A magnetic bead-based assay for the rapid detection of methicillin-resistant Staphylococcus aureus by using a microfluidic system with integrated loop-mediated isothermal amplification

This study reports a new diagnostic assay for the rapid detection of methicillin-resistant Staphylococcus aureus (MRSA) by combing nucleic acid extraction and isothermal amplification of target nucleic acids in a magnetic bead-based microfluidic system. By using specific probe-conjugated magnetic beads, the target deoxyribonucleic acid (DNA) of the MRSA can be specifically recognized and hybridized onto the surface of the magnetic beads which are then mixed with clinical sample lysates. This is followed by purifying and concentrating the target DNA from the clinical sample lysates by applying a magnetic field. Nucleic acid amplification of the target genes can then be performed by the use of a loop-mediated isothermal amplification (LAMP) process via the incorporation of a built-in micro temperature control module, followed by analyzing the optical density (OD) of the LAMP amplicons using a spectrophotometer. Significantly, experimental results show that the limit of detection (LOD) for MRSA in the clinical samples is approximately 10 fg μL(-1) by performing this diagnostic assay in the magnetic bead-based microfluidic system. In addition, the entire diagnostic protocol, from bio-sample pre-treatment to optical detection, can be automatically completed within 60 min. Consequently, this miniature diagnostic assay may become a powerful tool for the rapid purification and detection of MRSA and a potential point-of-care platform for detection of other types of infections.     

Real-time monitoring of strand-displacement DNA amplification by a contactless electrochemical microsystem using interdigitated electrodes

This paper reports the design and implementation of a contactless conductivity detection system which combines a thermal control cell, a data processing system and an electrochemical (EC) cell for label-free isothermal nucleic acid amplification and real-time monitoring. The EC cell consists of a microchamber and interdigitated electrodes as the contactless conductivity biosensor with a cover slip as insulation. In our work, contactless EC measurements, the effects of trehalose on amplification, and chip surface treatment are investigated. With the superior performance of the biosensor, the device can detect the amount of pure DNA at concentrations less than 0.1 pg μl(-1). The EC cell, integrated with a heater and a temperature sensor, has successfully implemented nicking-based strand-displacement amplification at an initial concentration of 2.5 μM and the yields are monitored directly (dismissing the use of probes or labels) on-line. This contactless detector carries important advantages: high anti-interference capability, long detector life, high reusability and low cost. In addition, the small size, low power consumption and portability of the detection cell give the system the potential to be highly integrated for use in field service and point of care applications.     

Ultrasensitive electrochemical biosensor for uranium using deoxyribozymes with amplification by gold nanoparticles

A novel highly sensitive and specific electrochemical biosensor for detecting uranium based on specific Deoxyribozymes and gold nanoparticles (AuNPs) is reported. In this work, AuNPs provide excellent electrochemical signal transduction and a large surface area for immobilising numerous Deoxyribozymes, so a low detection limit of 3.24 ng L^?1 uranium and a good linear relationship over the range 5.94–35.1 ng L^?1 (r = 0.994) were obtained. The proposed biosensor presents high specificity and selectivity for uranium and is not affected by other metal ions. Thus, the biosensor protocol offers good selectivity, rapid speed and operational convenience for detection uranium in liquid waste.     

Detection of influenza virus using a lateral flow immunoassay for amplified DNA by a microfluidic RT-PCR chip

Influenza virus RNA was amplified by a continuous-flow polydimethylsiloxane microfluidic RT-PCR chip within 15-20 min. The amplified influenza virus RNA was observed with the naked eye, as the red color at the test line, using a lateral flow immunoassay within 1 min.     

Ratiometric fluorescence transduction by hybridization after isothermal amplification for determination of zeptomole quantities of oligonucleotide biomarkers with a paper-based platform and camera-based detection

Paper is a promising platform for the development of decentralized diagnostic assays owing to the low cost and ease of use of paper-based analytical devices (PADs). It can be challenging to detect on PADs very low concentrations of nucleic acid biomarkers of lengths as used in clinical assays. Herein we report the use of thermophilic helicase-dependent amplification (tHDA) in combination with a paper-based platform for fluorescence detection of probe-target hybridization. Paper substrates were patterned using wax printing. The cellulosic fibers were chemically derivatized with imidazole groups for the assembly of the transduction interface that consisted of immobilized quantum dot (QD)–probe oligonucleotide conjugates. Green-emitting QDs (gQDs) served as donors with Cy3 as the acceptor dye in a fluorescence resonance energy transfer (FRET)-based transduction method. After probe-target hybridization, a further hybridization event with a reporter sequence brought the Cy3 acceptor dye in close proximity to the surface of immobilized gQDs, triggering a FRET sensitized emission that served as an analytical signal. Ratiometric detection was evaluated using both an epifluorescence microscope and a low-cost iPad camera as detectors. Addition of the tHDA method for target amplification to produce sequences of ∼100 base length allowed for the detection of zmol quantities of nucleic acid targets using the two detection platforms. The ratiometric QD-FRET transduction method not only offered improved assay precision, but also lowered the limit of detection of the assay when compared with the non-ratiometric QD-FRET transduction method. The selectivity of the hybridization assays was demonstrated by the detection of single nucleotide polymorphism.     

Development of a rapid process monitoring method for dry-coated tableting process by using near-infrared spectroscopy

A nondestructive transmittance near-infrared (NIR) method for detecting off-centered cores in dry-coated (DC) tablets was developed as a monitoring system in the DC tableting process. Caffeine anhydrate was used as a core active pharmaceutical ingredient (API), and DC tablets were made by the direct compression method. NIR spectra were obtained from these intact DC tablets using the transmittance method. The reference assay was performed with HPLC. Calibration models were generated by partial least squares (PLS) regression and principal component regression (PCR) utilizing external validations. Hierarchical cluster analysis (HCA) of the results confirmed that NIR spectroscopy correctly detected off-centered cores in DC tablets. We formulated and used the Centering Index (CI) to evaluate the precision of core alignment and generated an NIR calibration model that could correctly predict this index. The principal component (PC) 1 loading vector of the final calibration model indicated that it could specifically detect the misalignment of tablet cores. The model also had good linearity and accuracy. The CIs of unknown sample tablets predicted by the final calibration model and those calculated through the HPLC analysis were closely parallel with each other. These results demonstrate the validity of the final calibration model and the utility of the transmittance NIR spectroscopic method developed in this study as a monitoring system in DC tableting process.     

Supramolecular imprinted electrochemical sensor for the neonicotinoid insecticide imidacloprid based on double amplification by Pt-In catalytic nanoparticles and a Bromophenol blue doped molecularly imprinted film

The authors describe an electrochemical sensor for the neonicotinoid insecticide imidacloprid (IMI) based on Pt-In catalytic nanocomposite film and Bromophenol blue amplification. The Pt-In nanocomposite film was deposited on the surface of a modified glassy carbon electrode. The composite molecularly imprinted polymer (MIP) was prepared by electro-polymerization using bromophenol blue doped o-aminophenol as functional monomer and 4-tert-butylcalix[6]arene-IMI supramolecular inclusion complex as template molecule. The experimental results showed that the current intensity of IMI was clearly amplified in the potential range from ?0.3 to ?1.8 V, because of the double amplification, based on the Pt-In film and Bromophenol blue catalysis. Moreover, the double recognition ability of the sensor, which relied on the MIP and the vacuum structure of 4-tert-butylcalix[6]arene, effectively increased the specific recognition performance. The feasibility of its practical applications has been demonstrated by the analysis of vegetable samples.

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Graphical abstract A supramolecular imprinted electrochemical sensor for imidacloprid determination was prepared based on Pt-In nanocomposite film and bromophenol blue amplification. Because of the advantages of the specific recognition sites in MIPs and supramolecular chemistry, the sensor showed good selectivity for imidacloprid.

     

Voltammetric determination of attomolar levels of a sequence derived from the genom of hepatitis B virus by using molecular beacon mediated circular strand displacement and rolling circle amplification

The authors describe an electrochemical method for the determination of the single-stranded DNA (ssDNA) oligonucleotide with a sequence derived from the genom of hepatitis B virus (HBV). It is making use of circular strand displacement (CSD) and rolling circle amplification (RCA) strategies mediated by a molecular beacon (MB). This ssDNA hybridizes with the loop portion of the MB immobilized on the surface of a gold electrode, while primer DNA also hybridizes with the rest of partial DNA sequences of MB. This triggers the MB-mediated CSD. The RCA is then initiated to produce a long DNA strand with multiple tandem-repeat sequences, and this results in a significant increase of the differential pulse voltammetric response of the electrochemical probe Methylene Blue at a rather low working potential of ?0.24 V (vs. Ag/AgCl). Under optimal experimental conditions, the assay displays an ultrahigh sensitivity (with a 2.6 aM detection limit) and excellent selectivity. Response is linear in the 10 to 700 aM DNA concentration range.

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Graphical abstract Schematic of a voltammetric method for the determination of attomolar levels of target DNA. It is based on molecular beacon mediated circular strand displacement and rolling circle amplification strategies. Under optimal experimental conditions, the assay displays an ultrahigh sensitivity with a 2.6 aM detection limit and excellent selectivity.

     

A new time interval analysis system for the on-line monitoring of artificial radionuclides in airborne dust using a phosfitch type alpha/beta detector

A portable system for high-speed time interval analysis (TIA) method was constructed. The system installed with an original time counting board could record pulse incident time of 1 MHz precisely. During the measurement, calculation of time intervals for all the pulses and on-line display of the results were performed simultaneously. Using this system, the correlated decay events of ²¹⁴Bi to ²¹⁰Po in the airborne dust collected on filter paper were counted, together with a dual-phosphor type alpha/beta detector. A rapidly detective technique of artificial radioactivity under the variations of naturally occurring radioactivities is discussed.     

Improvement in performance of a flow electrochemical sensor by using carbamoyl-arms polyazamacrocycle for the preconcentration of lead ions onto the electrode

A flow electrochemical sensor for trace analysis of lead, using TETRAM-modified graphite felt electrode is reported here. TETRAM ligands are covalently immobilized on the graphite felt by chemical reactions on amino acid linkers, previously attached to the electrode by an electrochemical process. The detection is performed in two steps: the preconcentration of Pb²⁺ ions by complexation with immobilized TETRAM and the analysis by linear sweep stripping voltammetry. A calibration curve typical of at least two equilibrium processes is obtained. A limit of detection of 2.5 × 10^?8 mol L^?1 is reached for a total analysis time of 35 min. Interestingly, the flow sensor shows a good selectivity toward lead in presence of Cu²⁺, Cd²⁺, Ni²⁺, Zn²⁺ and Co²⁺ ions. This new sensor exhibits improved sensitivity and selectivity compared to the previously reported sensor using cyclam-modified electrode. It is stable after three uses, using strong acidic medium for the regeneration step.     

Fabricating a reversible and regenerable electrochemical biosensor for quantitative detection of antibody by using “triplex-stem” DNA molecular switch

A reversible and regenerable electrochemical biosensor is fabricated for quantitative detection of antibody based on “triplex-stem” molecular switches. A hairpin-shaped oligonucleotide (hairpin DNA) labeled with ferrocene (Fc) at the 3′-end is fixed on the gold electrode serving as a signal transduction probe. Its hairpin structure leads Fc close to the surface of gold electrode and produces a strong current signal (on-state). A single-strand oligonucleotide modified with two digoxin molecules on the two arm segments (capture DNA) interact with hairpin DNA with the help of Ag⁺ ions. The “triplex-stem” DNA forms, which separates Fc from the electrode and reduces the electrochemical signal (off-state). Binding of digoxin antibody to digoxin releases capture DNA from the hairpin DNA, creating an effective “off-on” current signal switch. The stability of the “triplex-stem” structure of hairpin/capture DNA is critical to the signal switch and the sensitivity of the method, which can be adjusted conveniently and efficiently by changing Ag⁺ concentrations. Based on the “off-on” current signal switch, this biosensor is used to detect digoxin antibody sensitively in blood serum. The linear range is 1.0–500 pg with a correlation coefficient of 0.996, and the detection limit is 0.4 pg. Also, this biosensor shows excellent reversibility and reproducibility, which are significant requirements for practical biosensor applications.     

The design and use of a simple System Suitability Test Mix for generic reverse phase high performance liquid chromatography-mass spectrometry systems and the implications for automated system monitoring using global software tracking

The development of a seven-component test mixture designed for use with a generic gradient and a reversed-phase high performance liquid chromatography-mass spectrometry (RP-HPLC-MS) system is discussed. Unlike many test mixtures formulated in order to characterise column quality at neutral pH, the test mixture reported here was designed to permit an overall suitability assessment of the whole liquid chromatography-mass spectrometry (LCMS) system. The mixture is designed to test the chromatographic performance of the column as well as certain aspects of the performance of the individual instrumental components of the system. The System Suitability Test Mix can be used for low and high pH generic reverse phase LCMS analysis. Four phthalates are used: diethyl phthalate (DEP), diamyl phthalate (DAP), di-n-hexyl phthalate (DHP) and dioctyl phthalate (DOP). Three other probes are employed: 8-bromoguanosine (8-BG), amitryptyline (Ami), and 4-chlorocinnamic acid (4-CCA). We show that analysis of this test mixture can alert the user when any part of the system (instrument or column) contributes to loss of overall performance and may require remedial action and demonstrate that it can provide information that enables us to document data quality control.     

Collection of selected reaction monitoring and full scan data on a time scale suitable for target compound quantitative analysis by liquid chromatography/tandem mass spectrometry

A hybrid linear ion trap/triple quadrupole mass spectrometer was used to demonstrate the value of collecting full scan qualitative data during quantitative analysis of target compounds. We present examples of the additional information that can be obtained from plasma samples analyzed primarily for target compound concentrations. This information includes detection of circulating metabolites, dosing vehicle, interfering matrix components, and potential interfering drug conjugates. Additionally, the quantitative results from selected reaction monitoring (SRM) analysis and from combined full scan and SRM analysis (SRM/EMS) were compared. The quantitative data in both scan modes are acceptable in terms of sensitivity, accuracy and precision. One can conclude from this work that the hybrid linear ion trap/triple quadrupole mass analyzer can provide in a single analysis both useful qualitative data, and accurate and precise quantitative data from the samples routinely prepared and analyzed for target drug concentrations.     

Drug monitoring: simultaneous analysis of lamotrigine, oxcarbazepine, 10-hydroxycarbazepine, and zonisamide by HPLC-UV and a rapid GC method using a nitrogen-phosphorus detector for levetiracetam

A high-performance liquid chromatography (HPLC) assay using UV detection is described for the simultaneous measurement of the newer generation anti-epileptic medications lamotrigine, oxcarbazepine (parent drug and active metabolite 10- hydroxycarbazepine), and zonisamide. Detection of all four compounds can be done at 230 nm; however, there is a potential interference with zonisamide in patients on clonazepam therapy. Therefore, the method uses dual wavelength detection: 230 nm for oxcarbazepine and 10-hydroxycarbazepine and 270 nm for lamotrigine and zonisamide. In addition, a simple gas chromatography method using a nitrogen-phosphorus detector is described for the measurement of levetiracetam, another of the recently approved anti-epileptic medications. For both methods, limits of quantitation, linearities, accuracies, and imprecisions cover the therapeutic range for drug monitoring of patients. A wide variety of clinical drugs, including other anti-epileptic drugs, do not interfere with these assays. These procedures would be of special interest to clinical laboratories, particularly due to the limited availability of immunoassays for newer generation anti-epileptic medications and that therapeutic uses of these drugs are expanding beyond epilepsy to other neurologic and psychiatric disorders.