielement analysis of biological samples by inductively coupled plasma-mass spectrometry

Interest in the biological behavior of a growing number of elements, along with increasing recognition of the importance of interactions among them, demands a versatile and reliable technique for multielement analysis of biological samples. Significant improvements over the sensitivity achieved with conventional inductively coupled plasma (ICP) optical emission spectrometries have been realized with the introduction of quadrupole mass spectrometry (MS) for detection of ions in the plasma. The hybrid technique of ICP-MS promises to be a method of rapid multielement analysis, at detection limits that approach or surpass those of other technologies. However, the application of ICP-MS to analyses of biological interest is truly in its infancy. Here we report the use of ICP-MS for the determination of more than 30 elements of biological interest in a tissue and a biological fluid (rat liver and serum, respectively). Experimental values of the elements serve as a basis for discussion of analytical protocols, performance criteria, and certain problems peculiar to ICP-MS.     

A Comparative Study of Methods To Validate Formaldehyde Decontamination of Biological Safety Cabinets

Methods of validation of formaldehyde decontamination of biological safety cabinets were compared. Decontamination of metal strips inoculated with Mycobacterium bovis, poliovirus, or Bacillus spp. spores was compared with the results obtained with three biological indicators. Conditions for successful decontamination, particularly relative humidity, were defined. The Attest 1291 biological indicator was the only biological indicator which was an aid in the detection of gross decontamination failure.     

Foldamers with hybrid biological and synthetic sequences as selective DNA fluorescent probes

Foldable polymers with alternating single-strand deoxyribonucleic acid (ssDNA) and planar fluorescent organic chromophores can self-organize into folded nanostructures and hence are hybrid foldamers with biological sequences and synthetic properties. The biological sequence provides highly specific molecular recognition properties, while the physical properties of synthetic chromophores offer sensitive fluorescence detection. In this paper, we describe that rational designed hybrid foldamers exhibit potential in the detection of polynucleotides. Under strictly controlled laboratory conditions, fluorescence measurements indicate that configuration change due to binding of polynucleotides with one or two mismatched bases can be readily distinguished. These results shed light on the design and construction of nanostructured foldamers with actuator and sensory properties, which may find important applications as biological probes.     

A facile method for the preparation of deuterium labeled salvinorin A: synthesis of [2,2,2-2H3]-salvinorin A

Salvinorin A is a novel hallucinogen isolated from the widely available leaves of Salvia divinorum. Based on its mechanism of action, salvinorin A has shown potential as a stimulant abuse therapeutic. However, there are no methods for the detection of salvinorin A or its metabolites in biological fluids. In order to begin developing salvinorin A as a potential therapeutic, an understanding of its metabolism is needed. Here, a straightforward synthesis of a deuterium labeled analog of salvinorin A and its utility as an internal standard for the detection of salvinorin A and its metabolites in biological fluids by LC-MS is described.     

Measurement of cytokine antibodies. Test development

Several assays have been used for detection of antibodies against cytokines. The choice of assay is greatly dependent on the intended goal, e.g. detection of naturally occurring antibodies or therapy induced antibodies. The different assays can be grouped in 2 categories. The interference or indirect assays are based on the detection of the test sample interference with the biological activity, with detection of the cytokine in EIA or with binding to cellular receptors. In direct assays cytokine antibodies are detected by binding to solid phase fixed cytokines, followed by incubation with a secondary enzyme-labelled anti-human Ig antibody or by binding to¹²⁵I-labelled cytokines in RIA.     

Rhodamine-based fluorogenic probe for imaging biological thiol

We have developed a new fluorescent probe for biological thiol. The probe was synthesized by the modification of the 2,4-dinitrobenzenesulfonyl group with rhodamine 110. The selective detection of thiol species such as cysteine or glutathione was achieved in biological conditions. Moreover, the probe was successfully applied to the imaging of thiol species in living human cells.     

微针及其在生物诊疗中的应用研究进展

微针在过去几十年中发展迅速,在透皮给药等领域已得到了广泛的研究.近些年随着微电子技术的蓬勃兴起,微针在生物诊疗中的应用越来越多的受到人们关注.通过微针提取血液和组织液进行检测分析以及微针作为电极在体液内直接对血糖、黑色素瘤、pH值等进行实时检测方面,都展现出了良好的实时检测应用前景.文中综述了微针的材料与结构设计及其在...     

Determination of illicit and/or abused drugs and compounds of forensic interest in biosamples by capillary electrophoretic/electrokinetic methods

The application of capillary electrophoresis (CE) methods in forensic toxicology for the determination of illicit and/or misused drugs in biological samples is reviewed in the present paper. Sample pretreatments and direct injection modes used in CE for analysis of drugs in biological fluids are briefly described. Besides, applications of separation methods based on capillary zone electrophoresis or micellar electrokinetic chromatography with UV absorbance detection to (i) analysis of drugs of abuse, (ii) analysis of other drugs and toxicants of potential forensic interest and (iii) for metabolism studies are reviewed. Also, alternative CE methods are briefly discussed, including capillary isotachophoresis and separation on mixed polymer networks. High sensitivity detection methods used for forensic drug analysis in biological samples are then presented, particularly those based on laser induced fluorescence. A glimpse of the first examples of application of CE–mass spectrometry in forensic toxicology is finally given.     

The assay of methylglyoxal in biological systems by derivatization with 1,2-diamino-4,5-dimethoxybenzene.

A procedure for the assay of methylglyoxal in biological systems is described, together with sample storage, sample processing procedures, and statistical evaluation. Specimen data are presented. Methylglyoxal was assayed by derivatization with 1,2-diamino-4,5-dimethoxybenzene and high-performance liquid chromatography (HPLC) of the resulting quinoxaline, 6,7-dimethoxy-2-methylquinoxaline, with spectrophotometric or fluorescence detection. Derivatization, solid-phase extraction, and HPLC were performed under acid conditions to prevent the spontaneous formation of methylglyoxal from glyceraldehyde 3-phosphate and dihydroxyacetone phosphate during the assay. The limits of detection in the biological matrix were 45 pmol (absorbance detection) and 10 pmol (fluorimetric detection), the recovery was 58%, and the intra- and interbatch coefficients of variance were 7.7 and 30.0%, respectively. The concentration of methylglyoxal in whole blood from normal healthy human individuals was (mean +/- SE, nM) 256 +/- 92 (n = 12) and that from diabetic patients was 479 +/- 49 (n = 55), showing a significant increase in diabetes mellitus (P < 0.01; Mann-Whitney U test). Sample processing under acidic conditions was essential to avoid interferences. Previous estimates of the concentration of methylglyoxal in biological samples require re-evaluation.     

Determination of plasma, urine, and bovine serum albumin low-molecular-weight carbonyl levels by capillary gas chromatography with electron-capture and mass-selective detection

Peroxidation of lipids produces low-molecular-weight carbonyl compounds, which are reactive with biological nucleophiles. The analysis of these compounds is often difficult. A multicomponent method for the determination of 11 of them in biological samples is reported. The samples are subjected to a pretreatment-derivatization procedure followed by gas chromatographic analysis with either electron-capture detection (ECD) or mass-selective detection (MSD) in the selected-ion monitoring mode. The procedure involves derivatization of the analyte with 2,4,6-trichlorophenylhydrazine, extraction with n-hexane, and separation of the derivatization products on a nonpolar gas chromatographic column. The concentration of the derivatization reagent, pH, reaction time, temperature, and presence of extraneous ions were investigated to determine the optimal derivatization conditions. Under these conditions, the method allows for the selective detection of low-molecular-weight carbonyl compounds at femtomole levels in several biological materials such as plasma, urine, and bovine serum albumin without interferences. The limits of detection were in the ranges 0.01-0.2 microM for ECD and 0.15-1.5 microM for MSD. The mean procedural recoveries obtained during the method validation were within the range 85-95% and the intra- and interassay standard deviations do not exceed 4.6 and 6.1%, respectively.     

High-performance liquid chromatography of monosaccharides and oligosaccharides in a complex biological matrix.

Analysis of oligosaccharides in complex biological matrices is hampered by the fact that oligosaccharides, closely related in structure, are difficult to separate from each other and that conventional detection procedures (refraction index and uv detection) are not specific enough for carbohydrates. Prepurification of samples by procedures like desalting or gel filtration is often used but can lead to the loss of specific oligosaccharides. We have used pellicular anion chromatography in combination with a postcolumn reaction for reducing carbohydrates based on 4-aminobenzoylhydrazide. This procedure not only detected normal mono- and oligosaccharides but N-acetylhexosamines and reducing N-acetylhexosamine containing oligosaccharides as well. A sensitivity of about 20-25 pmol for non-GlcNAc containing mono- or oligosaccharides and between 30-50 pmol for GlcNAc or oligosaccharides with GlcNAc at the reducing side was reached. The postcolumn detection was compared with pulsed amperometric detection and appeared to be more specific for mono- and oligosaccharides. Except for deproteination to protect the column, no further sample preparation was needed with this system for our application (urines). In this way pellicular anion chromatography in combination with this postcolumn reaction reaction to be a sensitive and specific HPLC procedure for analysis of monosaccharides and oligosaccharides in complex biological matrices.     

Controlled biological and biomimetic systems for landmine detection

Humanitarian demining requires to accurately detect, locate and deactivate every single landmine and other buried mine-like objects as safely and as quickly as possible, and in the most non-invasive manner. The quality of landmine detection affects directly the efficiency and safety of this process. Most of the available methods to detect explosives and landmines are limited by their sensitivity and/or operational complexities. All landmines leak with time small amounts of their explosives that can be found on surrounding ground and plant life. Hence, explosive signatures represent the robust primary indicator of landmines. Accordingly, developing innovative technologies and efficient techniques to identify in real-time explosives residue in mined areas represents an attractive and promising approach. Biological and biologically inspired detection technology has the potential to compete with or be used in conjunction with other artificial technology to complement performance strengths. Biological systems are sensitive to many different scents concurrently, a property that has proven difficult to replicate artificially. Understanding biological systems presents unique opportunities for developing new capabilities through direct use of trained bio-systems, integration of living and non-living components, or inspiring new design by mimicking biological capabilities. It is expected that controlled bio-systems, biotechnology and microbial techniques will contribute to the advancement of mine detection and other application domains. This paper provides directions, evaluation and analysis on the progress of controlled biological and biomimetic systems for landmine detection. It introduces and discusses different approaches developed, underlining their relative advantages and limitations, and highlighting trends, safety and ecology concern, and possible future directions.     

A review of fluorescence methods for assessing labile iron in cells and biological fluids

A variety of biochemical, pharmacological, and toxicological properties have been attributed to labile forms of iron that are associated with cells or with biological fluids. Unlike the major fraction of bioiron which is protein bound, the labile bioiron is chelatable and therefore amenable for detection by metal-sensing devices that are coupled to chelation moieties. The present review deals with the detection of various labile forms of iron present in living cells and in fluids of biological interest, in health and disease. The main focus of the review is on the design and application of fluorescent probes as analytical tools for assessing labile iron and iron transport mechanisms and the efficiency of iron chelators in solution and in cellular systems.     

The discovery approaches and detection methods of microRNAs

MicroRNAs (miRNAs) are small, highly conserved, non-coding RNAs that regulate gene expression of target mRNAs through cleavage or translational inhibition. Computer-based approaches for miRNA gene identification are being considered as indispensable in miRNAs research. Similarly, experimental approaches for detection of miRNAs are crucial to the testing and validating of computational algorithms. The detection of miRNAs in tissues or cells can supply valuable information for investigating the biological function of these molecules. Selective and highly sensitive detection methods will pave the way for extended understanding of miRNA function within organisms. In this review, we summarize the various computational methods for identification of miRNAs as well as the methodologies that have been developed to detection miRNAs.     

Selective quantitative bioanalysis of proteins in biological fluids by on-line immunoaffinity chromatography-protein digestion-liquid chromatography-mass spectrometry

A quantitative method for the determination of proteins in complex biological matrices has been developed based on the selectivity of antibodies for sample purification followed by proteolytic digestion and quantitative mass spectrometry. An immunosorbent of polyclonal anti-bovine serum albumin (BSA) antibodies immobilized on CNBR agarose is used in the on-line mode for selective sample pretreatment. Next, the purified sample is trypsin digested to obtain protein specific peptide markers. Subsequent analysis of the peptide mixture using a desalination procedure and a separation step coupled, on-line to an ion-trap mass spectrometer, reveals that this method enables selective determination of proteins in biological matrices like diluted human plasma. This approach enhances substantially the selectivity compared to common quantitative analysis executed with immunoassays and colorimetry, fluorimetry or luminescence detection. Hyphenation of the immunoaffinity chromatography with on-line digestion and chromatography-mass spectrometry is performed and a completely on-line quantification of the model protein BSA in bovine and human urine was established. A detection limit of 170 nmol/l and a quantification limit of 280 nmol/l is obtained using 50 microl of either standard or spiked biological matrix. The model system allows fully automated absolute quantitative mass spectrometric analysis of intact proteins in biological matrices without time-consuming labeling procedures.     

Conjugated polymers for enhanced bioimaging

Background

Conjugated polymers (CPs) have been used for creating bioimaging tools or biosensors that provide a direct link between spectral signal and different biological processes. The detection schemes of these sensors are mainly employing the efficient light harvesting properties or the conformation sensitive optical properties of the CPs. Hence, the presence of biomolecules or biological events can be detected through fluorescence resonance energy transfer (FRET) between the CP and an acceptor molecule, or through their impact on the conformation of the conjugated backbone, which is seen as an alteration of the optical properties of the CP.

Scope of the review

In this review, the utilization of CPs for sensitive detection of DNA and protein conformational changes will be presented. The main part will be focused on the specific binding of CPs to protein deposits associated with protein misfolding diseases, such as Alzheimer's disease (AD), and the discovery that tailor-made CPs can be used for in vivo optical imaging of protein aggregates will be discussed.

Major conclusions

The unique optical properties of CPs can be used as molecular tools for sensitive detection of genetic material and for characterization of the pathological hallmarks associated with protein misfolding disorders, such as AD.

General significance

CPs are novel molecular tools that can be used for sensitive bioimaging of biological processes and these tools offer the possibility to study biological events in a complementary fashion to conventional techniques.This article is part of a Special Issue entitled Nanotechnologies - Emerging Applications in Biomedicine.