Search

The proposed miniaturized biosensor, the dual-channel multi-analyte single polarization (DCSP) photonic crystal fiber (PCF) sensor, offers real-time measuring and high sensitivity, making it a promising candidate for future sensing devices. It was structured using the stack-and-draw technique to show the sensor practical feasibility. The DCSP PCF sensor offers maximum wavelength and amplitude sensitivities of 11,000 nm/RIU and 807 RIU−1, respectively in both channels for the analyte refractive index (RI) of na = 1.33 to 1.41. This DCSP PCF sensor has a birefringence of 11.2 × 10 - 4 . The use of DCSP PCF sensors improves the sensitivity to wavelength in detecting multiple analytes in real time. These sensors are suitable for biosensing applications, especially when working with small sample sizes. Additionally, DCSP PCF sensors operate at a single polarization, resulting in lower confinement loss and better wavelength resolution, which enhances their performance. These findings suggest that DCSP PCF sensors have the potential to enhance future sensing technology and improve the detection of diseases and liquid substances. [ABSTRACT FROM AUTHOR]

The potential of liquid biopsy for the prognosis and diagnosis of diseases is unquestionable. Within the evolving landscape of disease diagnostics and personalized medicine, circulating microRNAs (c-miRNAs) stand out among the biomarkers found in blood circulation and other biological fluids due to their stability, specificity, and non-invasive detection in biofluids. However, the complexity of human diseases and the limitations inherent in single-marker diagnostics highlight the need for a more integrative approach. It has been recently suggested that a multi-analyte approach offers advantages over the single-analyte approach in the prognosis and diagnosis of diseases. In this review, we explore the potential of combining three well-studied classes of biomarkers found in blood circulation and other biofluids—miRNAs, DNAs, and proteins—to enhance the accuracy and efficacy of disease detection and monitoring. Initially, we provide an overview of each biomarker class and discuss their main advantages and disadvantages highlighting the superiority of c-miRNAs over the other classes of biomarkers. Additionally, we discuss the challenges and future directions in integrating these biomarkers into clinical practice, emphasizing the need for standardized protocols and further validation studies. This integrated approach has the potential to revolutionize precision medicine by offering insights into disease mechanisms, facilitating early detection, and guiding personalized therapeutic strategies. The collaborative power of c-miRNAs with other biomarkers represents a promising frontier in the comprehensive understanding and management of complex diseases. Nevertheless, several challenges must be addressed before this approach can be translated into clinical practice. [ABSTRACT FROM AUTHOR]

The COVID-19 pandemic has emphasized the urgent need for point-of-care methods suitable for the rapid and reliable diagnosis of viral infections. To address this demand, we report the rapid, label-free simultaneous determination of two SARS-CoV-2 proteins, namely, the nucleoprotein and the receptor binding domain peptide of S1 protein, by implementing a bioanalytical device based on Multi Area Reflectance Spectroscopy. Simultaneous detection of these two proteins is achieved by using silicon chips with adjacent areas of different silicon dioxide thickness on top, each of which is modified with an antibody specific to either the nucleoprotein or the receptor binding domain of SARS-CoV-2. Both areas were illuminated by a single probe that also collected the reflected light, directing it to a spectrometer. The online conversion of the combined reflection spectra from the two silicon dioxide areas into the respective adlayer thickness enabled real-time monitoring of immunoreactions taking place on the two areas. Several antibodies have been tested to define the pair, providing the higher specific signal following a non-competitive immunoassay format. Biotinylated secondary antibodies and streptavidin were used to enhance the specific signal. Both proteins were detected in less than 12 min, with detection limits of 1.0 ng/mL. The assays demonstrated high repeatability with intra- and inter-assay coefficients of variation lower than 10%. Moreover, the recovery of both proteins from spiked samples prepared in extraction buffer from a commercial self-test kit for SARS-CoV-2 collection from nasopharyngeal swabs ranged from 90.0 to 110%. The short assay duration in combination with the excellent analytical performance and the compact instrument size render the proposed device and assay suitable for point-of-care applications. [ABSTRACT FROM AUTHOR]

The heterogeneity of each individual oncologic disease can be mirrored by molecular analysis of a simple blood draw in real time. Liquid biopsy testing has been shown useable for cancer detection, proof of minimal residual disease, therapy decision making and monitoring. However, an individual blood analyte does not present a comprehensive picture of the disease. It was recently shown that multi-modal/multi-parametric/multi-analyte liquid biopsy testing has the advantage of generating a high-resolution snapshot of the disease complexity. The different blood analytes such as circulating tumor cells, circulating immune cells, tumor-educated platelets, extracellular vesicles, cell-free DNA, cell-free RNA and circulating proteins complement each other and have additive value for clinical cancer management. We, here, like to review the studies leading to these promising conclusions and like to, at the end, mention that many challenges lie ahead before the translation into the clinic can be accomplished, including issues concerning clinical utility, method standardization, cost reimbursement and data management.

The COVID-19 pandemic has emphasized the urgent need for point-of-care methods suitable for the rapid and reliable diagnosis of viral infections. To address this demand, we report the rapid, label-free simultaneous determination of two SARS-CoV-2 proteins, namely, the nucleoprotein and the receptor binding domain peptide of S1 protein, by implementing a bioanalytical device based on Multi Area Reflectance Spectroscopy. Simultaneous detection of these two proteins is achieved by using silicon chips with adjacent areas of different silicon dioxide thickness on top, each of which is modified with an antibody specific to either the nucleoprotein or the receptor binding domain of SARS-CoV-2. Both areas were illuminated by a single probe that also collected the reflected light, directing it to a spectrometer. The online conversion of the combined reflection spectra from the two silicon dioxide areas into the respective adlayer thickness enabled real-time monitoring of immunoreactions taking place on the two areas. Several antibodies have been tested to define the pair, providing the higher specific signal following a non-competitive immunoassay format. Biotinylated secondary antibodies and streptavidin were used to enhance the specific signal. Both proteins were detected in less than 12 min, with detection limits of 1.0 ng/mL. The assays demonstrated high repeatability with intra- and inter-assay coefficients of variation lower than 10%. Moreover, the recovery of both proteins from spiked samples prepared in extraction buffer from a commercial self-test kit for SARS-CoV-2 collection from nasopharyngeal swabs ranged from 90.0 to 110%. The short assay duration in combination with the excellent analytical performance and the compact instrument size render the proposed device and assay suitable for point-of-care applications.

Surveillance testing is an essential component to ensuring safe, effective, and high-quality drug products are available in the commercially marketed US supply chain. Surveillance allows the agency to assess product quality and monitor for potential adulteration of drug products being used by consumers. Opioid drug products can be adulterated to enhance the effect of the intended active ingredient. Numerous accounts have been reported where fentanyl has been used as an adulterant in illicit street drugs such as heroin, cocaine, or methamphetamine. To efficiently surveil the legitimate opioid supply chain, an analytical method with the ability to simultaneously detect, identify and quantify opioid molecules is desired. In this study, a multi-opioid protocol (MOP) using liquid chromatography-high resolution mass spectrometry (HPLC-HRMS) technology was developed and validated for the detection and quantification of 27 opioid drugs. The MOP analytical procedure was applied to the analysis of drug substance and finished dosage forms. MOP was used to identify and quantify active pharmaceutical ingredients (API) listed on the label claim, and in the case of suspected economically motivated adulteration could identify and quantify undeclared opioid APIs. The analytical method analysis time was 16 minutes and the LOD and LOQ in full MS mode were (average) 0.3 and 0.8 ng/mL, respectively. The validation criteria parameters were satisfactory based on international guidelines (ICH). The MOP was successfully applied to the analysis of over 160 drug substances and finished products. For all samples tested in the study, their identities were confirmed, and assays met specifications. Overall, there was no evidence of illegal substitution or adulteration in any of the ingredients and products tested from the legitimate commercial marketed US supply chain. • Surveillance is an essential component to ensuring safe and high-quality drug products are available in the US supply chain. • Ensuring no economically motivated adulteration is taking place is a key component of quality ensuring. • A multi-opioid protocol using HPLC-HRMS was developed and validated for the detection and quantification of 27 opioid drugs. • The method analysis time was 16 minutes and the LOD and LOQ in full MS mode were (average) 0.3 and 0.8 ng/mL, respectively. • The multi-opioid protocol was successfully applied to the analysis of over 160 drug substances and finished products. [ABSTRACT FROM AUTHOR]

Abstract Background Single liquid biopsy analytes (LBAs) have been utilized for therapy selection in metastatic breast cancer (MBC). We performed integrative statistical analyses to examine the clinical relevance of using multiple LBAs: matched circulating tumor cell (CTC) mRNA, CTC genomic DNA (gDNA), extracellular vesicle (EV) mRNA, and cell-free DNA (cfDNA). Methods Blood was drawn from 26 hormone receptor-positive, HER2-negative MBC patients. CTC mRNA and EV mRNA were analyzed using a multi-marker qPCR. Plasma from CTC-depleted blood was utilized for cfDNA isolation. gDNA from CTCs was isolated from mRNA-depleted CTC lysates. CTC gDNA and cfDNA were analyzed by targeted sequencing. Hierarchical clustering was performed within each analyte, and its results were combined into a score termed Evaluation of multiple Liquid biopsy analytes In Metastatic breast cancer patients All from one blood sample (ELIMA.score), which calculates the contribution of each analyte to the overall survival prediction. Singular value decomposition (SVD), mutual information calculation, k-means clustering, and graph-theoretic analysis were conducted to elucidate the dependence between individual analytes. Results A combination of two/three/four LBAs increased the prevalence of patients with actionable signals. Aggregating the results of hierarchical clustering of individual LBAs into the ELIMA.score resulted in a highly significant correlation with overall survival, thereby bolstering evidence for the additive value of using multiple LBAs. Computation of mutual information indicated that none of the LBAs is independent of the others, but the ability of a single LBA to describe the others is rather limited—only CTC gDNA could partially describe the other three LBAs. SVD revealed that the strongest singular vectors originate from all four LBAs, but a majority originated from CTC gDNA. After k-means clustering of patients based on parameters of all four LBAs, the graph-theoretic analysis revealed CTC ERBB2 variants only in patients belonging to one particular cluster. Conclusions The additional benefits of using all four LBAs were objectively demonstrated in this pilot study, which also indicated a relative dominance of CTC gDNA over the other LBAs. Consequently, a multi-parametric liquid biopsy approach deconvolutes the genomic and transcriptomic complexity and should be considered in clinical practice.

Liquid biopsy is a promising technique for clinical management of oncological patients. The diversity of analytes circulating in the blood useable for liquid biopsy testing is enormous. Circulating tumor cells (CTCs), cell-free DNA (cfDNA) and extracellular vesicles (EVs), as well as blood cells and other soluble components in the plasma, were shown as liquid biopsy analytes. A few studies directly comparing two liquid biopsy analytes showed a benefit of one analyte over the other, while most authors concluded the benefit of the additional analyte. Only three years ago, the first studies to examine the value of a characterization of more than two liquid biopsy analytes from the same sample were conducted. We attempt to reflect on the recent development of multimodal liquid biopsy testing in this review. Although the analytes and clinical purposes of the published multimodal studies differed significantly, the additive value of the analytes was concluded in almost all projects. Thus, the blood components, as liquid biopsy reservoirs, are complementary rather than competitive, and orthogonal data sets were even shown to harbor synergistic effects. The unmistakable potential of multimodal liquid biopsy testing, however, is dampened by its clinical utility, which is yet to be proven, the lack of methodical standardization and insufficiently mature reimbursement, logistics and data handling. [ABSTRACT FROM AUTHOR]

Systemic sclerosis (SSc) is a heterogeneous autoimmune disease associated with several anti-nuclear antibodies (ANA), including those in the classification criteria (anti-centromere, anti-topoisomerase I (Scl-70), anti-RNA Pol III). However, the presence of less common antibodies such as anti-fibrillarin (U3-RNP) that generate a clumpy nucleolar pattern by HEp-2 indirect immunofluorescence assay (IFA, ICAP AC-9) are considered disease specific and are with clinical subsets of SSc, therefore playing a role in diagnosis and prognosis. A specific and sensitive anti-fibrillarin assay would be an important addition to serological diagnosis and evaluation of SSc. The goal of this study was to evaluate a new particle-based multi-analyte technology (PMAT) for the measurement of anti-fibrillarin antibodies. A total of 149 patient samples were collected including 47 samples from France (Lyon and Paris, n = 32) and Italy (Careggi Hospital, Florence, n = 15) selected based on AC-9 HEp-2 IFA staining (> 1:640, clumpy nucleolar pattern) and 102 non-SSc controls (inflammatory bowel disease (IBD) n = 20, Sjögren's syndrome (SjS) n = 20, infectious disease (ID) n = 7, systemic lupus erythematosus (SLE) n = 17, rheumatoid arthritis (RA) n = 17, and healthy individuals (HI) n = 21). All samples were tested on the anti-fibrillarin PMAT assay (research use only, Inova Diagnostics, USA). Additionally, the 47 anti-fibrillarin positive samples were also tested on PMAT assays for detecting other autoantibodies in ANA-associated rheumatic diseases (AARD). Anti-fibrillarin antibody data performed by fluorescence enzyme immunoassay (FEIA, Thermo Fisher, Germany) was available for 34 samples. The anti-fibrillarin PMAT assay was positive in 31/32 (96.9%, France) and 12/15 (80.0%, Italy) of samples preselected based on the AC-9 IIF pattern (difference p = 0.09). Collectively, the PMAT assay showed 91.5% (95% confidence interval (CI): 80.1–96.6%) sensitivity with 100.0% (95% CI: 96.4–100.0%) specificity in non-SSc controls. Strong agreement was found between PMAT and FEIA with 100.0% positive qualitative agreement (34/34) and quantitative agreement (Spearman's rho = 0.89, 95% CI: 0.77.9–0.95%, p < 0.0001). Although most anti-fibrillarin positive samples were mono-specific (69.8%), some expressed additional antibodies (namely Scl-70, centromere, dsDNA, Ro52, Ro60, SS-B, Ribo-P, DFS70, and EJ). In conclusion, this first study on anti-fibrillarin antibodies measured using a novel PMAT assay shows promising results where the new PMAT assay had high level of agreement to FEIA for the detection of anti-fibrillarin antibodies. The availability of novel AFA assays such as PMAT might facilitate the clinical deployment, additional studies, standardization efforts, and potentially consideration of AFA for next generations of the classification criteria. [ABSTRACT FROM AUTHOR]

Background: Single liquid biopsy analytes (LBAs) have been utilized for therapy selection in metastatic breast cancer (MBC). We performed integrative statistical analyses to examine the clinical relevance of using multiple LBAs: matched circulating tumor cell (CTC) mRNA, CTC genomic DNA (gDNA), extracellular vesicle (EV) mRNA, and cell-free DNA (cfDNA). Methods: Blood was drawn from 26 hormone receptor-positive, HER2-negative MBC patients. CTC mRNA and EV mRNA were analyzed using a multi-marker qPCR. Plasma from CTC-depleted blood was utilized for cfDNA isolation. gDNA from CTCs was isolated from mRNA-depleted CTC lysates. CTC gDNA and cfDNA were analyzed by targeted sequencing. Hierarchical clustering was performed within each analyte, and its results were combined into a score termed Evaluation of multiple Liquid biopsy analytes In Metastatic breast cancer patients All from one blood sample (ELIMA.score), which calculates the contribution of each analyte to the overall survival prediction. Singular value decomposition (SVD), mutual information calculation, k-means clustering, and graph-theoretic analysis were conducted to elucidate the dependence between individual analytes. Results: A combination of two/three/four LBAs increased the prevalence of patients with actionable signals. Aggregating the results of hierarchical clustering of individual LBAs into the ELIMA.score resulted in a highly significant correlation with overall survival, thereby bolstering evidence for the additive value of using multiple LBAs. Computation of mutual information indicated that none of the LBAs is independent of the others, but the ability of a single LBA to describe the others is rather limited—only CTC gDNA could partially describe the other three LBAs. SVD revealed that the strongest singular vectors originate from all four LBAs, but a majority originated from CTC gDNA. After k-means clustering of patients based on parameters of all four LBAs, the graph-theoretic analysis revealed CTC ERBB2 variants only in patients belonging to one particular cluster. Conclusions: The additional benefits of using all four LBAs were objectively demonstrated in this pilot study, which also indicated a relative dominance of CTC gDNA over the other LBAs. Consequently, a multi-parametric liquid biopsy approach deconvolutes the genomic and transcriptomic complexity and should be considered in clinical practice. [ABSTRACT FROM AUTHOR]

Driving under the influence of psychoactive substances is a major cause of motor vehicle crashes. The identification and quantification of substances most frequently involved in impaired-driving cases in a single analytic procedure could be an important asset in forensic toxicology. In this study, a highly sensitive and selective liquid chromatography (LC) approach hyphenated with Orbitrap high-resolution mass spectrometry (HRMS) was developed for the quantification of the main drugs present in the context of driving under the influence of drugs (DUID) using 100 μL of whole blood. This procedure involves a simple sample preparation and benefit from the selectivity brought by parallel reaction monitoring (PRM) allowing to solve most DUID cases using a single multi-analyte injection. The method was fully validated for the quantification of the major classes of psychoactive substances associated with impaired-driving (cannabinoids, cocaine and its metabolites, amphetamines, opiates and opioids, and the major benzodiazepines and z-drugs). The validation guidelines set by the “Société Française des Sciences et des Techniques Pharmaceutiques” (SFSTP) were respected for 22 psychoactive substances using 15 internal standards. Trueness was measured to be between 95.3 and 107.6% for all the tested concentrations. Precision represented by repeatability and intermediate precision was lower than 12% while recovery (RE) and matrix effect (ME) ranged from 49 to 105% and from −51 to 3%, respectively. The validated procedure provides an efficient approach for the simultaneous and simple quantification of the major drugs associated with impaired driving benefiting from the selectivity of PRM.

Keywords: Lung cancer; Diagnosis; Prognosis; Liquid biopsy; cfDNA; Mutation; DNA methylation; Multi-analyte EN Lung cancer Diagnosis Prognosis Liquid biopsy cfDNA Mutation DNA methylation Multi-analyte 1 7 7 02/02/21 20210129 NES 210129 Kezhong Chen, Jianlong Sun and Heng Zhao contributed equally to this work. In this study, we developed a set of experimental and computational tools to measure both genetic and epigenetic signals from plasma cfDNA of LC patients as well as patients bearing benign lung nodules (BLN) using high-throughput sequencing [[3]], aiming to explore the potential utility of blood-based biomarkers for LC diagnosis and prognosis. The percentages of cfDNA variants matching corresponding WBC sample were 20.7% (40 out of 193) for LC cfDNA and 39.1% (18 out of 46) for BLN cfDNA, suggesting that a significant portion of cfDNA variants was derived from CH, especially in BLN plasma ( I p i = 8.89E-03, chi-squared test). [Extracted from the article]

Recently, ultrasensitive detection and multi-guest sensing have received extensive attention due to their high sensitivity and efficiency. Herein, we report a novel approach to achieve ultrasensitive detection of multi-analyte. This approach is concluded as "rationally introduce Aggregation-Induced Emission (AIE) into chemosensor". According to this approach, by rationally introducing self-assembly moiety, the obtained chemosensor DNS could serve as a novel AIEgen and show strong AIE in DMSO/H 2 O (water fraction 80%) binary solution. Interestingly, a simple fluorescent sensor array based on the DNS has been developed. This sensor array could selectively sense Fe3+, Al3+, H 2 PO 4 − and L-Arg in water solution. More importantly, this sensor array shows ultrasensitive detection for Fe3+, Al3+ and L-Arg. The LODs of the sensor array for Fe3+, Al3+ and L-Arg are in the range of 3.54 × 10−9 M to 9.42 × 10−9 M. Moreover, H 2 PO 4 − could realize the reversible detection of Fe3+ in the DMSO/H 2 O (water fraction 80%) solution. Meanwhile, DNS -based test papers and thin films were prepared, which could serve as test kits for convenient detection Fe3+, Al3+, and L-Arg in water. In addition, they could also act as efficient erasable fluorescent display materials. Herein, we report a novel approach to achieving ultrasensitive detection of multi-analyte. This approach is concluded as "rationally introduce Aggregation-Induced Emission (AIE) into chemosensor". According to this approach, by rationally introducing self-assembly moiety, the obtained chemosensor DNS could serve as a novel AIEgen and show strong AIE in DMSO/H 2 O (water fraction 80%) binary solution. Interestingly, a simple fluorescent sensor array based on the DNS has been developed. This sensor array could selectively sense Fe3+, Al3+, H 2 PO 4 − and L-Arg in water solution. More importantly, this sensor array shows ultrasensitivity for Fe3+, Al3+ and L-Arg. The LODs of the sensor array for Fe3+, Al3+ and L-Arg are in the range of 3.54 × 10−9 M to 9.42 × 10−9 M. Moreover, H 2 PO 4 − could realize the reversible detection of Fe3+ in the DMSO/H 2 O (water fraction 80%) solution. Meanwhile, DNS -based test papers and thin films were prepared, which could serve as test kits for convenient detection of Fe3+, Al3+, and L-Arg in water. In addition, they could also act as efficient erasable fluorescent display materials. Unlabelled Image • Rationally induce AIE into the small molecular chemosensor. • A simple sensor array for Fe3+, Al3+, H 2 PO 4 - and L-Arg was created. • Ultrasensitive detection for Fe3+, Al3+ and L-Arg in aqueous solutions. • LODs of the sensor array for Fe3+, Al3+ and L-Arg reached 10-9 M. • Fe3+, Al3+, L-Arg test papers and fluorescent display materials were prepared. [ABSTRACT FROM AUTHOR]

A fast and simple approach to overcome challenges in emergency toxicological analysis, using ultra‐high performance liquid chromatography–tandem mass spectrometry (UHPLC‐MS/MS) has been developed, for the detection of analytes in blood and urine samples from the following drug classes: analgesics, benzodiazepines, antidepressants, anticonvulsants, drugs of abuse, and pesticides. These substances are relevant in the context of emergency toxicology in Brazil. The sample preparation procedure was relatively easy and fast to perform. The method was fully validated giving limits of in the range of 0.5 and 20 ng mL−1 for blood and urine samples. The intraday and interday precision and accuracy were considered adequate for all analytes once the relative standard deviation (RSD) (%) was lower than 20% for quality control (QC) low and lower than 15% for CQ medium and high. The developed method was successfully applied to 320 real samples collected at the Poison Control Center of São Paulo, and 89.1% have shown to be positive for some of the analytes. This confirms its applicability and importance to emergency toxicological analysis, and it could be very useful in both fields of clinical and forensic toxicology. [ABSTRACT FROM AUTHOR]

Abstract A new and simple quinoline-based fluorescent probe for discriminative sensing of Zn2+ and Cd2+ has been synthesized by inserting an amide group into the 8-aminoquinoline fluorophore and a propargylamine chelating site. This easily-available chemosensor displayed selective and distinct ratiometric fluorescence responses to Zn2+ in almost totally water solution through its amide tautomer binding form, and to Cd2+ in CH 3 CN aqueous medium through its imidic acid tautomer binding form, respectively. Moreover, the in situ prepared probe 1 –Zn2+ complex could act as a relay fluorescent sensor selectively toward pyrophosphate (PPi) and adenosine 5′-triphosphate (ATP) anions via further complexation. Thus, with good specificity, low detection limits and fast response time, a highly efficient fluorescence platform for simultaneous multi-analyte detection has been developed by using the uncomplicated single molecule. Finally, this multi-functional probe was applied successfully for analysis of all the target ions in the tap water sample and on test paper strips, and bioimaging of Zn2+ in mung bean sprouts with low phytotoxicity. Graphical abstract A new and simple quinoline-based fluorescent probe was constructed facilely for efficient multiple detection of Zn2+, Cd2+, PPi and ATP. Image 1 Highlights • A quinoline-based fluorescent probe was synthesized. • The probe displayed different ratiometric fluorescence responses to Zn2+ and Cd2+. • The Zn2+ complex of the probe behaved relay sensing for PPi and ATP. • The probe could analyze Zn2+, Cd2+, PPi and ATP in tap water and on test strips, and bioimage Zn2+ in bean sprouts. [ABSTRACT FROM AUTHOR]

Meat products are susceptible to food fraud due to their high commercial value. One possible adulteration is the undeclared substitution of high-priced meat protein by cheaper foreign proteins. A reliable detection of these added proteins is required in order to control food specification, especially regarding food fraud. A sensitive multi-method for the detection of 23 foreign protein sources (alfalfa, buckwheat, broad bean, chia, chickpea, coconut, egg, flaxseed, hemp, lentil, lupine blue, maize, milk, pea, peanut, potato, pumpkin, rapeseed, rice, sesame, sunflower, soy, and wheat) in meat products applying high performance liquid chromatography–tandem mass spectrometry (HPLC-MS/MS) has been developed. In this context, a new rapid defatting procedure, without carry-over effect, suitable for routine analysis, a robust protein extraction protocol (TRIS/HCl (1 M, pH 8.2) with 40% acetonitrile), suitable for various animal- and plant-based proteins including grain proteins, and new peptide markers for buckwheat and potato were established. A comprehensive investigation of the influence of cooking (emulsion-type sausages), grilling (hamburger patties) and maturation (salamis) showed that the detection method is robust against different types of processing. The limits of detection for all foreign protein sources were ≤ 0.02% protein and no false-positive or -negative results were obtained. • Simultaneous detection of 23 foreign protein sources in meat products. • Identification of new heat-stable peptide markers for buckwheat and potato. • Establishment of a robust extraction protocol for various foreign proteins. • Development of a new rapid defatting procedure for meat products without carry-over. • Method is suitable for differently processed meat products. [ABSTRACT FROM AUTHOR]

Dynamics of mRNA from circulating tumor cells (CTCs), mRNA from extracellular vesicles (EVs), and cell-free DNA (cfDNA) were assessed to examine the relevance of a longitudinal multi-parametric liquid biopsy strategy. Eighteen milliliters of blood was drawn from 27 hormone receptor-positive and human epidermal growth factor receptor 2 (HER2)-negative metastatic breast cancer (MBC) patients at disease progression and at two subsequent radiologic staging time points. CTC mRNA and EV mRNA were analyzed using multi-marker qPCR, and cfDNA was analyzed using targeted next-generation sequencing (NGS). The presence of ERBB2 or ERBB3 overexpression signals in CTCs significantly correlated with disease progression (87% specificity, 36% sensitivity, p-value = 0.023), and the presence of either ERBB3 signals in CTCs or EVs or cfDNA variants in ERBB3 also showed a significant association with progressive MBC. Fluctuations during treatment were detected in the EV fraction with the appearance of hitherto undetected ERCC1 signals correlating with progressive disease (97% specificity, 18% sensitivity, p-value = 0.030). Allele frequency development of ESR1 and PIK3CA variants detected at subsequent staging time points could be used as a predictor for therapy success and, importantly, might help guide therapy decisions. The three analytes, each with their own unique features for disease monitoring, were shown to be complementary, underlining the usefulness of the longitudinal multi-parametric liquid biopsy approach.

The following review focuses on progress made in the last five years with the NRL Array Biosensor, a portable instrument for rapid and simultaneous detection of multiple targets. Since 2003, the Array Biosensor has been automated and miniaturized for operation at the point-of-use. The Array Biosensor has also been used to demonstrate (1) quantitative immunoassays against an expanded number of toxins and toxin indicators in food and clinical fluids, and (2) the efficacy of semi-selective molecules as alternative recognition moieties. Blind trials, with unknown samples in a variety of matrices, have demonstrated the versatility, sensitivity, and reliability of the automated system.

Over the past 15 years, chromatographic techniques with mass spectrometric detection have been increasingly used to monitor the rapidly expanded list of regulated flavour and fragrance ingredients. This trend entails a need for good quantification practices suitable for complex media, especially for multi-analytes. In this article,we present experimental precautions needed to perform the analyses and ways to process the data according to the most recent approaches. This notably includes the identification of analytes during their quantification and method validation, when applied to real matrices, based on accuracy profiles. A brief survey of application studies based on such practices is given. This article is part of the themed issue 'Quantitative mass spectrometry'. [ABSTRACT FROM AUTHOR]

The diagnostic methods in an emergency scenario must be simple, fast, and efficient to provide an effectiveness and efficient treatment, thus reducing the consequences of exposure. Considering the sample analysis, the protein precipitation combined with LC-MS/MS has been shown to be a good strategy for the simultaneous determination of compounds of toxicological interest, such as medicines and drugs of abuse. In this study, a rapid and simple multi-analyte method was developed and validated for the quantification of 57 pharmaceuticals and illicit drugs in plasma samples. Sample pre-treatment consists of protein precipitation of 50 µL of the sample with 240 µL of organic solvent mixture (MeOH:ACN, 3:1, v/v), centrifugation, and injection into the LC-MS/MS, with a chromatographic run time of 7 min. The method was validated considering lower limit of quantification (LLOQ), interferences, linearity, precision, accuracy, dilution integrity, carryover, and matrix effect. The LLOQs ranged from 5 to 20 ng/mL and all analytes were linear (r 2 >0.99) in the tested concentration ranges. The method proved to be precise and accurate, presenting QC concentrations for all analytes within acceptable limits by the guideline used (CV % ≤20 % and bias ± 20 %). The developed method was successfully applied in 470 plasma samples of real cases of poisoning. A total of 80 % of the samples were positive for at least one substance, with acetaminophen (32.1 %), diazepam (25.1 %), and lidocaine (18.9 %) being the most detected. The most prevalent exposure circumstance among the cases was suicide attempt. The most frequent age groups were young adults between 20 and 29 years old and children under 5 years old. The methodology developed proved to be efficient in the simultaneous determination of 57 substances of toxicological interest, contributing to a correct diagnosis and, consequently, to the most appropriate management and treatment of the intoxicated patient. Furthermore, it is possible to observe the most commonly involved toxic agents in the Rio Grande do Sul, southern Brazil, helping to trace a profile of the poisoning patient, important in toxicovigilance actions. • A fast and simple LC-MS/MS screening of the 57 pharmaceutical and illicit drugs. • A quick, inexpensive, and efficient sample preparation that is easy to implement. • The method was successfully applied to 470 samples of poisoned patients. • 80 % of the samples were positive for at least one of the investigated substances. • A poisoning profile of a Brazilian state was observed based on toxicological analysis. [ABSTRACT FROM AUTHOR]

We present an electrochemical lab-on-a-chip (LOC) platform for the simultaneous detection of up to four different analytes. The possibility to separately immobilize different assays in a channel network, without active valves, was successfully demonstrated using a model assay linked to glucose oxidase. This enables the detection of various analytes even with different assay formats. For the assay immobilization, the channel surface, made out of dry film photoresist (DFR), could be activated by means of EDC/NHS-linker chemistry and used for the covalent binding of primary amines. Cross-sensitivity due to diffusion within the channel network could be experimentally excluded. [ABSTRACT FROM AUTHOR]

For the first time, a multi-analyte biosensor platform has been developed using the label-free 1-lambda-reflectometry technique. This platform is the first, which does not use imaging techniques, but is able to perform multi-analyte measurements. It is designed to be portable and cost-effective and therefore allows for point-of-need testing or onsite field-testing with possible applications in diagnostics. This work highlights the application possibilities of this platform in the field of animal testing, but is also relevant and transferable to human diagnostics. The performance of the platform has been evaluated using relevant reference systems like biomarker (C-reactive protein) and serology (anti- Salmonella antibodies) as well as a panel of real samples (animal sera). The comparison of the working range and limit of detection shows no loss of performance transferring the separate assays to the multi-analyte setup. Moreover, the new multi-analyte platform allows for discrimination between sera of animals infected with different Salmonella subtypes. [ABSTRACT FROM AUTHOR]

In the present study, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) multi-analyte approach using one single work-up approach in whole blood, plasma, serum, post-mortem blood, liver tissue, gastric content, hair, and urine was developed for fast target screening and reliable identification of 130 analytes often requested in clinical and forensic toxicology. Samples (500 μL each) of whole blood, plasma, serum, post-mortem blood, tissue (homogenized 1 + 4 with water), as well as 3 g of distilled gastric contents, 1 mL of urine, or 20 mg of pulverized hair were extracted at different pH values with an diethyl ether-ethyl acetate mixture (1:1). Separation and identification were performed using LC-QTRAP with electrospray ionization in positive mode. For identification 1 scheduled multi-reaction-mode (sMRM) method with 390 transitions was developed covering benzodiazepines, Z-drugs, antidepressants, neuroleptics, opioids, new synthetic drugs, and phosphodiesterase type 5 inhibitors. For positive sMRM transitions with intensities exceeding 5000 cps, dependent scans (EPI scan collision energy, 35 eV, collision energy spread, 15 eV) were performed for library search using our in-house library. The method was developed with respect to selectivity, matrix effects, recovery, process efficiency, limit of detection, and applicability. The simple work-up procedure was suitable for all biosamples with exception of urine in respect to low concentrated analytes, which showed median recovery values of 59%. The method was selective for 130 analytes in all 8 biosamples. For 106 analytes, the limit of detection in whole blood, plasma, and serum was lower than the lowest therapeutic concentration listed in blood level lists. Copyright © 2014 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

ABSTRACT Host cell proteins (HCPs) are among the process-related impurities monitored during recombinant protein pharmaceutical process development. The challenges of HCP detection include (1) low levels of residual HCPs present in large excess of product protein, (2) the assay must measure a large number of different protein analytes, and (3) the population of HCP species may change during process development. Suitable methods for measuring process-related impurities are needed to support process development, process validation, and control system testing. A multi-analyte enzyme-linked immunosorbent assay (ELISA) is the workhorse method for HCP testing due to its high throughput, sensitivity and selectivity. However, as the anti-HCP antibodies, the critical reagents for HCP ELISA, do not comprehensively recognize all the HCP species, it is especially important to ensure that weak and non-immunoreactive HCPs are not overlooked by the ELISA. In some cases limited amount of antibodies to HCP species or antigen excess causes dilution-dependent non-linearity with multi-product HCP ELISA. In our experience, correct interpretation of assay data can lead to isolation and identification of co-purifying HCP with the product in some cases. Moreover, even if the antibodies for a particular HCP are present in the reagent, the corresponding HCP may not be readily detected in the ELISA due to antibody/antigen binding conditions and availability of HCP epitopes. This report reviews the use of the HCP ELISA, discusses its limitations, and demonstrates the importance of orthogonal methods, including mass spectrometry, to complement the platform HCP ELISA for support of process development. In addition, risk and impact assessment for low-level HCPs is also outlined, with consideration of clinical information. Biotechnol. Bioeng. 2014;111: 2367-2379. © 2014 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Abstract: An ultrasensitive electrochemical immunoassay was developed for rapid detection of interleukin-6 (IL-6) and matrix metallopeptidase-9 (MMP-9); the method utilized PS@PDA-metal nanocomposites based on graphene nanoribbon (GNR)-modified heated screen-printed carbon electrode (HSPCE). Because of the good hydrophilicity and low toxicity, GNRs were used to immobilize antibodies (Ab) and amplify the electrochemical signal. PS@PDA-metal was used to label antibodies and generate a strong electrochemical signal in acetic buffer. A sandwich strategy was adopted to achieve simultaneous detection of MMP-9 and IL-6 based on HSPCE without cross-talk between adjacent electrodes in the range of 10−5 to 103 ngmL−1 with detection limits of 5fgmL−1 and 0.1pgmL−1 (S/N=3), respectively. The proposed method showed wide detection range, low detection limit, acceptable stability and good reproducibility. Satisfactory results were also obtained in the practical samples, thus showing this is a promising technique for simultaneous clinical detection of biocomponent proteins. [Copyright &y& Elsevier]

Abstract: A capillary electrophoresis competitive immunoassay was developed for the simultaneous quantitation of insulin, glucagon, and islet amyloid polypeptide (IAPP) secretion from islets of Langerhans. Separation buffers and conditions were optimized for the resolution of fluorescein isothiocyanate (FITC)-labeled glucagon and IAPP immunoassay reagents, which were excited with the 488nm line of an Ar+ laser and detected at 520nm with a photomultiplier tube (PMT). Cy5-labeled insulin immunoassay reagents were excited by a 635nm laser diode module and detected at 700nm with a separate PMT. Optimum resolution was achieved with a 20mM carbonate separation buffer at pH 9.0 using a 20cm effective separation length with an electric field of 500V/cm. Limits of detection for insulin, glucagon, and IAPP were 2, 3, and 3nM, respectively. This method was used to monitor the simultaneous secretion of these peptides from as few as 14 islets after incubation in 4, 11, and 20mM glucose for 6h. For insulin and IAPP, a statistically significant increase in secretion levels was observed, while glucagon levels were significantly reduced in the 4 and 11mM glucose conditions. To further demonstrate the utility of the assay, the Ca2+-dependent secretion of these peptides was demonstrated which agreed with published reports. The ability to examine the secretion of multiple peptides may allow for the determination of regulation of secretory processes within islets of Langerhans. [Copyright &y& Elsevier]

Background: Interest in biomarker patterns and disease has led to the development of immunoassays that evaluate multiple analytes in parallel while using little sample. However, there are no current standards for multiplex configuration, validation, and quality. Thus, validation by platform, population, and question of interest is recommended. We sought to determine the best blood fraction for multiplex evaluation of circulating biomarkers in post-menopausal women, and to explore body composition phenotype discrimination by biomarkers. Methods: Archived serum and plasma samples from a sample of healthy post-menopausal women with the highest (n=9) and lowest (n=11) percent lean mass, as determined by dual-energy X-ray absorptiometry, were used to measure 90 analytes using bead-based, suspension multiplex assays. Replicates of serum and plasma were analyzed in a random selection of four of these individuals. Results: Ninety percent of the analytes were detectable for ≥50% of samples; when limited to these well detected analytes, mean replicate correlations for serum and plasma were 0.87 and 0.85, respectively. Serum had lower error rates discriminating phenotypes; seven serum vs. two plasma analytes discriminated extreme body phenotypes. Conclusions: Serum and plasma performed similarly for the majority of the analytes. Serum showed a slight advantage in predicting extreme body composition phenotypes in postmenopausal women using parallel evaluation of analytes. [ABSTRACT FROM AUTHOR]

microfluidic device was developed to produce temporal concentration gradients of multiple analytes. Four on-chip pumps delivered pulses of three analytes and buffer to a 14-cm channel where the pulses were mixed to homogeneity. The final concentration of each analyte was dependent on the temporal density of the pulses from each pump. The concentration of each analyte was varied by changing the number of pump cycles from each reservoir while maintaining the total number of pump cycles per unit time to ensure a constant total flow rate in the device. To gauge the independent nature of each pump, sinusoidal waves of fluorescein concentration were produced from each pump with independent frequencies and amplitudes. The resulting fluorescence intensity was compared with a theoretical summation of the waves and the experimental data matched the theoretical waves within 1%, indicating that the pumps were operating independently and outputting the correct frequency and amplitude. The device was used to demonstrate the role of adenosine triphosphate-sensitive K channels in glucose-stimulated increases in intracellular [Ca] in islets of Langerhans. Perfusion of single islets of Langerhans with combinations of glucose, diazoxide, and K resulted in intracellular Ca patterns similar to what has been observed using conventional perfusion devices. The system will be useful in other studies with islets of Langerhans, as well as other assays that require the modulation of multiple analytes in time. [ABSTRACT FROM AUTHOR]

Abstract: In this work, we have applied particle manipulation based on negative dielectrophoresis (n-DEP) to develop rapid and separation-free immunosensing systems. Two widely used pesticides, atrazine and bromopropylate, were used as target molecules to demonstrate competitive immunosensing based on the rapid manipulation of microparticles. A suspension of the fluorescence microparticles modified with a specific antibody was injected into the n-DEP device consisting of the interdigitated microarray (IDA) electrode and indium-tin-oxide (ITO) substrate immobilized by protein conjugation with antigen. The application of 2MHz AC voltage (16V peak-to-peak) to the IDA forced most of the particles to form a line pattern on the upper ITO over the gaps of IDA within 60s. In the absence of analytes, patterned microparticles were irreversibly captured on the ITO by the construction of immuno-complexes. When the microparticles bearing anti-atrazine IgG antibody were suspended in an analyte (atrazine) solution, irreversible capturing of microparticles on the ITO was inhibited because of the occupation of the binding sites of the antibodies with free-atrazine. As a result, the analyte molecules were re-dispersed from the ITO to disintegrate the line formation after turning off the voltage. We could discriminatively detect the fluorescence intensity of the captured microparticles at the designated areas from that of the uncaptured microparticles suspended in the solution. Thus, the separation steps usually required for conventional immunoassay are eliminated in the present procedure. A pre-incubation of microparticles for 3min in an orange juice solution containing analyte allowed for the determination of the atrazine and bromopropylate concentrations with a limit of detection of 4 and 1.5μgL−1, respectively, providing sufficient detectability to achieve international regulations regarding pesticide residues in food samples. The assay was significantly accelerated by the rapid particle manipulation with n-DEP and totally accomplished within 5min. We also demonstrated the possibility of the simultaneous determination of two pesticide residues by using the DEP devices with two channels modified with specific competitors for atrazine and bromopropylate. [Copyright &y& Elsevier]

Abstract: Changes detected in the imprinting effect by OMNiMIPs imprinted with multiple templates appear to be a function of the maximum template loading. Below the maximum template loading, the polymers imprinted with multiple compounds provide molecular recognition close to the polymers imprinted with single compounds, for each template compound tested. However, template loading past this point can result in significant lowering of the imprinting effect. For example, 1,1′-bi-2-naphthol enantiomers showed nearly a 60% loss in enantioselectivity on OMNiMIP 8 (imprinted with four templates); yet still maintained a separation factor of 3.7 allowing baseline separation of enantiomers. Similar behavior was seen for the other three template molecules, although losses in enantioselectivity were less severe. The multi-analyte imprinted OMNiMIP 8 was shown to be capable of separating a template molecule individually from a mixture, including enantiomers, but not all four concurrently. With respect to physical properties of the different OMNiMIPs, gradual trends in porosity and surface area correlated to the concentration of the templates, independent of their molecular structure. [Copyright &y& Elsevier]

Abstract: The BioRad BioPlex™ 2200 Vasculitis kit demonstrates excellent relative sensitivity and relative specificity for the semi-quantitative detection of IgG autoantibodies to MPO, PR3 and GBM. The fully-automated platform simultaneously measures three analytes in a single tube, offering superior advantage in speed and ease of use over current assays. The availability of a fully-automated platform with 24-hour availability for these three antibodies may be of considerable value in the differential diagnosis of patients with rapidly progressive glomerulonephritis. [Copyright &y& Elsevier]

The following review focuses on progress made in the last five years with the NRL Array Biosensor, a portable instrument for rapid and simultaneous detection of multiple targets. Since 2003, the Array Biosensor has been automated and miniaturized for operation at the point-of-use. The Array Biosensor has also been used to demonstrate (1) quantitative immunoassays against an expanded number of toxins and toxin indicators in food and clinical fluids, and (2) the efficacy of semi-selective molecules as alternative recognition moieties. Blind trials, with unknown samples in a variety of matrices, have demonstrated the versatility, sensitivity, and reliability of the automated system. [ABSTRACT FROM AUTHOR]

Abstract: Patterning substrates with biological reagents is a critical component of biosensor development. Many applications require multi-analyte patterning capabilities, with a need to deposit several species reproducibly with a high degree of precision. We demonstrate a piezoelectric inkjet printing system that is capable of creating sub-millimeter (down to 150μm) patterns of aqueous and nonaqueous reagents with precise placement for biosensor applications. The size, shape, and density of the patterns may be modified by simple adjustments of the patterning parameters. Using this system, two methods of multi-analyte protein patterning for use in biosensor assays are demonstrated. The first method involves the deposition of multiple proteins directly onto a gold substrate. Specific binding of an antibody to the deposited antigen is demonstrated, although nonspecific adsorption of the antibody may limit the utility of this simple method in quantitative biosensor applications. A second, more sophisticated multi-analyte patterning method involves two sequential patterning steps, consisting of an initial deposition onto gold of a mixed thiol layer to provide oriented binding capabilities in a nonfouling background and a second deposition of multiple biotinylated proteins. Highly specific antibody binding to this patterned multi-analyte surface was demonstrated, with minimal nonspecific adsorption to the surrounding regions. Thus, this method produces high-quality, localized, and customizable sub-millimeter patterns in a nonfouling background for multi-analyte bioassay development. [Copyright &y& Elsevier]

Abstract: This work describes the application of an optical biosensor (RIver ANALyser, RIANA) to the simultaneous analysis of three relevant environmental organic pollutants, namely, the pesticides atrazine and isoproturon and the estrogen estrone, in real water samples. This biosensor is based on an indirect inhibition immunoassay which takes place at a chemically modified optical transducer chip. The spatially resolved modification of the transducer surface allows the simultaneous determination of selected target analytes by means of “total internal reflection fluorescence” (TIRF). The performance of the immunosensor method developed was evaluated against a well accepted traditional method based on solid-phase extraction followed by liquid chromatography–mass spectrometry (LC–MS). The chromatographic method was superior in terms of linearity, sensitivity and accuracy, and the biosensor method in terms of repeatability, speed, cost and automation. The application of both methods in parallel to determine the occurrence and removal of atrazine, isoproturon and estrone throughout the treatment process (sand filtration, ozonation, activated carbon filtration and chlorination) in a waterworks showed an overestimation of results in the case of the biosensor, which was partially attributed to matrix and cross-reactivity effects, in spite of the addition of ovalbumin to the sample to minimize matrix interferences. Based on the comparative performance of both techniques, the biosensor emerges as a suitable tool for fast, simple and automated screening of water pollutants without sample pretreatment. To the author''s knowledge, this is the first description of the application of the biosensor RIANA in the multi-analyte configuration to the regular monitoring of pollutants in a waterworks. [Copyright &y& Elsevier]

Abstract: Foodborne contaminants come in a variety of sizes ranging from simple chemical compounds to entire bacterial cells. Due to public health concerns, there is a current need in the food industry for a sensitive, specific and rapid method to monitor for the presence of these toxic species, either as a result of natural or deliberate contamination. The Array Biosensor developed at the NRL encompasses these qualities, including the ability to measure multiple analytes simultaneously on a single substrate. In this study, we demonstrate the Array Biosensor''s ability to measure both large pathogens, such as the bacteria Campylobacter jejuni (C. jejuni), and small toxins, including the mycotoxins ochratoxin A, fumonisin B, aflatoxin B1 and deoxynivalenol. Sandwich immunoassays were used to measure C. jejuni in buffer and a number of food matrices, while competitive immunoassays, taking only 15min, were developed for the simultaneous detection of multiple mycotoxins. The combination of sandwich and competitive immunoassay formats on a single substrate was demonstrated, allowing the simultaneous detection of both large (C. jejuni) and small (aflatoxin B1) food contaminants. [Copyright &y& Elsevier]

Abstract: A method was developed and validated for the simultaneous determination of five cannabinoids, viz. cannabidiol (CBD), cannabidiol acid (CBD-COOH), cannabinol (CBN), Δ9-tetrahydrocannabinol (THC), and 3′-carboxy-Δ9-all-trans-tetrahydrocannabinol (THC-COOH) in cannabis products. The cannabinoids were extracted from the grinded cannabis samples with a mixture of methanol–chloroform and analysed using liquid chromatography with ion-trap-mass-spectrometry (LC–IT-MSn). For quantification the two most abundant diagnostic MS–MS ions of the analyte in the sample and external standard were monitored. For confirmation purposes the EU criteria as described in Commission Decision 2002/657/EC were followed. Fully satisfactory results were obtained, that is, unequivocal confirmation according to the most stringent EU criteria was possible. The limits of quantification were 0.1g/kg for CBD, 0.04g/kg for CBD-COOH, 0.03g/kg for CBN, 0.28g/kg for THC and 9.9g/kg for THC-COOH. The repeatabilities, defined by R.S.D., were 2% for CBN, THC and THC-COOH at the concentration levels of respectively 0.023, 3.3 and 113g/kg and 5% for CBD-COOH at the level of 0.34g/kg (n = 6). [Copyright &y& Elsevier]

A multi-analyte biosensor based on nucleic acid hybridization and liposome signal amplification was developed for the rapid serotype-specific detection of Dengue virus. After RNA amplification, detection of Dengue virus specific serotypes can be accomplished using a single analysis within 25 min. The multi-analyte biosensor is based on single-analyte assays (see Baeumner et al (2002) Anal Chem 74:1442–1448) developed earlier in which four analyses were required for specific serotype identification of Dengue virus samples. The multi-analyte biosensor employs generic and serotype-specific DNA probes, which hybridize with Dengue RNA that is amplified by the isothermal nucleic acid sequence based amplification (NASBA) reaction. The generic probe (reporter probe) is coupled to dye-entrapping liposomes and can hybridize to all four Dengue serotypes, while the serotype-specific probes (capture probes) are immobilized through biotin—streptavidin interaction on the surface of a polyethersulfone membrane strip in separate locations. A mixture of amplified Dengue virus RNA sequences and liposomes is applied to the membrane and allowed to migrate up along the test strip. After the liposome-target sequence complexes hybridize to the specific probes immobilized in the capture zones of the membrane strip, the Dengue serotype present in the sample can be determined. The amount of liposomes immobilized in the various capture zones directly correlates to the amount of viral RNA in the sample and can be quantified by a portable reflectometer. The specific arrangement of the capture zones and the use of unlabeled oligonucleotides (cold probes) enabled us to dramatically reduce the cross-reactivity of Dengue virus serotypes. Therefore, a single biosensor can be used to detect the exact Dengue serotype present in the sample. In addition, the biosensor can simultaneously detect two serotypes and so it is useful for the identification of possible concurrent infections found in clinical samples. The various biosensor components have been optimized with respect to specificity and sensitivity, and the system has been ultimately tested using blind coded samples. The biosensor demonstrated 92% reliability in Dengue serotype determination. Following isothermal amplification of the target sequences, the biosensor had a detection limit of 50 RNA molecules for serotype 2, 500 RNA molecules for serotypes 3 and 4, and 50,000 molecules for serotype 1. The multi-analyte biosensor is portable, inexpensive, and very easy to use and represents an alternative to current detection methods coupled with nucleic acid amplification reactions such as electrochemiluminescence, or those based on more expensive and time consuming methods such as ELISA or tissue culture. [ABSTRACT FROM AUTHOR]

Protein assays provide direct access to biologically and pharmacologically relevant information. To obtain a maximum of information from the very smallest amounts of complex biological samples, highly multiplexed protein assays are needed. However, at present, cross-reactions of binding reagents restrict the use of such assays to selected cases and severely limit the potential for up-scaling the technology. Here we describe a double-chip format, which can effectively overcome this specificity problem for sandwich immunoassays. This format consists of a capture array and a reference array with fluorescent labeled detection antibodies coupled to the reference array via DNA duplexes. This format allows for the local application of the labeled detection antibodies onto their corresponding specific spots on the capture array. Here we show that this double-chip format allows for the use of cross-reactive antibodies without generating false positive signals, and an assay for the parallel detection of seven different cytokines was set up. Even without further optimization, the dynamic range and the limit of detection for interleukin 8 were found to be comparable to those obtained with other types of multiplexed sandwich immunoassays. [ABSTRACT FROM AUTHOR]

In most medical and environmental applications of biosensors, only single analytes are determined. However, the monitoring of several analytes is obviously preferable in order to gather more information about the sample under analysis. In line with this, different technologies are being developed to obtain multi-analyte sensors.In this paper, an analytical method for the simultaneous determination of three different contaminants—atrazine, isoproturon, and estrone—in natural waters by using an optical immunosensor prototype, the so-called “RIver ANAlyser” (RIANA), is described. RIANA is based on a rapid solid-phase fluoroimmunoassay that takes place at an optical transducer chip. The transducer surface is chemically modified with three analytes derivatives placed in different discrete locations. The sensor surface can be regenerated thus allowing the performance of several measurements with the same transducer. Each test cycle, including one regeneration step, is accomplished in 15 min. Detection limits achieved were 0.155, 0.046, and 0.084 μg/l, for atrazine, isoproturon, and estrone, respectively. Satisfactory repetition, with relative standard deviations between 1.06 and 6.98%, was obtained. Excluding a minor non-specifical binding of the isoproturon antibodies, no cross-reactivity effects were observed. Matrix effects were significant only in the case of wastewater samples. Biosensor measurements were validated using conventional liquid chromatography-mass spectrometry. The results obtained with both techniques were in good agreement. [Copyright &y& Elsevier]

The array biosensor is capable of detecting multiple targets rapidly and simultaneously on the surface of a single waveguide. Sandwich and competitive fluoroimmunoassays have been developed to detect high and low molecular weight toxins, respectively, in complex samples. Recognition molecules (usually antibodies) were first immobilized in specific locations on the waveguide and the resultant patterned array was used to interrogate up to 12 different samples for the presence of multiple different analytes. Upon binding of a fluorescent analyte or fluorescent immunocomplex, the pattern of fluorescent spots was detected using a CCD camera. Automated image analysis was used to determine a mean fluorescence value for each assay spot and to subtract the local background signal. The location of the spot and its mean fluorescence value were used to determine the toxin identity and concentration. Toxins were measured in clinical fluids, environmental samples and foods, with minimal sample preparation. Results are shown for rapid analyses of staphylococcal enterotoxin B, ricin, cholera toxin, botulinum toxoids, trinitrotoluene, and the mycotoxin fumonisin. Toxins were detected at levels as low as 0.5 ng mL-1. [ABSTRACT FROM AUTHOR]