Conventional Capillary Research Articles

Seq2Sat and SatAnalyzer toolkit: Towards comprehensive microsatellite genotyping from sequencing data.

Accurate and efficient microsatellite loci genotyping is an essential process in population genetics that is also used in various demographic analyses. Protocols for next-generation sequencing of microsatellite loci enable high-throughput and cross-compatible allele scoring, common issues that are not addressed by conventional capillary-based approaches. To improve this process, we have developed an all-in-one software, called Seq2Sat (sequence to microsatellite), in C++ to support automated microsatellite genotyping. It directly takes raw reads of microsatellite amplicons and conducts read quality control before inferring genotypes based on depth-of-read, read ratio, sequence composition and length. We have also developed a module for sex identification based on sex chromosome-specific locus amplicons. To allow for greater user access and complement autoscoring, we developed SatAnalyzer (microsatellite analyzer), a user-friendly web-based platform that conducts reads-to-report analyses by calling Seq2Sat for genotype autoscoring and produces interactive genotype graphs for manual editing. SatAnalyzer also allows users to troubleshoot multiplex optimization by analysing read quality and distribution across loci and samples in support of high-quality library preparation. To evaluate its performance, we benchmarked our toolkit Seq2Sat/SatAnalyzer against a conventional capillary gel method and existing microsatellite genotyping software, MEGASAT, using two datasets. Results showed that SatAnalyzer can achieve >99.70% genotyping accuracy and Seq2Sat is ~5 times faster than MEGASAT despite many more informative tables and figures being generated. Seq2Sat and SatAnalyzer are freely available on github (https://github.com/ecogenomicscanada/Seq2Sat) and dockerhub (https://hub.docker.com/r/rocpengliu/satanalyzer).

Comparative analytical characteristics of multicapillary chromatography columns with different capillary diameters

Straight gas chromatography multicapillary columns (MCC) with 40 µm diameter capillaries (hereafter – 40 µm MCC) have been known for quite a long time and are well studied; they are used in portable gas analyzers. Some chromatographic characteristics of 25 µm MCCs, which appeared relatively recently, were also studied, while commercially available 60 and 80 µm MCCs are poorly studied. In this work the main analytical characteristics of 60 and 80 µm MCCs were determined and compared with the characteristics of 25 and 40 µm MCCs. It was shown that the maximum specific efficiency of the columns decreased with increasing column capillary diameter and is approximately 24.8, 18.2, 13.7 and 9.5 thousand theoretical plates (t.p.) per meter for 25, 40, 60 and 80 µm MCCs, respectively. It was established that the height equivalent to a theoretical plate of 60 and 80 μm MCCs was not varied significantly over a wide range of carrier gas velocities (nitrogen and helium), which allowed operating MCCs at high carrier gas flows essentially without loss of their efficiency. Moreover, for all MCCs the separation rate for peaks with a retention factor over 10 exceeded 600 t.p./s, and for peaks with a lower retention factor could be several thousand t.p./s, which is significantly higher than for conventional capillary and packed columns. It was established that it was possible to create very high carrier gas flows (up to 1000 cm3/min or more) for 60 µm MCCs and especially 80 µm MCCs at a relatively low pressure drop across the column. Therefore they can work as a part of chromatographic systems that require high carrier gas flow.

Dynamic in situ growth of bonded-phase silica nanospheres on silica capillary inner walls for open-tubular liquid chromatography

Silica nanospheres (SNS) were grown on the inner walls of silica capillaries through a dynamic in situ nucleation process to prepare a highly porous and large accessible surface area substrate. The SNS were then functionalized with octadecyl (C18), 3-aminopropyltriethoxysilane (APTES), beta-cyclodextrin (β-CD), and amino groups to develop robust and efficient chromatographic stationary phases. The modified silica capillaries were exploited for open-tubular liquid chromatography (OT-LC) and open-tubular capillary electrochromatography (OT-CEC) applications. The prepared stationary phases were compared to conventional capillaries in terms of separation performance. The synthesis process was optimized, and the bonded-phase stationary phases were characterized by the electron microscopy technique. The effects of different solvents, additives, and functional groups on the geometry and chromatographic resolving power of the SNS were envisaged. The capillaries modified with octadecyl groups were evaluated for the separation of non-steroidal anti-inflammatory drugs, phenones, alkenylbenzenes, and enantiomers of chlorophenoxy herbicides. As an application instance, an SNS-C18-coated capillary was utilized for the separation of alkenylbenzenes from clove extract and protein digest medium, through OT-LC and OT-CEC techniques, respectively. The β-CD functionalized capillary was applied for the OT-CEC separation of a dichlorprop racemic mixture.Graphical abstract

Underfill Flow in Flip-Chip Encapsulation Process: A Review

Abstract The scope of review of this paper focused on the precuring underfilling flow stage of encapsulation process. A total of 80 related works has been reviewed and being classified into process type, method employed, and objective attained. Statistically showed that the conventional capillary is the most studied underfill process, while the numerical simulation was mainly adopted. Generally, the analyses on the flow dynamic and distribution of underfill fluids in the bump array aimed for the filling time determination as well as the predictions of void occurrence. Parametric design optimization was subsequently conducted to resolve the productivity issue of long filling time and reliability issue of void occurrence. The bump pitch was found to the most investigated parameter, consistent to the miniaturization demand. To enrich the design versatility and flow visualization aspects, experimental test vehicle was innovated using imitated chip and replacement fluid, or even being scaled-up. Nonetheless, the analytical filling time models became more accurate and sophiscasted over the years, despite still being scarce in number. With the technological advancement on analysis tools and further development of analytic skills, it was believed that the future researches on underfill flow will become more comprehensive, thereby leading to the production of better packages in terms of manufacturing feasibility, performances, and reliability. Finally, few potential future works were recommended, for instance, microscopic analysis on the bump–fluid interaction, consideration of filler particles, and incorporation of artificial intelligence.

Comparison of capillary, venous and buffy coat blood samples in detecting Plasmodium species among malaria suspected patients attending at Hamusite health center. A cross-sectional study

BackgroundBoth capillary and venous blood samples have been interchangeably used for the diagnosis of malaria in Ethiopia. However, Plasmodium parasites are thought to be more concentrated in capillary than in venous blood. Hence, selecting a sample source where parasites are more concentrated is indispensable approach in order to maximize the accuracy of blood film microscopy. Therefore, the present study aimed to compare the detection rate and the parasitemia level of Plasmodium species from conventional capillary and venous blood films, and buffy coat preparations.MethodsA facility based cross-sectional study was conducted from Feburary to March 2020 among 210 febrile patients attending Hamusite health center, northwest Ethiopia. Capillary and venous blood samples were collected and buffy coat was prepared from each sample. Thin and thick blood films were prepared, stained, and examined microscopically following standard protocol. Data were analysed using Statistical Package for Social Sciences Software version 20 and Med-Calc software version 19.3.ResultsCapillary blood buffy coat (61/210, 29.0%) had significantly higher detection rate as compared to capillary (48/210, 22.9%) and venous (42/210, 20.0%) blood films (p < 0.001). However, no significant difference was observed between capillary and venous blood films (p = 0.070) in detecting Plasmodium species. The highest and the lowest mean asexual stage parasite counts were found in capillary blood buffy coat (4692.88) and venous blood (631.43) films, respectively showing significant variations (p < 0.001). Mean gametocyte count was also highest in capillary blood buffy coat (3958.44). As compared to capillary blood buffy coat, the sensitivity of venous blood buffy coat, capillary blood film and venous blood film were 73.8, 78.7, 68.9%, respectively.ConclusionCapillary blood buffy coat samples showed the highest sensitivity in detecting and quantitating malaria parasites that its use should be promoted in clinical settings. However, conventional capillary and venous blood films could be used interchangeably.

Capacitively coupled contactless conductivity detection for analytical techniques – Developments from 2018 to 2020

The developments of analytical contactless conductivity measurements based on capacitive coupling over the two years from mid-2018 to mid-2020 are covered. This mostly concerns applications of the technique in zone electrophoresis employing conventional capillaries and to a lesser extent lab-on-chip devices. However, its use for the detection in several other flow-based analytical methods has also been reported. Detection of bubbles and measurements of flow rates in two-phase flows are also recurring themes. A few new applications in stagnant aqueous samples, e.g. endpoint detection in titrations and measurement on paper-based devices, have been reported. Some variations of the design of the measuring cells and their read-out electronics have also been described.

Development of Container Free Sample Exposure for Synchrotron X-ray Footprinting.

The method of X-ray footprinting and mass spectrometry (XFMS) on large protein assemblies and membrane protein samples requires high flux density to overcome the hydroxyl radical scavenging reactions produced by the buffer constituents and the total protein content. Previously, we successfully developed microsecond XFMS using microfluidic capillary flow and a microfocused broadband X-ray source at the Advanced Light Source synchrotron beamlines, but the excessive radiation damage incurred when using capillaries prevented the full usage of a high-flux density beam. Here we present another significant advance for the XFMS method: the instrumentation of a liquid injection jet to deliver container free samples to the X-ray beam. Our preliminary experiments with a liquid jet at a bending magnet X-ray beamline demonstrate the feasibility of the approach and show a significant improvement in the effective dose for both the Alexa fluorescence assay and protein samples compared to conventional capillary flow methods. The combination of precisely controlled high dose delivery, shorter exposure times, and elimination of radiation damage due to capillary effects significantly increases the signal quality of the hydroxyl radical modification products and the dose-response data. This new approach is the first application of container free sample handling for XFMS and opens up the method for even further advances, such as high-quality microsecond time-resolved XFMS studies.

Effect of Conventional and Granular Capillaries Usage on the Bondability, Reliability, and Manufacturability of Gold Wedge Bonds

The evaluation of the manufacturability, bondability, and reliability performances of the granular capillary is required to produce high-quality wire bonds. In this paper, two different types of wire bonding capillaries, namely, granular and conventional capillaries, were used for the gold (Au) wire bonding process. The wire pulling test was performed on the Au wire bonds created by both types of capillaries right after the wire bonding process and after the Au wire bonds were gone through accelerated tests of high-temperature storage and temperature cycling tests. It is noted that wire pull strengths were increased with the usage of the granular capillary compared to that of the conventional capillary for all five samples due to the occurrence of intimate bond between Au wedge bonds and bond pads. The increase of yield and the occurrence of consistence with one type of the failure mode for the Au wedge bonds that used granular capillary are due to the increment of scrubbing surface area between Au wire bonds and bond pads. The reduction of wire bonder stoppage with the usage of the granular capillary is due to the reduction of the short tail failure mode occurrence. This can be seen through the increment of the average yield percentage. It is observed that the granular capillary has the highest number of touchdowns or lifetimes compared to that of the conventional capillary. It is also found that the created wire bonds that used granular capillary have good bondability and reliability. These findings are based on the evaluation of the bonding interface thickness and distribution.

Capillary Scale Admittance and Conductance Detection.

Admittance detection, more commonly called capacitively coupled contactless conductivity detection, is widely used. While the true conductance of a solution is linear with concentration up to ∼1 mequiv/L, the admittance signal is nonlinear. In small-bore capillaries and highly resistive solutions, such as in suppressed open tubular ion chromatography (SOTIC), the admittance signal is exponentially related to concentration. Solution contact conductivity detection is common in microfluidics, but no true conductivity measurement efforts have been made with conventional capillaries in the last two decades. We examine five solution contact cell geometries: (A) wire/disk electrodes perpendicular to the flow direction, (B) annular tubular electrodes facing the flow (that exits through the center tube), (C) ring-disk electrodes facing the flow that exits through the annulus, (D) coplanar parallel wire/disk electrodes facing the flow that exits around the electrodes, and (E) planar electrodes separated by a thin insulating layer with a through-hole in which the liquid flows. Present limitations of the available components as well as the designs themselves do not allow type A and B cells to reach low enough dispersion levels. Types C-E all produce the same efficiency for chloride in a SOTIC setup (12000 ± 200 plates/m), limited by dispersion in the suppressor. Further choice and refinement will be dictated by the availability of a lower dispersion suppressor. All such contact conductance results were linear with concentration, and initial results suggest that attainable LODs will be competitive with those from benchtop ICs.

Injection system for fast capillary electrophoresis based on pressure regulation with flow restrictors.

A fast automated system for rapid electrophoretic separations in short conventional capillaries employing contactless conductivity detection is presented. The instrument is based on pneumatic pressurization and does not require a conventional pump. The required pressures and flow rates for the different steps of the injection and flushing processes are produced with the help of two flow restrictors. The device is implemented on a microfluidic breadboard with dimensions of ca. 13 × 20 cm and employs miniature valves. Nine inorganic cations, namely NH4+, K+ , Na+ , Ca2+ , Mg2+ , Mn2+ , Sr2+ , Li+ , and Ba2+ , could be separated in a capillary of 10 μm inner diameter and 6 cm effective length within 25 s. Following a reduction of the effective length to 4cm, still five inorganic cations could be separated in a time span of 12s. The repeatability of peak areas was better than 3.1% and limits of detection between 3.5 and 5.5 μM were achieved.

An experimental critique on the existence of fragile-to-strong transition in glass-forming liquids

A number of recent studies in the literature have reported the existence of unusual temperature dependence of the shear viscosity for a variety of glass-forming liquids that cannot be described with a single fragility index. Rather, these liquids display an apparent fragile-to-strong transition (FST) with lowering of temperature that was suggested to be indicative of a structural transition. In the present work we critique the accuracy of the high-temperature oscillation viscometry technique that was used almost exclusively for viscosity measurements of these liquids above their liquidus temperature. Viscosity measurements are carried out on binary Ge-Se chalcogenide liquids in their supercooled and stable states using conventional capillary and parallel-plate rheometry techniques and are compared with the oscillation viscometry data in the literature. Such comparisons conclusively demonstrate that the latter measurement technique often underestimates the viscosity by nearly an order of magnitude, especially at high temperatures. These results, when taken together, indicate that the observation of FST in glass-forming liquids is likely an artifact of the inaccuracy of the oscillation viscometry technique. Moreover, these results cast serious doubt on the reliability of the high-temperature oscillation viscometry data for metals and alloys that are prevalent in the literature.

Successive injection in microstructured-capillary electrophoresis for rapid pairwise comparisons. Application to questioned documents

Herein we propose a successive double injection in microstructured-capillary electrophoresis for a fast, simple and easy-to-interpret comparison of samples. First, injection was optimized through three capillaries (conventional capillary of 28.5μm id, microstructured capillary (MSC) of 6 holes of 32.8μm each, and MSC of 61 holes of 8.9μm each). The 6-hole MSC was selected, as it provided reproducible results, and higher volume capacity, which allowed larger injection volumes. Then, the method was applied to the analysis of different types of inks present in questioned documents. After the criterion to establish a MATCH or No match between two successive samples, a blind test with 20 pairwise comparisons between questioned signatures was finally made to demonstrate the potential of the method. The method proved to be useful in pairwise comparison analyses, as it: i) provides a fast and practical analysis; ii) maintains the same CE conditions for both samples; iii) uses simple data treatment which puts on easy interpretation on the results, interesting in court.

MTE23.01 Biomarker Characterization: Challenges and Perspectives

The treatment of lung adenocarcinomas has improved with the identification of driver gene mutations and the development of drugs tackling the altered driver gene proteins.1 The interrogation of EGFR mutations as well as ALK and ROS1 translocations is nowadays part of the routine diagnostic workup of lung adenocarcinomas. Further therapies aiming at mutations in driver genes like RET, HER2, BRAF and MET are emerging. The main techniques currently employed in molecular pathology laboratories for mutation detection are mutation specific PCR, conventional capillary (Sanger) sequencing and next generation sequencing (NGS).2 The basic principles of these methods, advantages and disadvantages with a special emphasis on NGS and its potency for cancer diagnostics will be discussed. Recently the detection of driver gene mutations in DNA that is derived from cancer cells and released into the blood has become feasible (so-called “liquid biopsy“).3 It has already entered routine diagnostics with the detection of the T790M mutation in circulating tumor DNA of primary EGFR mutated lung cancers, that developed resistance to first or second generation tyrosine kinase inhibitors.4 The verification of a T790M mutation either by liquid biopsy or the analysis of tumor tissue is a prerequisite for therapy of lung adenocarcinomas with third generation tyrosine kinase inhibitors like osimertinib. The principles of liquid biopsy, employed techniques and potential applications will be introduced. The recent advent of cancer immunotherapy that interferes with immune checkpoint molecules like programmed death 1 (PD-1) offers a new treatment for subgroups of lung cancer patients. A biomarker that would predict responsiveness to this expensive therapy is greatly needed. The expression of the PD-1 ligand (PD-L1) by tumor or immune/stromal cells is a potential biomarker, however its utility is heavily debated.5 The confusion on PD-L1 as a biomarker is partly caused by technical difficulties in the determination of expression, such as the antibody used for immunohistochemistry and the determination of a threshold of expression that correlates with response.6 A further biomarker that is determined by immunohistochemistry is the expression of EGFR in lung squamous cell carcinoma. A therapy with the recently approved anti-EGFR antibody necitumumab requires the demonstration of EGFR expression by the cancer cells.7 The methods and pitfalls in PD-1 and EGFR immunohistochemistry will be presented. The requirement for biomarkers increases. This poses a challenge for diagnostics in respect to availability of techniques, infrastructure and budget. Particularly in lung cancer often very little tissue is available, but different assays should be run. To fulfill the increasing demand for a plethora of biomarker analysis multiplexing techniques that simultaneously interrogate a large number of gene mutations, gene fusions, gene amplification and deletions, as well as RNA and protein expression will be needed. An outlook on available and emerging multiplexing techniques will be provided. 1. The Cancer Genome Atlas Research N (2014) Comprehensive molecular profiling of lung adenocarcinoma. Nature 511 (7511):543-550. http://dx.doi.org/10.1038/nature13385 2. Buermans HP, den Dunnen JT (2014) Next generation sequencing technology: Advances and applications. Biochimica et biophysica acta 1842 (10):1932-1941. http://dx.doi.org/10.1016/j.bbadis.2014.06.015 3. Diaz LA, Jr., Bardelli A (2014) Liquid biopsies: genotyping circulating tumor DNA. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 32 (6):579-586. http://dx.doi.org/10.1200/JCO.2012.45.2011 4. Thress KS, Paweletz CP, Felip E, Cho BC, Stetson D, Dougherty B, Lai Z, Markovets A, Vivancos A, Kuang Y, Ercan D, Matthews SE, Cantarini M, Barrett JC, Janne PA, Oxnard GR (2015) Acquired EGFR C797S mutation mediates resistance to AZD9291 in non-small cell lung cancer harboring EGFR T790M. Nat Med 21 (6):560-562. http://dx.doi.org/10.1038/nm.3854 http://www.nature.com/nm/journal/v21/n6/abs/nm.3854.html - supplementary-information 5. Sunshine J, Taube JM (2015) PD-1/PD-L1 inhibitors. Current opinion in pharmacology 23:32-38. http://dx.doi.org/10.1016/j.coph.2015.05.011 6. Garon EB, Rizvi NA, Hui R, Leighl N, Balmanoukian AS, Eder JP, Patnaik A, Aggarwal C, Gubens M, Horn L, Carcereny E, Ahn M-J, Felip E, Lee J-S, Hellmann MD, Hamid O, Goldman JW, Soria J-C, Dolled-Filhart M, Rutledge RZ, Zhang J, Lunceford JK, Rangwala R, Lubiniecki GM, Roach C, Emancipator K, Gandhi L (2015) Pembrolizumab for the Treatment of Non–Small-Cell Lung Cancer. New England Journal of Medicine 372 (21):2018-2028. http://dx.doi.org/10.1056/NEJMoa1501824 7. Thatcher N, Hirsch FR, Luft AV, Szczesna A, Ciuleanu TE, Dediu M, Ramlau R, Galiulin RK, Balint B, Losonczy G, Kazarnowicz A, Park K, Schumann C, Reck M, Depenbrock H, Nanda S, Kruljac-Letunic A, Kurek R, Paz-Ares L, Socinski MA (2015) Necitumumab plus gemcitabine and cisplatin versus gemcitabine and cisplatin alone as first-line therapy in patients with stage IV squamous non-small-cell lung cancer (SQUIRE): an open-label, randomised, controlled phase 3 trial. The Lancet Oncology 16 (7):763-774. http://dx.doi.org/10.1016/s1470-2045(15)00021-2

Contactless conductivity detection for analytical techniques- Developments from 2014 to 2016.

The development of capacitively coupled contactless conductivity detection for the two-year period from mid-2014 to mid-2016 is covered in this review. This includes a survey of fundamental studies and further developments of the measuring technique reported as well as a discussion of new applications. These mostly concern capillary electrophoresis carried out in conventional capillaries as well as on microchip electrophoresis devices. The main focus is on the determination of small non-UV-absorbing organic ions and inorganic ions in different types of samples of clinical, nutritional or environmental interest. Outside of electrophoresis contactless conductivity detection is finding uses in detection in column chromatography, flow-injection analysis and industrial applications.

STUDY OF PRESSURE CHANGE IN THE CAVITY WITH A MICROPOROUS FILLER

The aim of the study is to determine influence of composition and properties of filler (vacuum insulation, sorbent) of pumping time and pressure distribution in the pumping cavity. In the work accepted assumptions: molecular flow regime of gas, microporous structure is presented of capillary equivalent length and diameter, gassing from the surface independent of pressure and are constant along the length of conventional capillary. Pumping process has been considered for steady pumping process and pumping process with constant pumped gas flow. Accordingly were obtained analytical dependence of pressure distribution in capillary and time of pumping process for design pressure ranges.

Fundamentals on new capillaries inspired by photonic crystal fibers as optofluidic separation systems in CE

Two prototypes of microstructured capillaries (MSCs) were designed, manufactured, and used to carry out different experiments. MSC-1 consisted of six holes of ≈28μm id whereas MSC-2 consisted of 85 holes of ≈7.7μm id. A fundamental study on the hydrodynamic injection through a commercial CE equipment was conducted. Experimental times to flush specific volumes were approximately three times larger than the theoretical values. Then, the detection of starch was carried out by using the MSCs and conventional capillaries, and the electropherograms were compared on the basis of analytical parameters employed in CE. An improvement in peak asymmetry was obtained for the MSC-1 compared to the conventional capillaries. S/N was one order of magnitude increased with the MSC, improving ten times the sensitivity. Considering this advantage, the separation and detection of nitrostarch was performed as a first application of the MSC-1. Minimal sample amounts of nitrostarch (1.7μg) were detected. Results present a real interest in forensics since this substance had not been previously detected through CE, leading to new investigations in the design of new capillaries capable of enhancing CE performance.

Photonic crystal fibres as efficient separation component in capillary electrophoresis

Telecommunication developments are leading to original and new fibre designs which indeed are being used in a huge variety of fields. In this manuscript we show that Photonic Crystal Fibres (PCFs) can be used as efficient chemical separation devices in commercial capillary electrophoresis (CE) equipment. A comparison of conventional capillaries, PCFs and new devices inspired on PCFs named Smart Micro-Structured Capillaries (SMSCs) is made. Results showed the usefulness and efficiency of these physical supports in CE through the detection of fluorescein. In addition, some considerations to take into account for SMSCs manufacturing are discussed. Further work should allow coupling the separation advantages achieved by these elements with the wide variety of detection techniques available in fibre-optics.

Sex identification of ancient DNA samples using a microfluidic device

A microfluidic device has been developed for the sex identification of ancient DNA samples and works by manipulating liquids within an environment of micrometer dimensions. In this work a range of microfluidic DNA extraction methods were evaluated for their compatibility with ancient DNA samples, and the use of streptavidin-coated super paramagnetic particles to isolate biotin-labeled abasic sites within damaged DNA was shown to be the most reproducible. Polymerase chain reaction-based DNA amplification was possible on the microfluidic device when less than 50 pg of template DNA was added. As a proof-of-principle, powdered bone samples were analysed using the integrated methodology developed. Following conventional capillary gel electrophoresis, two out of the three samples produced positive amplification results and were successfully identified as female. These sex identifications were corroborated by independent Amelogenin, anthropological and Y chromosome analysis. The work reported here is the first step in the development of a complete miniaturized microfluidic system that would enable on-site ancient DNA analysis.

Rapid Preparation of C18 Monoliths for Micro‐column Separation Using Ultraviolet and Microwave Irradiation

AbstractAn organic‐based monolith with long alkyl chain ligands was prepared by UV photo‐initiation using (a) 1‐octadecene as a functional monomer, (b) ethylene glycol dimethacrylate (EDMA) as the cross‐linking agent, (c) 1‐propanol, 1,4‐butanediol and dimethylformamide as triporogenic solvents, and (d) Irgacure 1800 as the initiator. The monoliths containing a high fraction of 1‐octadecene possessed a better total porosity, improved permeability, and result in faster separation. Similar monolithic capillary was quickly fabricated in 3 min by microwave irradiation using azobisisobutyronitrile as the thermal initiator. Conventional polyimide‐coated capillaries were used instead of expensive UV‐transparent capillaries in both methods.

Combinable poly(dimethyl siloxane) capillary sensor array for single-step and multiple enzyme inhibitor assays

We describe a new method for fabricating a capillary-type sensor, called a combinable poly(dimethyl siloxane) (PDMS) capillary (CPC) sensor. The method for preparing the CPC simplifies enzyme inhibitor assays into a simple, single step assay. The sample inhibitor solution is introduced by capillary action. This triggers the spontaneous dissolution of physically adsorbed fluorescent substrates, and the substrate mixes with the inhibitor. This is followed by competitive reaction with insoluble enzyme to give a fluorescence response. CPC is composed of a convex-shaped PDMS stick containing reagents immobilized in an insoluble coating, and a concave-shaped PDMS stick containing reagents immobilized in a soluble coating. Since the concave-shaped PDMS has a deeper channel than the convex structure, combining these PDMS sticks is like closing the zipper of a "freezer bag". This allows easy fabrication of "thin and long" capillary structures containing different reagents inside the same capillary, without the need for precise alignment. This method allows the immobilization of two reactive reagents, such as enzyme and substrate required for a single step assay, which are typically very difficult to immobilize using commercially available conventional capillaries. Furthermore, by simply arraying various CPCs, the CPC sensor allows multiple assays. Here, we carried out a single-step enzyme inhibitor assay using the CPC. In addition, two independent CPCs were arrayed to demonstrate multiple assaying of a protease inhibitor.