Development of a solid‐phase microextraction LC–MS/MS method for determination of oxidative stress biomarkers in biofluids

Recently the connection between oxidative stress and various diseases, including cancer and Alzheimer's, attracts notice as a pathway suitable for diagnostic purposes. 8‐Oxo‐deoxyguanosine and 8‐oxo‐deoxyadenosine produced from the interaction of reactive oxygen species with DNA become prominent as biomarkers. Several methods have been developed for their determination in biofluids, including solid‐phase extraction and enzyme‐linked immunosorbent assays. However, still, there is a need for reliable and fast analytical methods. In this context, solid‐phase microextraction offers many advantages such as flexibility in geometry and applicable sample volume, as well as high adaptability to high‐throughput sampling. In this study, a solid‐phase microextraction method was developed for the determination of 8‐oxo‐deoxyguanosine and 8‐oxo‐deoxyadenosine in biofluids. The extractive phase of solid‐phase microextraction consisted of hydrophilic–lipophilic balanced polymeric particles. In order to develop a solid‐phase microextraction method suitable for the determination of the analytes in saliva and urine, several parameters, including desorption solvent, desorption time, sample pH, and ionic strength, were scrutinized. Analytical figures of merit indicated that the developed method provides reasonable interday and intraday precisions (<15% in both biofluids) with acceptable accuracy. The method provides a limit of quantification for both biomarkers at 5.0 and 10.0 ng/mL levels in saliva and urine matrices, respectively.     

Analysis of bile acids in human biological samples by microcolumn separation techniques: A review

Bile acids are a group of compounds essential for lipid digestion and absorption with a steroid skeleton and a carboxylate side chain usually conjugated to glycine or taurine. Bile acids are regulatory molecules for a number of metabolic processes and can be used as biomarkers of various disorders. Since the middle of the twentieth century, the detection of bile acids has evolved from simple qualitative analysis to accurate quantification in complicated mixtures. Advanced methods are required to characterize and quantify individual bile acids in these mixtures. This article overviews the literature from the last two decades (2000–2020) and focuses on bile acid analysis in various human biological samples. The methods for sample preparation, including the sample treatment of conventional (blood plasma, blood serum, and urine) and unconventional samples (bile, saliva, duodenal/gastric juice, feces, etc.) are shortly discussed. Eventually, the focus is on novel analytical approaches and methods for each particular biological sample, providing an overview of the microcolumn separation techniques, such as high-performance liquid chromatography, gas chromatography, and capillary electrophoresis, used in their analysis. This is followed by a discussion on selected clinical applications.     

Liquid-phase microextraction of biomarkers: A review on current methods

The detection and quantification of biomarkers have gained more attention in the medical discipline to evaluating disease progression to manage medical treatment. Biomarkers range from gases to biological macromolecules. Because of the nanomolar range levels of typical biomarkers in plasma, blood, urine, exhalation samples, and other biological fluids as well as complex matrix of biological media, adequate sample preparation methods should be used for quantification of biomarkers. Biomarkers are discussed here generally classified mainly into two subgroups which arisen from disease or exposure compounds. The analytical method is critical for the validity/reliability of a biomarker. Accuracy, precision, reproducibility, recovery, sensitivity, and specificity all have high influence to the consistency with the limit and reference values concerned. In this paper, developments in well-established liquid-phase microextraction techniques for the clinical analysis of biological samples will be reviewed and discussed. This article presents an overview of microextraction methods for biological samples, focusing especially on biomarkers.     

Simultaneous Quantification of Multiple Cancer Biomarkers in Blood Samples through DNA‐Assisted Nanopore Sensing

Protein biomarkers in blood have been widely used in the early diagnosis of disease. However, simultaneous detection of many biomarkers in a single sample remains challenging. Herein, we show that the combination of a sandwich assay and DNA‐assisted nanopore sensing could unambiguously identify and quantify several antigens in a mixture. We use five barcode DNAs to label different gold nanoparticles that can selectively bind specific antigens. After the completion of the sandwich assay, barcode DNAs are released and subject to nanopore translocation tests. The distinct current signatures generated by each barcode DNA allow simultaneous quantification of biomarkers at picomolar level in clinical samples. This approach would be very useful for accurate and multiplexed quantification of cancer‐associated biomarkers within a very small sample volume, which is critical for non‐invasive early diagnosis of cancer.     

Ultrasonic frequency analysis of antibody-linked hydrogel biosensors for rapid point of care testing

Analyte quantification in highly scattering media such as tissue, blood, and other biological fluids is challenging using conventional spectroscopic methods. Ultrasound easily penetrates these opaque samples, yet currently provides little chemical information. We have developed a general approach for creating hydrogel biosensors based on antibody-linked cellulose polymers. Target recognition induces changes to the sensor stiffness and size, which is accompanied by characteristic changes to a measured ultrasonic frequency profile. Using this technique, nM sensitivity for acetaminophen is demonstrated in a series of biofluids including whole blood, blood plasma, saliva, and urine. Likewise, this methodology is attractive for point of care diagnostics due to the short measurement time, simple methodology which excludes pretreatment of samples, and has minimal chemical or buffer requirements.     

Quantitative proteomics for cancer biomarker discovery

The mass spectrometry (MS)-based quantitative proteomics is powerful to discover disease biomarkers that can provide diagnostic, prognostic and therapeutic targets, and it also can address important problems in clinical and translational medical research. The current status of MS-based quantification strategy and technical advances of several main quantitative assays (two-dimensional (2-D) gel-based methods, stable isotope labeling with amino acids in cell culture (SILAC), isotope-coded affinity tag (ICAT), the isobaric tags for relative and absolute quantification (iTRAQ), 1?O labeling, absolute quantitation and label-free quantitation) have been summarized and reviewed. At present, except 2-D gel-based methods, several stable isotope labeling quantitative techniques, including SILAC, ICAT and iTRAQ, etc, have been widely applied in identification of differential expression of proteins, post-translational modifications and protein-protein interactions in order to look for novel candidate cancer biomarkers from different physiological states of cells, body fluids or tissue samples. Also, the advantages and challenges of different quantitative proteomic approaches are discussed in identification and validation of candidate targets.     

微流控芯片系统在循环肿瘤细胞分离检测中的应用进展

循环肿瘤细胞(CTCs)的分离分析一直是肿瘤相关研究中的热点方向,作为液体活检的重要标志物之一,其在外周血中的含量与癌症病发状况密切相关.然而人体血液中CTCs的含量非常低,通常来说仅有0～10个/mL,因此在开展临床血液样本中CTCs的检测前,往往需要对样本进行前处理,以实现CTCs的分离和富集.微流控芯片技术凭借样...     

Application of NMR metabolomics to search for human disease biomarkers

Since antiquity, humans have used body fluids like saliva, urine and sweat for the diagnosis of diseases. The amount, color and smell of body fluids are still used in many traditional medical practices to evaluate an illness and make a diagnosis. The development and application of analytical methods for the detailed analysis of body fluids has led to the discovery of numerous disease biomarkers. Recently, mass spectrometry (MS), nuclear magnetic resonance spectroscopy (NMR), and multivariate statistical techniques have been incorporated into a multidisciplinary approach to profile changes in small molecules associated with the onset and progression of human diseases. The goal of these efforts is to identify metabolites that are uniquely correlated with a specific human disease in order to accurately diagnose and treat the malady. In this review we will discuss recent developments in sample preparation, experimental techniques, the identification and quantification of metabolites, and the chemometric tools used to search for biomarkers of human diseases using NMR.     

Coupling Sensitive Nucleic Acid Amplification with Commercial Pregnancy Test Strips

The detection of nucleic acid biomarkers for point‐of‐care (POC) diagnostics is currently limited by technical complexity, cost, and time constraints. To overcome these shortcomings, we have combined loop‐mediated isothermal amplification (LAMP), programmable toehold‐mediated strand‐exchange signal transduction, and standard pregnancy test strips. The incorporation of an engineered hCG–SNAP fusion reporter protein (human chorionic gonadotropin‐O⁶‐alkylguanine‐DNA alkyltransferase) led to LAMP‐to‐hCG signal transduction on low‐cost, commercially available pregnancy test strips. Our assay reliably detected as few as 20 copies of Ebola virus templates in both human serum and saliva and could be adapted to distinguish a common melanoma‐associated SNP allele (BRAF V600E) from the wild‐type sequence. The methods described are completely generalizable to many nucleic acid biomarkers, and could be adapted to provide POC diagnostics for a range of pathogens.     

醛类标志物的化学衍生化色谱-质谱分析方法进展

人体接触环境中的化学污染物会导致多种疾病,包括癌症、糖尿病、心血管疾病、神经退行性疾病(阿尔茨海默症、帕金森病等)等。作为一类具有高反应活性的亲电化合物,醛类(包括外源性醛类或环境污染物暴露后产生的内源性醛类)可与人体中多种重要生物分子形成共价修饰产物而产生毒害作用。暴露组研究自2005年被首次提出以来一直是一个前沿热门领域,暴露组研究可绘制生物标志物与疾病风险之间的复杂关系,因此,所有生物标志物的可测量的和特征性的变化共同构成了暴露组研究的关键基础。醛类是化学暴露组的主要成分之一。由于醛类化合物自身物理化学性质和样品大量基质干扰存在,对它们进行分析和表征特别困难。醛类化合物的分析检测方法主要有传感分析法、电化学法、荧光成像、色谱法、质谱法、色谱-质谱联用法等。基于色谱-质谱的分析技术已成为化学暴露组研究的主要方法之一。化学衍生化,特别是稳定同位素标记衍生化(亦称化学同位素标记)结合液相色谱-质谱(LC-MS)技术能够解决靶向和非靶向代谢组和暴露组分析工作中的诸多问题。化学衍生化联合色谱-质谱的分析策略是复杂体系中醛类精准分析非常重要的解决方案之一。特别是近5年,基于化学衍生化的色谱-质谱分析方法开发与应用已成为醛类分析方法中的热点和亮点。该文主要总结与评述了近5年基于化学衍生化的气相色谱-质谱(GC-MS)和LC-MS最新进展,重点关注生物基质(血液、尿液、唾液、生物组织等)中醛类暴露标志物的分析方法进展。通过探讨标记小分子醛的各种衍生试剂、定性/定量分析方法及应用价值,评述醛类暴露标志物不同分析方法的优缺点以及未来发展趋势,为暴露组学、代谢组学、脂质组学的整合发展和环境生态健康研究提供一定的帮助。为了阐明外源性和内源性醛类化合物在生理和病理事件中所起的复杂作用,需要大力改进研究醛组学(aldehydome)的分析表征技术和工具。随着更先进的质谱仪的研发和使用,以及高效色谱分离和不断进步的生物信息学手段,并同时伴随着单细胞分析、质谱成像的兴起,未来的醛类暴露组分析方法会具有更高的灵敏度、更高的分析通量,更有希望筛选鉴定未知醛类化合物并发现新的暴露组生物标志物。     

Integrated dielectrophoretic and impedimetric biosensor provides a template for universal biomarker sensing in clinical samples

The detection and quantification of nucleic acid and proteomic biomarkers in bodily fluids is a critical part of many medical screening and diagnoses. However, majority of the current detection platforms are not ideal for routine, rapid, and low-cost testing in point-of-care settings. To address this issue, we developed a concept for a disposable universal point-of-care biosensor that can detect and quantify nucleic acid and proteomic biomarkers in diluted serum samples. The central tenet of sensing is the use of dielectrophoresis, electrothermal effects, and thermophoresis to selectively and rapidly isolate the biomarkers of interest in electrodes and then quantify using electrical impedance. When the sensor was applied to quantify microRNA and antigen biomarker molecules directly in diluted serum samples, it produced a LOD values in the fM range and sensitivity values from 10¹² to 10¹⁵ Ω/M with a 30 min assay time and assay cost of less than $50 per assay.     

Strategy for identification and detection of multiple oxidative modifications within proteins applied on persulfate-oxidized hemoglobin and human serum albumin

Oxidative stress has been suggested as an underlying mechanism of many human diseases. However, definitive evidence for this association has not been presented due to different shortcomings of the methods used to measure biomarkers of oxidative stress. Persulfates are oxidizing agents known to elicit hypersensitive reactions from the airways and skin. Despite a frequent use of persulfates at many work places, no biomarkers for persulfate exposure are available. The aim of this study was to develop a strategy for the identification and detection of multiple oxidative modifications within proteins. This strategy was applied on persulfate-oxidized proteins to identify oxidized peptides suitable for further investigation as biomarkers of persulfate exposure or oxidative stress. A strategy for the identification and the relative quantification of multiple oxidative modifications within proteins was developed. The usage of two software packages facilitated the search for modified peptides to a great extent. Oxidized peptides were relatively quantified using liquid chromatography/tandem mass spectrometry in selected reaction monitoring mode. The result showed that persulfates oxidize tryptophans and methionines resulting in mass shifts of 16 and/or 32 Da. Also, oxidized albumin peptides in nasal lavage fluid samples from subjects challenged with persulfate were detected. The oxidation degree before and after challenge remained constant for peptides containing methionine sulfoxide. For peptides containing oxidized tryptophan the oxidation degree increased after exposure. Some of these oxidized peptides may be suitable as biomarkers; however, further evaluation is required.     

Identifying critical states of hepatocellular carcinoma based on landscape dynamic network biomarkers

Hepatocellular carcinoma (HCC) is the major histological form of primary liver cancer. It has usually reached the disease state once the patient is diagnosed since there are no specific symptoms in the early stages of HCC. This fact increases the difficulty of curing HCC. Recently, quantities of evidence have shown that many mathematical methods (such as dynamic network biomarkers, DNB) can be used to detect critical states or tipping points of complex diseases. However, it is difficult to apply the DNB theory to the clinic since multiple samples are generally unavailable for individual patient. This paper constructs a novel method based on landscape dynamic network biomarkers (L-DNB), which aims to detect early warning signals from cirrhosis state to very advanced HCC state in individual patient. The selected dataset contains multiple samples for each HCC state. A score that indicates the disease characteristics is calculated for each sample by RNA-seq data, and several scores constitute a distribution in the same state. Quantifying the statistical characteristics of these distributions and determining that low-grade dysplastic and high-grade dysplastic are the critical states of HCC. These results can provide scientific advice for early warning indicators and optimal treatment time for HCC.     

Measurement of xenobiotics in saliva: is saliva an attractive alternative matrix? Case studies and analytical perspectives

The use of saliva for measuring xenobiotic concentrations has been practiced for a number of years. While the use of saliva has been generally reserved for the analysis of diagnostic and forensic/toxicology samples, attempts have been made to further enhance the value of saliva as an alternate matrix to those of plasma and serum. It is understood that saliva represents a handy tool for therapeutic drug monitoring (TDM) as it offers certain distinctive advantages. This scope of this review encompasses the following: (a) a comprehensive view of saliva as an alternate matrix for either plasma or serum to understand the pharmacokinetic/pharmacodynamic (PK/PD) characteristics; (b) an account of the factors contributing to the observed variability in salivary monitoring; (c) a tabular compilation of diverse case studies of xenobitoics belonging to different therapeutic classes with emphasis on assay methodology and applicable analytical/biopharmaceutical/pharmacokinetic findings; (d) relevant thoughts on assay procedures as they relate to salivary monitoring; and (e) some representative case studies highlighting the new thinking on the use of saliva outside of traditional TDM. Overall, based on the review, saliva represents a valuable TDM tool for a number of xenobiotics. While parent compound and phase I metabolite(s) for many xenobiotics have been generally quantifiable in saliva, phase II metabolites have not generally been detected in saliva. Therefore saliva samples could also be used to answer some specific PK/PD questions during the drug development process, if applicable. However, the development and validation of the assay in saliva needs to be carried out carefully with particular focus on proper sample collection, processing and storage to ensure the stability of the xenobiotics and with the same rigor as applied to plasma, serum and urine matrices.     

Label-free nanoplasmonic sensing of tumor-associate autoantibodies for early diagnosis of colorectal cancer

Colorectal cancer is treatable and curable when detected at early stages. However there is a lack of less invasive and more specific screening and diagnosis methods which would facilitate its prompt identification. Blood circulating autoantibodies which are immediately produced by the immune system at tumor appearance have become valuable biomarkers for preclinical diagnosis of cancer. In this work, we present the rapid and label-free detection of colorectal cancer autoantibodies directly in blood serum or plasma using a recently developed nanoplasmonic biosensor. Our nanoplasmonic device offers sensitive and real-time quantification of autoantibodies with excellent selectivity and reproducibility, achieving limits of detection around 1 nM (150–160 ng mL⁻¹). A preliminary evaluation of clinical samples of colorectal cancer patients has shown good correlation with ELISA. These results demonstrate the reliability of the nanobiosensor strategy and pave the way towards the achievement of a sensitive diagnostic tool for early detection of colorectal cancer.     

High-throughput biomarker discovery and identification by mass spectrometry

Native peptides and proteins are of increasing interest in biomedical research because they hold promise to represent a large number of useful diagnostic and therapeutic biomarkers. Discovery attempts from patient samples have to deal with the complexity of biology from a disease perspective as well as with a high individual variability. High throughput screening of samples is therefore the strategy of choice to detect relevant peptidic biomarkers, and requires a high order of automation particularly in the detection process. In this contribution, a novel technical approach employing a fully automated MALDI-TOF/TOF mass spectrometer is described. This approach combines high throughput biomarker discovery with the identification of corresponding endogenous peptides in one instrument and from the same set of samples. The degree of automation allows the analysis of thousands of chromatographic fractions corresponding to up to one hundred patient samples per day. The applied relative quantification via Differential Peptide Display((R)) is performed in a label-free way and shows a dynamic range of up to four orders of magnitude in the accessible peptide concentrations. The typical limit of detection is in the mid- to low-picomolar range for body fluids such as blood plasma, urine and cerebrospinal fluid. Sequence assignment via MALDI-TOF/TOF mass spectrometry is carried out either in an overview approach, characterizing rapidly the peptide composition e.g. of a novel sample, or in a directed approach, analyzing a list of biomarker candidates deduced from statistically significant abundance differences from the biomarker discovery process.     

cyc-DEP: Cyclic immunofluorescence profiling of particles collected using dielectrophoresis

Cancer is a highly heterogenous disease that requires precise detection tools and active surveillance methods. Liquid biopsy assays provide an agnostic way to follow the complex trajectory of cancer, providing better patient stratification tools for optimized treatment. Here, we present the development of a low-volume liquid biopsy assay called cyc-DEP (cyclic immunofluorescent imaging on dielectrophoretic chip) to profile biomarkers collected on a dielectrophoretic microfluidic chip platform. To enable on-chip cyclic imaging, we optimized a fluorophore quenching method and sequential rounds of on-chip staining with fluorescently conjugated primary antibodies. cyc-DEP allows for the quantification of a multiplex array of proteins using 25 µl of a patient plasma sample. We utilized nanoparticles from a prostate adenocarcinoma (LNCaP) cell line and a panel of six target proteins to develop our proof-of-concept technique. We then used cyc-DEP to quantify blood plasma levels of target proteins from healthy individuals, low-grade and high-grade prostate cancer patients (n = 3 each) in order to demonstrate that our platform is suitable for liquid biopsy analysis in its present form. To ensure accurate quantification of signal intensities and comparisons between different samples, we incorporated a signal intensity normalization method (fluorescent beads) and a custom signal intensity quantification algorithm that account for the distribution of signal across hundreds of collection regions on each chip. Our technique enabled a threefold improvement in multiplicity for detecting proteins associated with fluid samples, opening doors for early detection, and active surveillance through quantification of a multiplex array of biomarkers from low-volume liquid biopsies.     

Validity of extracellular water assessment with saliva samples using plasma as the reference biological fluid

Extracellular water (ECW) assessment is based on dilution techniques, commonly using blood sampling. However, plasma collection is an invasive procedure. We aimed to validate the use of saliva for ECW estimation by the bromide dilution technique using plasma as the reference method, in a sample of elite athletes. A total of 89 elite athletes with a mean age of 20.4 ± 4.4 years were evaluated. Baseline samples were collected before sodium bromide oral dose administration, and enriched samples were collected 3 h post‐dose administration. The bromide concentration was assessed by high‐performance liquid chromatography. Comparison of means, concordance coefficient correlation (CCC), multiple regression and Bland–Altman analysis were performed. The ECW from saliva explained 91% of the variance in ECW by plasma with a standard error of estimation of 0.91 kg. The CCC between alternative and reference methods was 0.952. No significant trend was observed between the mean and difference of the methods, with limits of agreement ranging between ?1.5 and 2.1 kg. These findings reveal that bromide dilution volume calculated from saliva samples is a valid noninvasive method for ECW assessment in elite athletes. Copyright © 2012 John Wiley & Sons, Ltd.     

Quantitative and sensitive detection of rare mutations using droplet-based microfluidics

Somatic mutations within tumoral DNA can be used as highly specific biomarkers to distinguish cancer cells from their normal counterparts. These DNA biomarkers are potentially useful for the diagnosis, prognosis, treatment and follow-up of patients. In order to have the required sensitivity and specificity to detect rare tumoral DNA in stool, blood, lymph and other patient samples, a simple, sensitive and quantitative procedure to measure the ratio of mutant to wild-type genes is required. However, techniques such as dual probe TaqMan(?) assays and pyrosequencing, while quantitative, cannot detect less than ～1% mutant genes in a background of non-mutated DNA from normal cells. Here we describe a procedure allowing the highly sensitive detection of mutated DNA in a quantitative manner within complex mixtures of DNA. The method is based on using a droplet-based microfluidic system to perform digital PCR in millions of picolitre droplets. Genomic DNA (gDNA) is compartmentalized in droplets at a concentration of less than one genome equivalent per droplet together with two TaqMan(?) probes, one specific for the mutant and the other for the wild-type DNA, which generate green and red fluorescent signals, respectively. After thermocycling, the ratio of mutant to wild-type genes is determined by counting the ratio of green to red droplets. We demonstrate the accurate and sensitive quantification of mutated KRAS oncogene in gDNA. The technique enabled the determination of mutant allelic specific imbalance (MASI) in several cancer cell-lines and the precise quantification of a mutated KRAS gene in the presence of a 200,000-fold excess of unmutated KRAS genes. The sensitivity is only limited by the number of droplets analyzed. Furthermore, by one-to-one fusion of drops containing gDNA with any one of seven different types of droplets, each containing a TaqMan(?) probe specific for a different KRAS mutation, or wild-type KRAS, and an optical code, it was possible to screen the six common mutations in KRAS codon 12 in parallel in a single experiment.