Miniaturization and optimization of 384-well compatible RNA sequencing library preparation.

Preparation of high-quality sequencing libraries is a costly and time-consuming component of metagenomic next generation sequencing (mNGS). While the overall cost of sequencing has dropped significantly over recent years, the reagents needed to prepare sequencing samples are likely to become the dominant expense in the process. Furthermore, libraries prepared by hand are subject to human variability and needless waste due to limitations of manual pipetting volumes. Reduction of reaction volumes, combined with sub-microliter automated dispensing of reagents without consumable pipette tips, has the potential to provide significant advantages. Here, we describe the integration of several instruments, including the Labcyte Echo 525 acoustic liquid handler and the iSeq and NovaSeq Illumina sequencing platforms, to miniaturize and automate mNGS library preparation, significantly reducing the cost and the time required to prepare samples. Through the use of External RNA Controls Consortium (ERCC) spike-in RNAs, we demonstrated the fidelity of the miniaturized preparation to be equivalent to full volume reactions. Furthermore, detection of viral and microbial species from cell culture and patient samples was also maintained in the miniaturized libraries. For 384-well mNGS library preparations, we achieved cost savings of over 80% in materials and reagents alone, and reduced preparation time by 90% compared to manual approaches, without compromising quality or representation within the library.

Recent advances in the capillary electrophoresis analysis of antibiotics with capacitively coupled contactless conductivity detection.

This review describes briefly the high rate of counterfeiting of antimicrobial drugs with focus upon its immediate health consequences. The major part of this review encompasses accounts of the improvements achieved in the domain of miniaturization of capillary electrophoresis with capacitively coupled contactless conductivity detection (CE-C4D). The application of this principle into the development of portable devices as well as its application to counter the health-system-crippling phenomenon of counterfeit antibiotic formulations, are discussed in the context of developing countries.

A robust method for testing urinary iodine using a microtitre robotic system.

PROJECT: Iodine deficiency disorders are due to inadequate thyroid hormone production and 2 billion individuals worldwide are estimated to have insufficient iodine intake. Laboratory assessment methods include urinary iodine (UI) concentration, blood FT3, FT4, TSH and Thyroglobulin. The aim of this study was to set up a robust method for testing urinary iodine using a microtitre robotic system. PROCEDURE: The UI method described is based on the Sandell-Kolthoff reaction, which utilizes the catalytic role of iodine in the reduction of ceric ammonium sulphate in the presence of arsenious acid. This method was automated for use on microtitre robotic system. RESULTS: The method was compared with the currently employed manual Sandell-Kolthoff reaction method in our laboratory as reference. The two methods correlated well using weighted Deming regression analysis (slope=1.066, intercept=6.5, r=0.994; n=211). Interassay and intraassay variations were similar to the reference method, but cost analysis indicated a large reduction in costs related to increased throughput, and reduced consumables and labour. CONCLUSIONS: We have successfully adapted UI testing to an automated method, permitting cheaper, faster and robust screening of large numbers of patients and populations. The described protocol can be used on different microtitre robotic systems permitting up to 372 patient samples per run for 4 microtitre plate systems.

Microscale chemistry-based design of eco-friendly, reagent-saving and efficient pharmaceutical analysis: a miniaturized Volhard's titration for the assay of sodium chloride.

This work demonstrates the extended application of microscale chemistry which has been used in the educational discipline to the real analytical purposes. Using Volhard's titration for the determination of sodium chloride as a paradigm, the reaction was downscaled to less than 2 mL conducted in commercially available microcentrifuge tubes and using micropipettes for the measurement and transfer of reagents. The equivalence point was determined spectrophotometrically on the microplates which quickened the multi-sample measurements. After the validation and evaluation with bulk and dosage forms, the downsized method showed good accuracy comparable to the British Pharmacopeial macroscale method and gave satisfactory precision (intra-day, inter-day, inter-analyst and inter-equipment) with the relative standard deviation of less than 0.5%. Interestingly, the amount of nitric acid, silver nitrate, ferric alum and ammonium thiocyanate consumed in the miniaturized titration was reduced by the factors of 25, 50, 50 and 215 times, respectively. The use of environmentally dangerous dibutyl phthalate was absolutely eliminated in the proposed method. Furthermore, the release of solid waste silver chloride was drastically reduced by about 25 folds. Therefore, microscale chemistry is an attractive, facile and powerful green strategy for the development of eco-friendly, safe, and cost-effective analytical methods suitable for a sustainable environment.

Practical approach for macroporous structure embedded microfluidic system and the catalytic microchemical application.

We present a low cost and practical approach to integrate 3D ordered macroporous polyfluoropolyether (PFPE) patterns into a microchannel by a series of porous pattern fabrication processes and subsequent photolithography in a site- and shape-selective manner. The 3D ordered macroporous patterns with high-resolution edges were firstly fabricated by microtransfer molding (µ-TM) of the sacrificial polystyrene (PS) template infiltrated with PFPE as a non-adhesive and solvent-resistant skeletal material. The resulting robust PFPE porous structures with high solvent resistance on a silicon wafer can easily be embedded into the microchannel with the aid of conventional photolithography, leading to a microfluidic system with a built-in microstructure. Moreover, catalytic Pd nanoparticles implanted on the surface of the porous structure were obtained by use of Pd nanoparticle deposited PS spheres, the porous structure embedded channel was utilized to perform a Suzuki coupling reaction.

Integrated fluidic systems on a nanometer scale and the study on behavior of liquids in small confinement.

Nanofluidic systems and the studies on the behavior of liquids confined in nanometer-sized space are reviewed. Miniaturized chemical systems having nanometer-sized structures are fabricated by using advanced nanofabrication techniques. The size-confinement effect is expected to be applied in well-controlled chemical and biochemical analysis. While electroosmosis and electrokinetic migration in small-sized channels have been investigated extensively, there have been few reports on pressure-driven flow systems having nanometer-sized structures, which are widely used in laboratory-scale and micrometer-sized systems. In this review, fundamental technologies that can be used in integrated chemical analysis systems having nanometer-sized structures are introduced. In addition to the technological investigations, important topics in the fundamental research on the properties of liquids confined in nanometer-sized space are also presented.

Chemical reactions in liquid-phase microextraction.

In recent years, liquid-phase microextraction (LPME), a microscale implementation of liquid-liquid extraction, has become a very popular sample pretreatment technique because it combines extraction and enrichment, and is inexpensive, easy to operate and nearly solvent-free. Especially so in hollow fiber-protected LPME, sample cleanup is also effected. Essentially, owing to its high sample-to-extracting solvent volume ratio, LPME can achieve high analyte enrichment. Since its advent, the technique has been widely used, and applied to environmental, pharmaceutical, biological and forensic analyses. This review focuses on developments relating to chemical reactions associated with LPME applications, in contrast to conventional, straightforward extractions in which analytes remain as they are during the extraction process. Chemical reactions brought about during LPME serve to promote the extractability of the analytes (thus expanding the scope of applicability of the technique), facilitate their (analyte) compatibility with the analytical system and/or improve detection sensitivity. The reactions that are usually enabled during LPME include ion-pair extraction (carrier-mediated membrane transport), complexation, chemical (pre-extraction, in situ, and post-extraction) derivatization, phase-transfer catalysis and other "special affinity" reactions. Strategies on chemical reactions in LPME are overviewed in this report.

A micromethod for the quantification of atenolol in plasma using high-performance liquid chromatography with fluorescence detection: therapeutic drug monitoring of two patients with severe coronary insufficiency before cardiac surgery.

A simple, rapid, selective, and sensitive analytical method was developed for the quantification of atenolol in small volumes of plasma, by high-performance liquid chromatography with fluorescence detection. Only 200 microL of plasma was used for chromatographic analysis. Separation was performed on a C18 reverse-phase column (4 microm) using a binary mobile phase consisting of 0.05 M of phosphate buffer, pH 5.5, and methanol (80:20, vol/vol) at a flow rate of 0.7 mL/minute. The retention times of atenolol and of the internal standard (sotalol) were 12.7 and 10.4 minutes, respectively. Validation of this analytical method showed a good linear correlation (8-2000 ng/mL), high sensitivity (quantification limit: 8 ng/ml and detection limit: 4 ng/mL), accuracy of 99.3%, and intraday and interday precision of 5.3% and 6.9%, respectively. Absolute recovery was 93.7%. The method was found to be robust, with acceptable stability. The analytical method was validated by the quantification of atenolol in plasma obtained from 2 patients with unstable angina, scheduled for myocardium revascularization surgery, who were chronically treated with 50 mg of atenolol administered per os once a day. The method developed was found to be adequate for use in pharmacokinetic studies and in adjusted dose pharmacotherapy.

Genome sequencing in microfabricated high-density picolitre reactors.

The proliferation of large-scale DNA-sequencing projects in recent years has driven a search for alternative methods to reduce time and cost. Here we describe a scalable, highly parallel sequencing system with raw throughput significantly greater than that of state-of-the-art capillary electrophoresis instruments. The apparatus uses a novel fibre-optic slide of individual wells and is able to sequence 25 million bases, at 99% or better accuracy, in one four-hour run. To achieve an approximately 100-fold increase in throughput over current Sanger sequencing technology, we have developed an emulsion method for DNA amplification and an instrument for sequencing by synthesis using a pyrosequencing protocol optimized for solid support and picolitre-scale volumes. Here we show the utility, throughput, accuracy and robustness of this system by shotgun sequencing and de novo assembly of the Mycoplasma genitalium genome with 96% coverage at 99.96% accuracy in one run of the machine.

Quantitative nanopipettor for miniaturized chemical and biochemical reaction sampling.

A novel and readily available pipettor capable of nanoliter-sized volume manipulation was developed to improve and increase the flexibility of small-scale reaction processing. The volume delivery was found to be reproducible, with typical relative standard deviations of 1-5%, and easily tunable over a range of nanoliter-sized aliquots. The nanopipettor was combined with capillary electrophoresis using laser-induced fluorescence detection to monitor a small-scale enzyme reaction (beta-galactosidase) using a tetramethylrhodamine-labeled substrate. The results were in good agreement with a standard enzyme assay using a micropipet, thus demonstrating the nanopipettor's potential in developing new nanoscale utrasensitive enzyme assays.

A method for parallel, automated, thermal cycling of submicroliter samples.

A large fraction of the cost of DNA sequencing and other DNA-analysis processes results from the reagent costs incurred during cycle sequencing or PCR. In particular, the high cost of the enzymes and dyes used in these processes often results in thermal cycling costs exceeding $0.50 per sample. In the case of high-throughput DNA sequencing, this is a significant and unnecessary expense. Improved detection efficiency of new sequencing instrumentation allows the reaction volumes for cycle sequencing to be scaled down to one-tenth of presently used volumes, resulting in at least a 10-fold decrease in the cost of this process. However, commercially available thermal cyclers and automated reaction setup devices have inherent design limitations which make handling volumes of <1 microL extremely difficult. In this paper, we describe a method for thermal cycling aimed at reliable, automated cycling of submicroliter reaction volumes.

System for preparing microhybridization arrays on glass slides.

We describe the construction and operation of an arrayer system to produce patterns of DNA sequences for analytical uses such as microarrays of oligonucleotide on microchips. Detailed documentation on construction is provided, as well as added electronic circuitry and the software for the instrument, including programs to machine its own working surface as well as those to operate it as an arrayer. Its cost is modest, and with a single droplet tip it can deposit 96 spots per slide on 32 slides in about 200 min (readily upgraded to higher speeds). As currently operated, it can place 400 spots in 1 cm2, and this density, too, can be increased easily. We discuss design features and performance to demonstrate utility and flexibility.

Microsampling homogeneous immunoassay with Cedia digoxin reagents on the Technicon CHEM 1 chemistry analyzer.

We report the determination of digoxin concentration in serum with Microgenics Cedia digoxin reagents on the Technicon CHEM 1. The Technicon CHEM 1 clinical chemistry analyzer has a throughput of 720 tests per hour and uses only 7 microliters each of two reagents. A 100 test kit can perform 2,640 tests. The within-run coefficient of variation (CV) range is 2.3-0.9% and the total CV is 6.3-2.9% at concentrations tested ranging from 1.10 to 2.94 ng/ml. The results of the Technicon CHEM 1 (y) assay correlated well with those by the Technicon RA 1000 system (x) with 31 clinical serum samples (y = -0.03 + 1.11x, r = 0.96). We concluded that the Cedia digoxin assay on the Technicon CHEM 1 provides a very cost-effective, precise, rapid, and accurate means to determine digoxin concentration in serum.

Fluorometric method for phenylalanine microplate assay adapted for phenylketonuria screening.

We adapted the method of McCaman and Robins for fluorometry of phenylalanine to a microplate assay for routine phenylketonuria screening. Sensitivity is 15 mumol/L for the plasma assay and 30 mumol/L for the dried blood-spot assay, with CV less than 10% for both assays. Results for human plasma by microplate assay correlated well (r = 0.99) with results of amino acid analyzer determination of phenylalanine. When measurements are performed in an automated reader, the microplate assay has considerable advantages over conventional measurements in cuvettes: smaller volumes of reagents and automation enabling high throughput and convenience. Because the described method is a quantitative one, we can postulate that, compared with the semiquantitative Guthrie inhibition bioassay, this microplate assay is more reliable, easier to perform, and about twofold less costly.