Components In Biological Samples Research Articles

Classification of multiple cancer types by combination of plasma-based near-infrared spectroscopy analysis and machine learning modeling

Background and aimNear-infrared spectroscopy (NIRS) is a non-invasive and convenient tool, which gains features related to chemical components in biological samples. Machine learning (ML) has been popularized in medical diagnosis. This study aimed at investigating a novel cancer diagnosis strategy using NIRS data based ML modeling. MethodsPlasma samples were collected from a total of 247 participants, including lung cancer, cervical cancer, nasopharyngeal cancer, and healthy control, and were randomly split into train set and test set. After performing NIRS analysis, the train dataset was utilized to train ML models, including partial least-squares (PLS), random forest (RF), gradient boosting machine (GBM), and support-vector machine (SVM). Subsequently, these models were tested for their prediction performance by the test set. ResultsAll ML models demonstrated high prediction performance in differentiating cancers from controls, and SVM had high prediction accuracy for different types of cancers. SVM was considered as the most suitable model for its minimal computational cost and high accuracies for both binary and quaternary classification. ConclusionsThis strategy coupling NIRS with ML is insightful that may aid in clinic cancer diagnosis, while further studies should test our results in a larger cohort with better representativeness.

How Conventional Approaches to the Use of Polymer-Containing Composites in Molecular Diagnostics Are Changing

Known approaches to the isolation of biopolymers, in particular nucleic acids (NAs), from biological samples are based on the binding of molecules to a sorbent (“positive selection”) with their subsequent elution using a suitable eluent. The review article discusses the physicochemical processes underlying the development of methods for isolating of NAs from biological samples. It has been shown that methods including the selective solid-phase extraction (i.e., reversible sorption) provide the possibility of miniaturization and automation of the corresponding processes. The review discusses the advantages of an alternative approach to NAs isolation based on the use of special sorbents that bind proteins and other components of biological samples, while these sorbents exhibit sorption inertness with respect to NAs (“negative selection”). Approaches providing the creation of such composite polymer-containing sorbents, which are designed for the sample preparation during molecular diagnostics, as well as the methods of their effective use are considered. It has been demonstrated that due to an interdisciplinary approach using a complex of synthetic and analytical methods, it is possible to combine as a single object of study the composite materials that are very different in structure and properties. Such nanostructured composites (based on porous silica, synthetic membranes, glass multicapillaries) contain fluoropolymers and polyanilines. The results of the use of such composites for selective isolation of NAs and/or proteins from biological samples are discussed. Alternative applications of such composites in molecular diagnostics, in particular, in mass spectrometry, are considered. Directions for expanding of the field of application of polymer-containing composites due to simultaneous use of the sorption properties of the obtained composites surface and the properties of sorbate molecules are outlined. It has been shown that both polymeric and low molecular weight modifiers of the same chemical nature are suitable for the technological preparation of such composites.

Sample preparation for structural mass spectrometry via polyacrylamide gel electrophoresis.

Mass spectrometry is an analytical technique that can detect protein molecules with high sensitivity. Its use is not limited to the mere identification of protein components in biological samples, but is recently being utilized for large-scale analysis of protein structures in vivo as well. Top-down mass spectrometry with an ultra-high resolution mass spectrometer, for example, ionizes proteins in their intact state and allows rapid analysis of their chemical structure, which is used to determine proteoform profiles. Furthermore, cross-linking mass spectrometry, which analyzes enzyme-digested fragments of chemically cross-linked protein complexes, allows acquisition of conformational information on protein complexes in multimolecular crowding environments. In the analysis workflow of structural mass spectrometry, prior fractionation of crude biological samples is an effective way to obtain more detailed structural information. Polyacrylamide gel electrophoresis (PAGE), known as a simple and reproducible means of protein separation in biochemistry, is one example of an excellent high-resolution sample prefractionation tool for structural mass spectrometry. This chapter describes elemental technologies for PAGE-based sample prefractionation including Passively Eluting Proteins from Polyacrylamide gels as Intact species for Mass Spectrometry (PEPPI-MS), a highly efficient method for intact in-gel protein recovery, and Anion-Exchange disk-assisted Sequential sample Preparation (AnExSP), a rapid enzymatic digestion method using a solid-phase extraction microspin column for gel-recovered proteins, in addition to presenting detailed experimental protocols and examples of their use for structural mass spectrometry.

Screening Method for 23 Alkaloids in Human Serum Using LC/MS/MS with a Pentafluorophenyl Column in Dynamic Multiple Reaction Monitoring Mode

Alkaloids are nitrogen-containing organic compounds, generally basic, and found in plants, fungi, and bacteria. Some alkaloids are used in medicine, but some compounds are highly toxic. Accidental ingestion, homicide, and suicide have occurred due to plants containing alkaloids. The identification of toxic components in biological samples is important for the diagnosis and/or treatment of poisoning cases in forensic and emergency medicine. Alkaloids have a wide variety of structures, such as isoquinoline alkaloid, indole alkaloid, tropane alkaloid, and diterpene alkaloid; therefore, there are few reports of simultaneous analysis methods. We have established a method for the simultaneous analysis of 23 alkaloids in human serum with a liquid chromatograph-tandem mass spectrometer (LC/MS/MS). A liquid-liquid extraction which was modified from the first step of the QuEChERS AOAC method was used for serum pretreatment. The separation of the compounds was performed using a pentafluorophenyl (PFP) column, CAPCELL CORE PFP (2.1 mm I.D. × 100 mm, 2.7 μm) in gradient mode. Mobile phase A consisted of 10 mM ammonium formate and 0.1% formic acid in ultrapure water, and mobile phase B was 10 mM ammonium formate and 0.1% formic acid in methanol. Simultaneous analysis was performed in dynamic multiple reaction monitoring mode. The separation of 23 alkaloids was satisfactory, as PFP columns exhibited different retention behaviors than alkyl phase columns. The PFP column effectively retained polar aromatic compounds; therefore, it was suitable for alkaloid analysis. The validated method was applied to a forensic case of aconite poisoning. The present method was useful in LC/MS/MS screening for 23 alkaloids in human serum.

Direct Liquid Extraction and Ionization Techniques for Understanding Multimolecular Environments in Biological Systems (Secondary Publication).

A combination of direct liquid extraction using a small volume of solvent and electrospray ionization allows the rapid measurement of complex chemical components in biological samples and visualization of their distribution in tissue sections. This review describes the development of such techniques and their application to biological research since the first reports in the early 2000s. An overview of electrospray ionization, ion suppression in samples, and the acceleration of specific chemical reactions in charged droplets is also presented. Potential future applications for visualizing multimolecular environments in biological systems are discussed.

Enhancing enrichment ability of ZIF-8 mixed matrix membrane microextraction by reverse micelle strategy for analysis of multiple ionizable bioactive components in biological samples

Recent research aimed at the design of mixed-matrix membrane (MMM) to be used for microextraction emphasized on membrane extraction phase with high surface area and porosity. This study explored the influence that surfactants have on MMM extraction efficiency for the first time. The zeolitic imidazolate framework 8-based MMM (ZIF-8-MMM) was synthesized by in situ self-assembly of ZIF-8 on the inner wall of a hollow fiber membrane with the aim of fabricating a microextraction device. By prompting the encapsulation of ionizable analytes in the polar core of reverse micelles, the presence of surfactants in extraction solvent assisted the dissolution of analytes in the fiber membrane lumen and enhanced their adsorption onto ZIF-8. Notably, hereby a microextraction method based on the novel ZIF-8-MMM–reverse micelle (ZIF-8-MMM-RM) system was developed and employed for the extraction and quantitation of two alkaloids (berberine and jatrorrhizine) and two flavonoids (wogonin and wogonoside) in biological samples. The main factors affecting microextraction performance, identity of the extraction solvent, surfactant concentration, sample solution pH and extraction time, were investigated in detail. The method showed good linearity (r2 > 0.99) and repeatability (RSD < 10%), low limits of detection (0.10–0.31 ng mL−1) and high relative recoveries (90.03–98.84%). The enrichment factor values ranged between 48.47 and 54.96. Reverse micelle formation prompted by surfactant addition was demonstrated to effectively assist the extraction of multiple ionizable analytes from biological samples, resulting in a marked improvement of ZIF-8-MMM extraction performance.

Gold nanoparticle-based colorimetric method for the detection of prostate-specific antigen

BackgroundProstate-specific antigen (PSA), a serine protease, is a biomarker for preoperative diagnosis and screening of prostate cancer and monitoring of its posttreatment.MethodsIn this work, we reported a colorimetric method for clinical detection of PSA using gold nanoparticles (AuNPs) as the reporters. The method is based on ascorbic acid (AA)-induced in situ formation of AuNPs and Cu2+-catalyzed oxidation of AA. Specifically, HAuCl4 can be reduced into AuNPs by AA; Cu2+ ion can catalyze the oxidation of AA by O2 to inhibit the formation of AuNPs. In the presence of the PSA-specific peptide (DAHSSKLQLAPP)-modified gold-coated magnetic microbeads (MMBs; denoted as DAHSSKLQLAPP-MMBs), complexation of Cu2+ by the MMBs through the DAH–Cu2+ interaction depressed the catalyzed oxidation of AA and thus allowed for the formation of red AuNPs. However, once the peptide immobilized on the MMB surface was cleaved by PSA, the DAHSSKLQ segment would be released. The resultant LAPP fragment remaining on the MMB surface could not sequestrate Cu2+ to depress its catalytic activity toward AA oxidation. Consequently, no or less AuNPs were generated.ResultsThe linear range for PSA detection was found to be 0~0.8 ng/mL with a detection limit of 0.02 ng/mL. Because of the separation of cleavage step and measurement step, the interference of matrix components in biological samples was avoided.ConclusionThe high extinction coefficient of AuNPs facilitates the colorimetric analysis of PSA in serum samples. This work is helpful for designing of other protease biosensors by matching specific peptide substrates.

Development of a nano-SiO2 based enzyme-linked ligand binding assay for the determination of ibuprofen in human urine

The application domains of classic enzyme-linked ligand binding assay (ELBA) is relatively narrow due to the high cost and hardly available binding receptor. In here, we described for the first time the possibility of developing a new ELBA based on silica nanoparticles (nano-SiO2) to assess the ibuprofen in human urine. Nano-SiO2 with a large surface area was introduced as stationary phase to improve the analytical performance. In the experiment, a competitively binding procedure with human serum albumin (HSA) was performed between the ibuprofen presented in sample and horseradish peroxidase labeled ibuprofen (HRP-ibuprofen) subsequently added. After centrifugal separation, the HRP/ibuprofen/nano-SiO2 composite catalyzed the substrate solution (TMB/H2O2) with a color change from colorless to yellow for quantitative measurement via an ultraviolet spectrophotometer. As a validation of the new principle, the developed nano-ELBA method was applied in the determination of ibuprofen excreted in human urine with excellent performance. This detection range only depends on the solubility of ligand and sensitivity of UV spectrophotometer. Our results indicate that this new method demonstrated to be able to rapidly and adequately determine the concentration of components in biological samples and advocate its effectiveness for various applications.

Development of Highly Sensitive Analytical Methods for Biologically Relevant Materials and Their Pharmaceutical Applications

One important aspect of analytical chemistry research in the pharmaceutical sciences is the development of diagnostic and therapeutic analyses for disease, and the development of analytical methods for elucidating the causes of disease. I have been focusing on developing a highly sensitive method for measuring trace amounts of specific components in biological samples. This research can be roughly divided into three approaches: the use of immunoassays and DNA hybridization as methods utilizing specific affinities, the use of capillary electrophoresis as a highly sensitive and rapid separation method, and the use of chemiluminescence and bioluminescence reactions. The components being measured are compounds such as hormones, tumor markers, drugs, reactive oxygen species and genes in biological samples for the purpose of developing therapies for the prevention and treatment of diseases.

Determination of Glycoproteins by a Self-Assembled 4-Mercaptophenylboronic Acid Film on a Quartz Crystal Microbalance.

Glycosylation plays an important part in many biological processes. However, many glycoproteins are either of low abundance or covered by other components in biological samples. Hence, developing new methods to measure the glycoproteins with both high efficiency and low detection limit is important. In this work, a self-assembled 4-mercaptophenylboronic acid film was coated on a quartz crystal microbalance chip. By optimizing the reaction time and the concentration of 4-mercaptophenylboronic acid, a sensor that specifically responded to glycoproteins was created. Then, several parameters for the prepared sensor were investigated and the working curve for representative glycoprotein-transferrin was established. The linearity range was from 50 to 400 ng/mL and the detection limit was 21.0 ng/mL. The sensor was used to detect transferrin in artificial urine samples. This sensor has a low detection limit of glycoproteins requiring only a small amount of samples, and thus has potential applications in both pharmaceutical and medical areas.

Automatic flow injection analysis (FIA) determination of total reducing capacity in serum and urine samples.

Automation of total antioxidant capacity assessment can substantially increase the determination throughput, allowing large scale studies and screening experiments. Total reducing capacity evaluation can be implemented under different chemistries, including the CUPRAC-Cupric Ion Reducing Antioxidant Capacity -assay. This assay is based on reduction of Cu(II)-neocuproine complex to highly colored Cu(I)-neocuproine complex by reducing (antioxidant) components of biological samples. In this chapter, we propose an automatic flow injection method for evaluation of total reducing capacity in serum and urine samples, attaining end-point data within 4 min using a kinetic matching strategy.

Self‐organizing maps: A versatile tool for the automatic analysis of untargeted imaging datasets

MS-based imaging approaches allow for location-specific identification of chemical components in biological samples, opening up possibilities of much more detailed understanding of biological processes and mechanisms. Data analysis, however, is challenging, mainly because of the sheer size of such datasets. This article presents a novel approach based on self-organizing maps, extending previous work in order to be able to handle the large number of variables present in high-resolution mass spectra. The key idea is to generate prototype images, representing spatial distributions of ions, rather than prototypical mass spectra. This allows for a two-stage approach, first generating typical spatial distributions and associated m/z bins, and later analyzing the interesting bins in more detail using accurate masses. The possibilities and advantages of the new approach are illustrated on an in-house dataset of apple slices.

Characterization of Peucedani Radix extract-derived angular-type pyranocoumarins in rats using ultra-high performance liquid chromatography coupled with hybrid triple quadrupole-linear ion trap mass spectrometry

The method adopting the pMIM-IDA-EPI system was demonstrated to be reliable and can be introduced as an alternative procedure for rapid characterization of herbal medicine-derived components in biological samples.

Systematic Analysis of Main Constituents in Rat Biological Samples after Oral Administration of the Methanol Extract of Fructus Aurantii by HPLC-ESI-MS/MS.

High performance liquid chromatography (HPLC) with diode array detection (DAD) and electrospray ionization tandem mass spectrometry (ESI/MS/MS) was used to analyze the main components in the methanol extract of Fructus Aurantii (FA) and the metabolites in rat biological samples after oral administration of the methanol extract of FA. There were 31 constituents identified in the extract of FA including 2 alkaloids, 1 coumarin, 10 flavonoid glycosides and 18 ploymethoxylated flavones. According to the UV spectrum and MS fragment character of main components in the methanol extract of FA, 18 parent constituents and 11 metabolites were tentatively identified in rat biological samples. Three groups of components in biological samples detected included flavonoid glycosides, their glucuronides and ploymethoxylated flavones. It was interested that flavonoid glycosides, their glucuronides and ploymethoxylated flavones can be investigated in rat plasma and urine, while in rat feces samples only flavonoid glycosides were detected. Triglycosyl, naringenin, neoeriocitrin, neoeriocitrin narirutin and hesperidin were the main components in rat feces which were found either in the plasma or in urine samples. However, naringin and neohesperidin were the main flavonoid glycosides which absorbed after oral administration. Except flavonoid glycosides and their glucuronides, ploymethoxylated flavones also the constituents absorbed because it was investigated mainly in rat plasma and urine but not in feces samples. The identification and elucidation of parent and metabolism components analyzed in biological samples provided the data for further pharmacological and clinical research on FA.

Application of gas chromatography–triple quadrupole mass spectrometry to the determination of sterol components in biological samples in consideration of the ionization mode

The hyphenation of gas chromatography (GC) and triple quadrupole mass spectrometry is a promising approach to increase sensitivity and selectivity as compared to single quad mass spectrometry. We present in this paper the application of GC-triple quadrupole mass spectrometry for determination of sterol components in biological samples. Due to the fact that sterols are quite small molecules an appropriate ionization mode has to be found for advantageous exploitation of the triple quad function. Electron ionization (EI), positive and negative chemical ionization (PCI, NCI) have been tested regarding sensitivity improvement in oxysterol and bile acid analysis in plasma samples. Target analytes were 24-, 25- and 27-hydroxycholesterol, 7β-hydroxycholesterol, 7-ketocholesterol, 3β,5α,6β-cholestanetriol, cholic acid, chenodeoxycholic acid, deoxycholic acid and lithocholic acid. In contrast to bile acids, oxysterols could be analyzed with the highest degree of sensitivity by application of PCI in multiple reaction monitoring mode whereas 7β-hydroxycholesterol and 7-ketocholesterol showed even better results with NCI.

Influence of salts and pcr inhibitors on the amplification capacity of three thermostable DNA polymerases

The full potential of PCR as a rapid DNA detection method, is limited by inhibition of thermostable DNA polymerases by components in biological samples and substances used for purification of template DNA. We compared the inhibition effect of blood, phenol and various ions on Taq, Tth and Tne thermostable DNA polymerases prepared by us. The amplification capacity of DNA polymerases was tested on the amplification of a 631 bp fragment of a â- globin gene from a 100 ng DNA template under the optimal PCR buffer. Blood above 1 % (v/v) and phenol above 0.1 % (v/v) inhibited Taq, while Tne and Tth tolerated 10 to 30 times more. The inhibitory effect of ions is lowest for the Tth DNA polymerase, followed by Taq and Tne DNA polymerase. We conclude that the PCR inhibiting effect of some substances to Taq DNA polymerase can be eliminated by the use of a more resistant thermostable DNA polymerase, such as Tth or Tne DNA polymerases.

Long‐term stability of serum components in the Janus Serum Bank

Objective. Biobank material is frequently used in epidemiological studies, but long‐term storage of serum at −25°C may reduce the quality of the samples. Knowledge about the stability of components in biological samples is fundamental for the interpretation of such studies. Material and methods. We investigated the stability of seven biological components in serum samples stored at −25°C for 25 and 2 years compared with 1‐month‐old samples. Specimens from 130 blood donors from each group were randomly selected among men without a cancer diagnosis during the follow‐up time. We compared the distribution, dispersion and localization of medians and means, and established reference intervals of the components. Results. The study demonstrated non‐significant and numerically small differences in the levels of sodium, calcium, iron and creatinine over time. Differences between mean values for uric acid (−7.6%), potassium (+26.4%) and bilirubin (−59.4%) between fresh and 25‐year‐old samples indicated that sample handling before freezing and degradation during long‐term storage may introduce a considerable bias for vulnerable components. Conclusions. There is large variation in level stability between different serum components in serum stored at −25°C. Differences in sample handling before freezing may introduce bias on vulnerable components. The present study supports a routine of careful matching of cases and controls on storage time in epidemiological studies when biobank material is used.

Sensitive HPLC method for quantitation of paclitaxel (Genexol ®) in biological samples with application to preclinical pharmacokinetics and biodistribution

A sensitive, specific and reproducible HPLC method has been developed and validated for the quantitative determination of paclitaxel in plasma, tissues and tumor of mice. Tissue specimens including liver, kidneys, spleen, lungs, heart and tumor were separately homogenized in bovine serum albumin (BSA, 40 g/l) in water. Plasma or tissue homogenates (0.1 ml) containing paclitaxel and internal standard (dimethyl-4,4′-dimethoxy-5,6,5′,6′-dimethylene dioxy biphenyl-2′,2′ dicarboxylate (DDB), I.S.) were extracted by ethyl acetate (10 ml). A 4.6 mm × 250 mm ODS column was used to separate the components in biological samples with UV detection at 227 nm and gradient system was applied to a quantitation of paclitaxel consisting of acetonitrile-deionized water. The I.S. and paclitaxel were eluted at 13.7 and 18.0 min, respectively, and no interfering peaks were observed. Linear relationships ( r 2 > 0.999) were obtained between the peak height ratios and the corresponding biological sample concentrations over the range of 0.1–20 μg/ml. The average intra- and inter-day variations (% R.S.D.s and % deviations) of the assay for biological samples were less than 10%. The LOD and LOQ were 5 and 10 ng/ml, respectively, for paclitaxel using a microsample volume (100 μl) of plasma sample. This HPLC method has been successfully applied for the determination of paclitaxel in pharmacokinetic and biodistribution study in after administration of 50 mg equivalent paclitaxel/kg dose of paclitaxel-loaded polymeric micelle and 20 mg equivalent paclitaxel/kg dose of Taxol ® to female SPF C57BL/6 mice.

Characterization of N-Terminal processing of group VIA phospholipase A 2 and of potential cleavage sites of amyloid precursor protein constructs by automated identification of signature peptides in LC/MS/MS analyses of proteolytic digests

Dysregulation of proteolytic processing of the amyloid precursor protein (APP) contributes to the pathogenesis of Alzheimer's Disease, and the Group VIA phospholipase A(2) (iPLA(2)beta) is the dominant PLA(2) enzyme in the central nervous system and is subject to regulatory proteolytic processing. We have identified novel N-terminal variants of iPLA(2)beta and previously unrecognized proteolysis sites in APP constructs with a C-terminal 6-myc tag by automated identification of signature peptides in LC/MS/MS analyses of proteolytic digests. We have developed a Signature-Discovery (SD) program to characterize protein isoforms by identifying signature peptides that arise from proteolytic processing in vivo. This program analyzes MS/MS data from LC analyses of proteolytic digests of protein mixtures that can include incompletely resolved components in biological samples. This reduces requirements for purification and thereby minimizes artifactual modifications during sample processing. A new algorithm to generate the theoretical signature peptide set and to calculate similarity scores between predicted and observed mass spectra has been tested and optimized with model proteins. The program has been applied to the identification of variants of proteins of biological interest, including APP cleavage products and iPLA(2)beta, and such applications demonstrate the utility of this approach.

The levels of kerosene components in biological samples after repeated dermal exposure to kerosene in rats

The current study was experimentally investigated using rats whether or not kerosene components are accumulated from daily repeated dermal exposure. Rats received daily 1 h-exposure to kerosene for 5 days (5K), daily 1 h-exposure for 4 days and left for 1 day (4KL), a single 1 h-exposure (1K), a single 1 h-exposure and left for 1 day (1KL), or a single 1 h-exposure, sacrificed and left dead for 1 day (1KLD). Kerosene components, trimethylbenzenes (TMBs) and aliphatic hydrocarbons (AHCs) in blood and tissues were determined by GC-MS. In blood, almost the same concentrations of TMBs were detected in the rats sacrificed immediately after exposure (5K, 1K and 1KLD), and only trace levels were detected in the rats sacrificed 1 day after exposure (4 and 1KL). Almost the same levels of AHCs in blood were detected among groups except for the rats sacrificed 1 day after a single exposure (1KL), in which AHCs were slightly lower. These results suggest that (1) AHCs tend to be accumulated from daily exposure, while TMBs do not, (2) the proportions of detected kerosene components in blood can be an indicator of whether the last exposure occurred just before death or not, (3) the kerosene levels last at least 1 day without blood circulation.