Multiplex identification of drug‐resistant Gram‐positive pathogens using stuffer‐free MLPA system

Early detection of pathogens from blood and identification of their drug resistance are essential for sepsis management. However, conventional culture‐based methods require relatively longer time to identify drug‐resistant pathogens, which delays therapeutic decisions. For precise multiplex detection of drug‐resistant Gram‐positive pathogens, we developed a method by using stuffer‐free multiplex ligation‐dependent probe amplification (MLPA) coupled with high‐resolution CE single‐strand conformation polymorphisms (CE‐SSCP) system. We designed three probe sets for genes specific to Gram‐positive species (Staphylococcus aureus: nuc, Enterococcus faecium: sodA, and Streptococcus pneumoniae: lytA) and two sets for genes associated with drug resistance (mecA and vanA) to discriminate major Gram‐positive pathogens with the resistance. A total of 94 different strains (34 reference strains and 60 clinical isolates) were used to validate this method and strain‐specific peaks were successfully observed for all the strains. To improve sensitivity of the method, a target‐specific preamplification step was introduced and, consequently, the sensitivity increased from 10 pg to 100 fg. We also reduced a total assay time to 8 h by optimizing hybridization time without compromising test sensitivity. Taken together, our multiplex detection system can improve detection of drug‐resistant Gram‐positive pathogens from sepsis patients’ blood.     

Application of thin‐layer chromatography/infrared matrix‐assisted laser desorption/ionization orthogonal time‐of‐flight mass spectrometry to structural analysis of bacteria‐binding glycosphingolipids selected by affinity detection

Glycosphingolipids (GSLs) play key roles in the manifestation of infectious diseases as attachment sites for pathogens. The thin‐layer chromatography (TLC) overlay assay represents one of the most powerful approaches for the detection of GSL receptors of microorganisms. Here we report on the direct structural characterization of microbial GSL receptors by employment of the TLC overlay assay combined with infrared matrix‐assisted laser desorption/ionization orthogonal time‐of‐flight mass spectrometry (IR‐MALDI‐o‐TOF‐MS). The procedure includes TLC separation of GSL mixtures, overlay of the chromatogram with GSL‐specific bacteria, detection of bound microbes with primary antibodies against bacterial surface proteins and appropriate alkaline phosphatase labeled secondary antibodies, and in situ MS analysis of bacteria‐specific GSL receptors. The combined method works on microgram scale of GSL mixtures and is advantageous in that it omits laborious and time‐consuming GSL extraction from the silica gel layer. This technique was successfully applied to the compositional analysis of globo‐series neutral GSLs recognized by P‐fimbriated Escherichia coli bacteria, which were used as model microorganisms for infection of the human urinary tract. Thus, direct TLC/IR‐MALDI‐o‐TOF‐MS adds a novel facet to this fast and sensitive method offering a wide range of applications for the investigation of carbohydrate‐specific pathogens involved in human infectious diseases. Copyright © 2010 John Wiley & Sons, Ltd.     

Rapid Colorimetric Identification and Targeted Photothermal Lysis of Salmonella Bacteria by Using Bioconjugated Oval‐Shaped Gold Nanoparticles

Salmonella bacteria are the major cause for the infection of 16 million people worldwide with typhoid fever each year. Antibiotic‐resistant Salmonella strains have been isolated from various food products. As a result, the development of ultrasensitive sensing technology for detection and new approaches for the treatment of infectious bacterial pathogens that do not rely on traditional therapeutic regimes is very urgent for public health, food safety, and the world economy. Driven by this need, we report herein a nanotechnology‐driven approach that uses antibody‐conjugated oval‐shaped gold nanoparticles to selectively target and destroy pathogenic bacteria. Our experiments have shown the use of a simple colorimetric assay, with an anti‐salmonella antibody conjugated to oval‐shaped gold nanoparticles, for the label‐free detection of S. typhimurium with an excellent detection limit (10⁴ bacteria per mL) and high selectivity over other pathogens. When bacteria conjugated to oval‐shaped gold nanoparticles were exposed to near‐infrared radiation, a highly significant reduction in bacterial cell viability was observed due to photothermal lysis. Ideally, this nanotechnology‐based assay would have enormous potential for rapid, on‐site pathogen detection to avoid the distribution of contaminated foods.     

Multiplex identification of sepsis‐causing Gram‐negative pathogens from the plasma of infected blood

Early and accurate detection of bacterial pathogens in the blood is the most crucial step for sepsis management. Gram‐negative bacteria are the most common organisms causing severe sepsis and responsible for high morbidity and mortality. We aimed to develop a method for rapid multiplex identification of clinically important Gram‐negative pathogens and also validated whether our system can identify Gram‐negative pathogens with the cell‐free plasm DNA from infected blood. We designed five MLPA probe sets targeting the genes specific to major Gram‐negative pathogens (uidA and lacY for E. coli, ompA for A. baumannii, phoE for K. pneumoniae, and ecfX for P. aeruginosa) and one set targeting the CTX‐M group 1 to identify the ESBL producing Gram‐negative pathogens. All six target‐specific peaks were clearly separated without any non‐specific peaks in a multiplex reaction condition. The minimum detection limit was 100 fg of pathogen DNA. When we tested 28 Gram‐negative clinical isolates, all of them were successfully identified without any non‐specific peaks. To evaluate the clinical applicability, we tested seven blood samples from febrile patients. Three blood culture positive cases showed E. coli specific peaks, while no peak was detected in the other four culture negative samples. This technology can be useful for detection of major sepsis‐causing, drug‐resistant Gram‐negative pathogens and also the major ESBL producing Gram‐negatives from the blood of sepsis patients in a clinical setting. This system can help early initiation of effective antimicrobial treatment against Gram‐negative pathogens for sepsis patients, which is very crucial for better treatment outcomes.     

Harnessing Effective Molarity to Design an Electrochemical DNA‐based Platform for Clinically Relevant Antibody Detection

Easy‐to‐use platforms for rapid antibody detection are likely to improve molecular diagnostics and immunotherapy monitoring. However, current technologies require multi‐step, time‐consuming procedures that limit their applicability in these fields. Herein, we demonstrate effective molarity‐driven electrochemical DNA‐based detection of target antibodies. We show a highly selective, signal‐on DNA‐based sensor that takes advantage of antibody‐binding‐induced increase of local concentration to detect clinically relevant antibodies in blood serum. The sensing platform is modular, rapid, and versatile and allows the detection of both IgG and IgE antibodies. We also demonstrate the possible use of this strategy for the monitoring of therapeutic monoclonal antibodies in body fluids. Our approach highlights the potential of harnessing effective molarity for the design of electrochemical sensing strategies.     

A Mix‐and‐Read Fluorescence Strategy for the Switch‐On Probing of Kinase Activity Based on an Aptameric‐Peptide/Graphene‐Oxide Platform

Protein kinase plays a vital role in regulating signal‐transduction pathways and its simple and quick detection is highly desirable because traditional kinase assays typically rely on a time‐consuming kinase‐phosphorylation process (ca. 1 h). Herein, we report a new and rapid fluorescence‐based sensing platform for probing the activity of protein kinase that is based on the super‐quenching capacity of graphene oxide (GO) nanosheets and specific recognition of the aptameric peptide (FITC‐IP₂₀). On the GO/peptide platform, the fluorescence quenching of FITC‐IP₂₀ that is adsorbed onto GO can be restored by selective binding of active protein kinase to the aptameric peptide, thereby resulting in the fast switch‐on detection of kinase activity (ca. 15 min). The feasibility of this method has been demonstrated by the sensitive measurement of the activity of cAMP‐dependent protein kinase (PKA), with a detection limit of 0.053 mU μL^?1. This assay technique was also successfully applied to the detection of kinase activation in cell lysate.     

Thin‐layer chromatography—an image‐processing method for the determination of acidic catecholamine metabolites

A sensitive and convenient method for acidic catecholamine metabolites (including homovanillic acid, vanillylmandelic acid, 3,4‐dihydroxymandelic acid, and 3,4‐dihydroxyphenylacetic acid) determination was developed based on thin‐layer chromatography and image‐processing analysis. The metabolites were separated without a prederivatization step using reversed phase RP‐18W high‐performance plates. The mobile phase composition, detection, and quantification conditions were systematically investigated through several trials. The reaction with 2,2‐diphenyl‐1‐picrylhydrazyl radical allowed specific detection of acidic catecholamine metabolites with a high sensitivity and a wide linear range. The limit of detection and the limit of quantification were in the range of 13–103 and 18–120 ng/spot, respectively, in all cases. Mean recoveries determined were in the range 95–106% for all of the investigated compounds. The proposed method allowed rapid simultaneous determination of acidic catecholamine metabolites from spiked human urine sample.     

Quantitation of Femtomolar‐Level Protein Biomarkers Using a Simple Microbubbling Digital Assay and Bright‐Field Smartphone Imaging

Quantitating ultra‐low concentrations of protein biomarkers is critical for early disease diagnosis and treatment. However, most current point‐of‐care (POC) assays are limited in sensitivity. Herein, we introduce an ultra‐sensitive and facile microbubbling assay for the quantification of protein biomarkers with a digital‐readout method that requires only a smartphone camera. We used machine learning to develop a smartphone application for automated image analysis to facilitate accurate and robust counting. Using this method, post‐prostatectomy surveillance of prostate specific antigen (PSA) can be achieved with a detection limit (LOD) of 2.1 fm (0.060 pg mL^?1), and early pregnancy detection using βhCG can be achieved with a of 0.034 mIU mL^?1 (2.84 pg mL^?1). This work provides the proof‐of‐principle of the microbubbling assay with a digital readout as an ultra‐sensitive technology with minimal requirement for power and accessories, facilitating future POC applications.     

A new single‐step quantitative pathogen detection system: Template‐tagging followed by multiplex asymmetric PCR using common primers and CE‐SSCP

Rapid diagnosis of bacterial infection is important for patient management and appropriate therapy during the early phase of bacteria‐induced disease. Among the existing techniques for identifying microbial, CE‐SSCP combined with 16S ribosomal RNA gene‐specific PCR has the benefits of excellent sensitivity, resolution, and reproducibility. However, even though CE‐SSCP can separate PCR products with high‐resolution, multiplex detection and quantification are complicated by primer‐dimer formation and non‐specific amplification. Here, we describe a novel technique for multiplex detection and quantification of pathogens by template‐tagging followed by multiplex asymmetric PCR and subsequent CE‐SSCP. More specifically, we reverse transcribed 16S ribosomal RNAs from seven septicemia‐inducing pathogens, tagged the templates with common end sequences, and amplified them using common primers. The resulting amplicons could be successfully separated by CE‐SSCP and quantified by comparison to an internal standard. This method yielded results that illustrate the potential of this system for diagnosing infectious disease.     

Preventive doping control screening analysis of prohibited substances in human urine using rapid‐resolution liquid chromatography/high‐resolution time‐of‐flight mass spectrometry

Unification of the screening protocols for a wide range of doping agents has become an important issue for doping control laboratories. This study presents the development and validation of a generic liquid chromatography/time‐of‐flight mass spectrometry (LC/TOFMS) screening method of 241 small molecule analytes from various categories of prohibited substances (stimulants, narcotics, diuretics, β₂‐agonists, β‐blockers, hormone antagonists and modulators, glucocorticosteroids and anabolic agents). It is based on a single‐step liquid‐liquid extraction of hydrolyzed urine and the use of a rapid‐resolution liquid chromatography/high‐resolution time‐of‐flight mass spectrometric system acquiring continuous full scan data. Electrospray ionization in the positive mode was used. Validation parameters consisted of identification capability, limit of detection, specificity, ion suppression, extraction recovery, repeatability and mass accuracy. Detection criteria were established on the basis of retention time reproducibility and mass accuracy. The suitability of the methodology for doping control was demonstrated with positive urine samples. The preventive role of the method was proved by the case where full scan acquisition with accurate mass measurement allowed the retrospective reprocessing of acquired data from past doping control samples for the detection of a designer drug, the stimulant 4‐methyl‐2‐hexanamine, which resulted in re‐reporting a number of stored samples as positives for this particular substance, when, initially, they had been reported as negatives. Copyright © 2010 John Wiley & Sons, Ltd.     

Fluorescence Polarization Based Nucleic Acid Testing for Rapid and Cost‐Effective Diagnosis of Infectious Disease

A new nucleic acid detection method was developed for a rapid and cost‐effective diagnosis of infectious disease. This approach relies on the three unique elements: 1) detection probes that regulate DNA polymerase activity in response to the complementary target DNA; 2) universal reporters conjugated with a single fluorophore; and 3) fluorescence polarization (FP) detection. As a proof‐of‐concept, the assay was used to detect and sub‐type Salmonella bacteria with sensitivities down to a single bacterium in less than three hours.     

Analysis of microcystins using high‐performance liquid chromatography and magnetic solid‐phase extraction with silica‐coated magnetite with cetylpyridinium chloride

Microcystins (MCs), produced by freshwater cyanobacteria, can be serious water pollutants, so it is important to monitor their concentration in drinking water. We have developed a method for rapid and accurate determination of microcystin levels in environmental water, using magnetic solid‐phase extraction and high‐performance liquid chromatography with UV detection. The magnetic composite material, which was combined with cetylpyridinium chloride, was prepared by hydrothermal synthesis. The optimal extraction of microcystins in water sample was achieved by optimizing the amount of adsorbent, time of adsorption, ratio of eluting solvent, and volume of eluent. Under the optimal conditions, the limit of detection of MC‐LR was 0.001 μg/L, and the limit of quantification was 0.0028 μg/L. The limit of detection of MC‐RR was 0.001 μg/L, and the limit of quantification was 0.003 μg/L. These values are far lower than those established by the International Health Organization for the maximum concentration of microcystins in drinking water. The magnetic solid‐phase extraction adsorbent used in this method has the advantages of simple preparation, low price, and easy solid–liquid separation, and it can be used for the rapid and sensitive monitoring of trace microcystins in environmental water samples.     

Two‐step signal amplification for high‐sensitivity detection of biomarkers using gold nanoparticle–based conjugates

The measurement of biomarkers in bodily fluids is extremely important for diagnosing disease, monitoring disease progression, and evaluating treatment efficacy. In this paper, we present a highly sensitive and compatible gold nanoparticle (AuNP)‐based, two‐step signal amplification system for biomarker detection. First, AuNPs were coated onto the surfaces of 96‐well plates to generate rough surfaces, which enable immobilization of many more capture antibodies than a smooth substrate. As a result, detection sensitivity was enhanced significantly. Second, the horseradish peroxidase (HRP)‐conjugated detection antibodies were labeled on large‐size AuNPs, which increase the localized amounts of HRP and thus further lower the detection limit. Based on the consecutive signal amplification system, a high‐sensitivity assay was achieved, with a LOD of 0.07 ng/mL for prostate‐specific antigen (PSA). This assay was allowed to detect the PSA levels in clinical samples without changing the current standard immunoassay setups, showing great potential in many settings where immunoassays are needed.     

Voltammetric Determination of Urinary 1‐Hydroxypyrene Using Molecularly Imprinted Polymer‐Modified Screen‐Printed Carbon Electrodes

A two step non‐competitive affinity method for the trace determination of 1‐hydroxypyrene (1‐OHP) using a disposable molecularly imprinted polymer (MIP) modified screen‐printed carbon electrode (MIP‐SPCE) has been developed. The MIP was synthesized according to a novel strategy, which is described, and is capable of rebinding the phenolic analyte, 1‐hydroxypyrene (1‐OHP), from high pH aqueous organic media, via ionic interactions. In the first step of our method 1‐OHP was accumulated at the MIP‐SPCE from 35% aqueous methanol containing 0.014 M NaOH and 0.14 M NaCl, at open circuit. In the second step, the resulting SPCE with accumulated 1‐OHP was then transferred to fresh, clean phosphate buffered aqueous methanol, and subjected to cyclic voltammetry (CV) or differential pulse voltammetry (DPV). The latter technique proved to be more sensitive at detecting 1‐OHP, with a limit of detection of 182 nM and a linear range to 125 μM on unmodified electrodes. The possible effects of interference by related phenolic compounds in the MIP‐SPCE of 1‐OHP were investigated. Finally the method was evaluated by carrying out 1‐OHP determinations on spiked human urine samples; the recovery of 1‐OHP was 79.4% and the coefficient of variation was found to be 7.7% (n= 4) using a separate MIP‐SPCE for each determination. Therefore, the performance data suggests that the method is reliable at the concentrations examined in this study. The method was found to be superior to the direct determination of 1‐OHP in human urine by DPV alone, which was greatly affected by interference from uric acid.     

Acetylcholinesterase Sensor with Patterned Structure for Detecting Organophosphorus Pesticides Based on Titanium Dioxide Sol‐gel Carrier

In this paper, novel and stable acetylcholinesterase (AChE) sensor with patterned structure for detecting organophosphorus pesticides (OPs) based on titanium dioxide sol‐gel carrier was proposed and prepared. Biosensor was assembled by dropping titanium oxide, chitosan (CS) and enzyme to the surface of the glass carbon step by step. The concentration range of the sensor detection for dichlorvos (DDVP) is 1.13 nM to 22.6 μM, and the limit of detection (LOD) is 0.23 nM. It can also detect dichlorvos in cabbage juice samples accurately. The preparation of biosensor adopted a patterned novel structure for the first time, which opens a new way for the structure optimization of organophosphorus pesticide sensor.     

Nanocatalyst/Nanoplasmon‐Enabled Detection of Organic Mercury: A One‐Minute Visual Test

We present a fast and sensitive nanosensor that can detect organic mercury, exploiting the combination of the catalytic and plasmonic properties of gold nanoparticles (AuNPs). The method is one‐step and completely instrument‐free, and has a colorimetric readout clearly detectable by simple visual inspection. The AuNPs catalyze efficient organic mercury reduction to the metallic form (Hg⁰), allowing its nucleation and amalgam formation on particle surface, with consequent aggregation‐induced plasmon shift. This leads to very rapid (1 min) and specific colorimetric detection of mercury species. The achieved limit of detection (20 ppb) is compliant with current regulatory limits in food.     

Rapid determination of rifaximin on dried blood spots by LC‐ESI‐MS

The use of blood spot collection cards is a simple way to obtain specimens for therapeutic drug monitoring, assessing adherence to medications and preventing toxicity in a clinical setting. A high‐throughput liquid chromatography–electrospray ionization mass spectrometric (LC‐ESI‐MS) method for determination of rifaximin on dried blood spots (DBS) was developed and validated. It involves solvent extraction of a punch of DBS followed by reversed‐phase LC on a monolithic column consisting of a silica rod with bimodal pore structure and detection by ESI‐MS. Rifampicin was used as an internal standard (IS). The run time was within 5.0 min with a very low back‐pressure at a flow rate of 0.5 mL/min. The assay was linear from 0.1 to 10 ng/mL. The mean recovery was 98.42%. The developed method is very simple, rapid and useful for clinical applications. Copyright © 2011 John Wiley & Sons, Ltd.     

Fast and sensitive analysis of beta blockers by ultra‐high‐performance liquid chromatography coupled with ultra‐high‐resolution TOF mass spectrometry

This paper presents a method for the determination of acebutolol, betaxolol, bisoprolol, metoprolol, nebivolol and sotalol in human serum by liquid–liquid extraction and ultra‐high‐performance liquid chromatography coupled with ultra‐high‐resolution TOF mass spectrometry. After liquid–liquid extraction, beta blockers were separated on a reverse‐phase analytical column (Acclaim RS 120; 100 × 2.1 mm, 2.2 μm). The total run time was 6 min for each sample. Linearity, limit of detection, limit of quantification, matrix effects, specificity, precision, accuracy, recovery and sample stability were evaluated. The method was successfully applied to the therapeutic drug monitoring of 108 patients with hypertension. This method was also used for determination of beta blockers in 33 intoxicated patients.     

Facile Synthesis of Leaf‐Like CuO Nanoparticles and Their Application on Glucose Biosensor

An efficient amperometric biosensor based on well‐crystallized leaf‐like CuO nanoparticles for detecting glucose has been proposed. The leaf‐like CuO nanoparticles, synthesized by a simple one‐step hydrothermal method, were characterized by X‐ray diffraction (XRD), scanning electron microscope (SEM) and transmission electron microscopy (TEM) for the morphology study. Under the optimal condition, the electrochemical behaviour of the leaf‐like CuO nanoparticles modified electrode for detection of glucose exhibited high sensitivity of 246 µA/mM/cm², short response time (within 5 s), linear dynamic range from 1.0 to 170 µM (R²=0.9995), and low limit of detection (LOD) (S/N=3) of 0.91 µM. The high sensitivity, good reproducibility, stability, and fast amperometric sensing towards oxidation of glucose, make this biosensor promising for future application.