Effects of Heavy Metal Co-Exposure on the formation of DNA Adducts from Aristolochic Acid I: Implications for Balkan Endemic Nephropathy Development.

Accumulated evidence has shown that Balkan endemic nephropathy (BEN) is a multifactorial environmental disease, with exposure to aristolochic acids (AA), and the associated DNA adduct formation, as a key causative factor of BEN development. Here, we show that coexposure to arsenic, cadmium, and iron increases the DNA adduct formation of AA in cultured kidney cells, while exhibiting both an exposure concentration and duration dependence. In contrast, coexposure to calcium and copper showed a decreasing DNA adduct formation. Because DNA damage is responsible for both the nephrotoxicity and carcinogenicity of AA, these results shed greater light on the endemic nature of BEN.

Rapid microfluidics prototyping through variotherm desktop injection molding for multiplex diagnostics.

In this work, we demonstrate an inexpensive method of prototyping microfluidics using a desktop injection molding machine. A centrifugal microfluidic device with a novel central filling mechanism was developed to demonstrate the technique. We overcame the limitations of desktop machines in replicating microfluidic features by variotherm heating and cooling the mold between 50 °C and 110 °C within two minutes. Variotherm heating enabled good replication of microfeatures, with a coefficient of variation averaging only 3.6% attained for the measured widths of 100 µm wide molded channels. Using this methodology, we produced functional polystyrene centrifugal microfluidic chips, capable of aliquoting fluids into 5.0 µL reaction chambers with 97.5% accuracy. We performed allele-specific loop-mediated isothermal amplification (AS-LAMP) reactions for genotyping CYP2C19 alleles on these chips. Readouts were generated using optical pH sensors integrated onto chips, by drop-casting sensor precursor solutions into reaction chambers before final chip assembly. Positive reactions could be discerned by decreases in pH sensor fluorescence, thresholded against negative control reactions lacking the primers for nucleic acid amplification and with time-to-results averaging 38 minutes. Variotherm desktop injection molding can enable researchers to prototype microfluidic devices more cost-effectively, in an iterative fashion, due to reduced costs of smaller, in-house molds. Designs prototyped this way can be directly translated to mass production, enhancing their commercialization potential and positive impacts.

Optical pH Monitoring in Microdroplet Platforms for Live Cell Experiments Using Colloidal Surfactants.

Droplet microfluidic technology facilitates the development of high-throughput screening applications in nanoliter volumes. Surfactants provide stability for emulsified monodisperse droplets to carry out compartmentalization. Fluorinated silica-based nanoparticles are used; they can minimize crosstalk in microdroplets and provide further functionalities by surface labeling. Here we describe a protocol for monitoring pH changes in live single cells by fluorinated silica nanoparticles, for their synthesis, chip fabrication, and optical monitoring on the microscale. The nanoparticles are doped with ruthenium-tris-1,10-phenanthroline dichloride on the inside and conjugated with fluorescein isothiocyanate on the surface. This protocol may be used more generally to detect pH changes in microdroplets. The fluorinated silica nanoparticles can also be used as droplet stabilizers with an integrated luminescent sensor for other applications.

Uncovering mechanisms of RT-LAMP colorimetric SARS-CoV-2 detection to improve assay reliability.

Improved diagnostics are needed to manage the ongoing COVID-19 pandemic. In this study, we enhanced the color changes and sensitivity of colorimetric SARS-CoV-2 RT-LAMP assays based on triarylmethane dyes. We determined a mechanism for the color changes and obtained sensitivities of 10 RNA copies per microliter.

Automated Miniaturized Digital Microfluidic Antimicrobial Susceptibility Test Using a Chip-Integrated Optical Oxygen Sensor.

We present the first digital microfluidic (DMF) antimicrobial susceptibility test (AST) using an optical oxygen sensor film for in-situ and real-time continuous measurement of extracellular dissolved oxygen (DO). The device allows one to monitor bacterial growth across the entire cell culture area, and the fabricated device was utilized for a miniaturized and automated AST. The oxygen-sensitive probe platinum(II)-5,10,15,20-tetrakis-(2,3,4,5,6-pentafluorophenyl)-porphyrin was embedded in a Hyflon AD 60 polymer and spin-coated as a 100 nm thick layer onto an ITO glass serving as the DMF ground electrode. This DMF-integrated oxygen sensing film was found to cause no negative effects to the droplet manipulation or cell growth on the chip. The developed DMF platform was used to monitor the DO consumption during Escherichia coli(E. coli) growth caused by cellular respiration. A rapid and reliable twofold dilution procedure was developed and performed, and the AST with E. coli ATCC 25922 in the presence of ampicillin, chloramphenicol, and tetracycline at different concentrations from 0.5 to 8 µg mL-1 was investigated. All sample dispensation, dilution, and mixing were performed automatically on the chip within 10 min. The minimum inhibitory concentration values measured from the DMF chip were consistent with those from the standard broth microdilution method but requiring only minimal sample handling and working with much smaller sample volumes.

A microfluidic chip for rapid analysis of DNA melting curves for BRCA2 mutation screening.

A microfluidic chip integrated with a microheater and a luminescent temperature sensor for rapid, spatial melting curve analysis was developed and applied for the screening of a breast cancer gene fragment. The method could detect genetic differences in around 3 minutes total for the whole procedure, which is much faster than established procedures. A microfabrication technique was developed to allow for bonding of a temperature sensing thin film and a Pt microheater with PDMS and the chips could be employed to generate and measure thermal gradients and the fluorescence intensity of stained DNA through multispectral optical imaging. The sensing layer consisting of poly(styrene-co-acrylonitrile) and a tris(1,10-phenanthroline)ruthenium(ii) temperature probe was generated by blade coating on a glass substrate with an attached Pt microheater. Calibration of the temperature between 20 and 90 °C yielded an overall resolution of around 0.13 K. The chip was employed for the screening of the BRCA 2 breast cancer gene; BRCA2 exon 5 was differentiated by its mutant rs80359463 by a 1.1 K difference in melting temperature and two fragments of BRCA2 exon 11 were differentiated by their mutants rs276174826 and rs876660311 by 0.7 K and 2.0 K, respectively. The standard deviations were between 0.1 and 0.5 K. Capable of detecting fluorescence in the DNA and temperature simultaneously and being imaged in a customized assembly, this microchip can be used to screen for mutations in a variety of DNA samples in disease diagnosis and prognosis.

Composite Films of CsPbBr3 Perovskite Nanocrystals in a Hydrophobic Fluoropolymer for Temperature Imaging in Digital Microfluidics.

A composite film material that combines CsPbBr3 perovskite nanocrystals with a Hyflon AD 60 fluoropolymer was developed and utilized for high-resolution optical temperature imaging. It exhibited bright luminescence and, most importantly, long-term stability in an aqueous medium. CsPbBr3 nanocrystal-Hyflon films immersed in aqueous solutions showed stable luminescence over at least 4 months and exhibited a fully reversible pronounced temperature sensitivity of 1.2% K-1 between 20 and 80 °C. They were incorporated into a digital microfluidic (electrowetting on dielectric) platform and were used for spatially resolved temperature measurements during droplet movements. Thermal mapping with a CsPbBr3 nanocrystal-Hyflon sensing layer in a room temperature environment (22.0 °C) revealed an increase in local temperatures of up to 40.2 °C upon voltage-driven droplet manipulations in a digital microfluidic system, corresponding to a local temperature change of up to 18.2 °C.

Microfluidic Free-Flow Isoelectric Focusing with Real-Time pI Determination.

Free-flow electrophoresis (FFE) may be used for continuous and preparative separation of a wide variety of biomolecules. Isoelectric focusing (IEF) provides for the separation of compounds according to their isoelectric point (pI). Here we describe a microfluidic chip-based protocol for the fabrication, application, and optical monitoring of free-flow isoelectric focusing (FFIEF) of proteins and peptides on the microscale with optical surveillance of the microscopic pH gradient provided by an integrated pH sensing layer. This protocol may be used with modifications also for the FFIEF of other biomolecules and may serve as template for the fabrication of microfluidic chips with integrated fluorescent or luminescent pH sensor layers for FFE and other applications.

Micro free-flow isoelectric focusing with integrated optical pH sensors.

Recently, a new observation method for monitoring of pH gradients in microfluidic free-flow electrophoresis has emerged. It is based on the use of chip-integrated fluorescent or luminescent micro sensor layers. These are able to monitor pH gradients in miniaturized separations in real time and spatially resolved; this is particularly useful in isoelectric focusing. Here these multifunctional microdevices that feature continuous separation, monitoring, and in some instances other functionalities, are reviewed. The employed microfabrication procedures to produce these devices are discussed and the different pH sensor matrices that were integrated and their applications in the separation of different types of biomolecules. The procedures for obtaining spatially resolved information about the separated molecules and the pH at the same time and different detection modalities to achieve this such as deep UV fluorescence as well as time-resolved referenced pH sensing and the integration of a precolumn labeling step into these platforms are also highlighted.

Continuous purification of reaction products by micro free-flow electrophoresis enabled by large area deep-UV fluorescence imaging.

Microreactors have gained increasing attention in their application toward continuous micro flow synthesis. An unsolved problem of continuous flow synthesis is the lack of techniques for continuous product purification. Herein, we present a micro free-flow electrophoresis device and accompanying setup that enables the continuous separation and purification of unlabeled organic synthesis products. The system is applied to the separation and purification of triarylmethanes. For imaging of the unlabeled analytes on-chip a novel setup for large area (3.6 cm2) deep ultra violet excitation fluorescence detection was developed. Suitable separation conditions based on low conductivity electrophoresis buffers were devised to purify the product. With the optimized conditions, starting materials and product of the synthesis were well separated (R > 1.2). The separation was found to be very stable with relative standard deviations of the peak positions smaller than 3.5% over 15 min. The stable conditions enabled collection of the separated compounds, and purity of the product fraction was confirmed using capillary electrophoresis and mass spectrometry. This result demonstrates the great potential of free-flow electrophoresis as a technique for product purification or continuous clean-up in flow synthesis. Graphical Abstract Micro free-flow electrophoresis (µFFE) allows continuous separation and purification of small organic synthesis products. Enabled by a novel deep-UV imaging setup starting materials and product of a recently developed synthesis for triarylmethanes could be purified. Thereby demonstrating the potential of µFFE as continuous purification technique for micro flow synthesis.

On-Chip Photothermal Analyte Detection Using Integrated Luminescent Temperature Sensors.

Optical absorbance detection based on attenuated light transmission is limited in sensitivity due to short path lengths in microfluidic and other miniaturized platforms. An alternative is detection using the photothermal effect. Herein we introduce a new kind of photothermal absorbance measurement using integrated luminescent temperature sensor spots inside microfluidic channels. The temperature sensors were photopolymerized inside the channels from NOA 81 UV-curable thiolene prepolymer doped with a tris(1,10-phenanthroline)ruthenium(II) temperature probe. The polymerized sensing structures were as small as 26 ± 3 µm in diameter and displayed a temperature resolution of better than 0.3 K between 20 and 50 °C. The absorbance from 532 nm laser excitation of the food dye Amaranth as a model analyte was quantified using these spots, and the influence of the flow rate, laser power, and concentration was investigated. Calibration yielded a linear relationship between analyte concentration and the temperature signal in the channels. The limit of detection for the azo-dye Amaranth (E123) in this setup was 13 µM. A minimal detectable absorbance of 3.2 × 10-3 AU was obtained using an optical path length of 125 µm in this initial study. A microreactor with integrated temperature sensors was then employed for an absorbance-based miniaturized nitrite analysis, yielding a detection limit of 26 µM at a total assay time of only 75 s. This technique is very promising for sensitive, and potentially spatially resolved, optical absorbance detection on the micro- and nanoscale.

Continuous on-chip fluorescence labelling, free-flow isoelectric focusing and marker-free isoelectric point determination of proteins and peptides.

We present a microfluidic platform that contains a micro flow reactor for on-chip biomolecule labelling that is directly followed by a separation bed for continuous free-flow electrophoresis and has an integrated hydrogel-based near-infrared fluorescent pH sensor layer. Using this assembly, labelling of protein and peptide mixtures, their separation via free-flow isoelectric focusing and the determination of the isoelectric point (pI) of the separated products via the integrated sensor layer could be carried out within typically around 5 minutes. Spatially-resolved immobilization of fluidic and sensing structures was carried out via multistep photolithography. The assembly was characterized and optimized with respect to their fluidic and pH sensing properties and applied in the IEF of model proteins, peptides and a tryptic digest from physalaemine. We have therefore realized continuous sample preparation and preparative separation, analyte detection, process observation and analyte assignment capability based on pI on a single platform the size of a microscope slide.

Microchamber arrays with an integrated long luminescence lifetime pH sensor.

A pH probe with a microsecond luminescence lifetime was obtained via covalent coupling of 6-carboxynaphthofluorescein (CNF) moieties to ruthenium-tris-(1,10-phenanthroline)(2+). The probe was covalently attached to amino-modified poly-(2-hydroxyethyl)methacrylate (pHEMA) and showed a pH-dependent FRET with luminescence lifetimes of 681 to 1260 ns and a working range from ca. pH 6.5 to 9.0 with a pKa of 7.79 ± 0.14. The pH sensor matrix was integrated via spin coating as ca. 1- to 2-µm-thick layer into "CytoCapture" cell culture dishes of 6 mm in diameter. These contained a microcavity array of square-shaped regions of 40 µm length and width and 15 µm depth that was homogeneously coated with the pH sensor matrix. The sensor layer showed fast response times in both directions. A microscopic setup was developed that enabled imaging of the pH inside the microchamber arrays over many hours. As a proof of principle, we monitored the pH of Escherichia coli cell cultures grown in the microchamber arrays. The integrated sensor matrix allowed pH monitoring spatially resolved in every microchamber, and the differences in cell growth between individual chambers could be resolved and quantified.

Label-free microfluidic free-flow isoelectric focusing, pH gradient sensing and near real-time isoelectric point determination of biomolecules and blood plasma fractions.

We demonstrate the fabrication, characterization and application of microfluidic chips capable of continuous electrophoretic separation via free flow isoelectric focussing (FFIEF). By integration of a near-infrared (NIR) fluorescent pH sensor layer under the whole separation bed, on-line observation of the pH gradient and determination of biomolecular isoelectric points (pI) was achieved within a few seconds. Using an optical setup for imaging of the intrinsic fluorescence of biomolecules at 266 nm excitation, labelling steps could be avoided and the native biomolecules could be separated, collected and analysed for their pI. The fabricated microchip was successfully used for the monitoring of the separation and simultaneous observation of the pH gradient during the isoelectric focussing of the proteins α-lactalbumin and ß-lactoglobulin, blood plasma proteins and the antibiotics ampicillin and ofloxacin. The obtained pIs are in good agreement with literature data, demonstrating the applicability of the system. Mass spectra from the separated antibiotics taken after 15 minutes of continuous separation from different fractions at the end of the microchip validated the separation via microfluidic isoelectric focussing and indicate the possibility of further on- or off-chip processing steps.

An integrated microfluidic chip enabling control and spatially resolved monitoring of temperature in micro flow reactors.

A strength of microfluidic chip laboratories is the rapid heat transfer that, in principle, enables a very homogeneous temperature distribution in chemical processes. In order to exploit this potential, we present an integrated chip system where the temperature is precisely controlled and monitored at the microfluidic channel level. This is realized by integration of a luminescent temperature sensor layer into the fluidic structure together with inkjet-printed micro heating elements. This allows steering of the temperature at the microchannel level and monitoring of the reaction progress simultaneously. A fabrication procedure is presented that allows for straightforward integration of thin polymer layers with optical sensing functionality in microchannels of glass-polydimethylsiloxane (PDMS) chips of only 150 µm width and 29 µm height. Sensor layers consisting of polyacrylonitrile and a temperature-sensitive ruthenium tris-phenanthroline probe with film thicknesses of about 0.5 to 6 µm were generated by combining blade coating and abrasion techniques. Optimal coating procedures were developed and evaluated. The chip-integrated sensor layers were calibrated and investigated with respect to stability, reproducibility, and response times. These microchips allowed observation of temperature in a wide range with a signal change of around 1.6 % per K and a maximum resolution of around 0.07 K. The device is employed to study temperature-controlled continuous micro flow reactions. This is demonstrated exemplarily for the tryptic cleavage of coumarin-modified peptides via fluorescence detection.

Microfluidic platforms employing integrated fluorescent or luminescent chemical sensors: a review of methods, scope and applications.

Herein we critically review microfluidic platforms that contain integrated fluorescent or luminescent chemical sensor assemblies. These were employed in particular for miniaturized oxygen and pH sensing. Microchips with optical temperature sensing capability are also covered since these share many concepts and applications. Other analytes and derived parameters from the above analytes are found in some sensing approaches in microfluidics.After an introduction, the work is structured into three main chapters dealing with the fabrication and microintegration of these sensors, readout and detection strategies, and applications of these microsystems. The fabrication is discussed with a focus on soft lithography-based approaches in polydimethylsiloxane (PDMS) or PDMS and glass hybrid devices that form the majority of work so far. Alternative approaches, particularly using glass or quartz as the main chip material are also covered. Detection techniques employed to date are the subject of the next chapter, where simple intensity as well as lifetime- or wavelength-referenced schemes are presented and the utility of image-based sensing on the microscale is discussed.Lastly, exciting applications of these microfluidic chips are highlighted. Luminescent oxygen and pH sensing has been of particular interest in the field of microbioreactors but other areas are also of interest, particularly chemical reactors and electrophoresis. Optical temperature sensing is discussed and its use in fundamental studies as well as in enzyme reactors. Integrated microsystems with biosensing capabilities and some for monitoring of metal ions and other analytes are also presented.

Rapid isoelectric point determination in a miniaturized preparative separation using jet-dispensed optical pH sensors and micro free-flow electrophoresis.

Herein, the fabrication, characterization, calibration, and application of integrated microfluidic platforms for fast isoelectric point (pI) determinations via free-flow electrophoresis with integrated inkjet-printed fluorescent pH sensor microstructures are presented. These devices allow one to determine the pI of a biomolecule from a sample mixture with moderately good precision and without addition of markers in typically less than 10 s total separation and analysis time. Polyhydroxyethyl methacrylate (pHEMA) hydrogels were covalently coupled with fluorescein and hydroxypyrene trisulfonic acid (HPTS)-based pH probes. These were piezoelectrically jet-dispensed onto acrylate-modified glass as pH sensor microarrays with a diameter of 300-600 µm and thicknesses of 0.4-2.4 µm with high spatial accuracy. Microchip fabrication and integration of these pH sensor arrays was realized by multistep liquid-phase photolithography from oligoethylene glycol precursors resulting in glass-based microfluidic free-flow isoelectric focusing (µFFIEF) chips with integrated pH observation capabilities. The microchips were characterized with regard to pH sensitivity, response times, photo-, and flow stability. Depending on the sensor matrix, they allowed IEF within a pH range of roughly 5.5-10.5 with good sensitivity and fast response times. These microchips were used for FFIEF of small molecule markers and several protein mixtures with simultaneous monitoring of local pH. This allowed the determination of their pI via multispectral imaging of protein and pH sensor fluorescence without addition of external markers. Obtained pI's were generally in good agreement with known data, demonstrating the applicability of the method for pI determination in micropreparative procedures within a time frame of a few seconds only.

Integrating continuous microflow reactions with subsequent micropreparative separations on a single microfluidic chip.

A current challenge in continuous flow chemistry using microfluidic devices is the combination with a micropreparative separation technique for online product clean-up and analysis. Herein we describe an approach for seamless on-chip integration of continuous flow reactors with a downstream separation functionality based on free-flow electrophoresis. A prototype device was fabricated via liquid phase lithography in a one-step procedure. This proof of concept was successfully validated by performing a model reaction of amino acids with ortho-phthaldialdehyde and continuous separation of the resulting products via micro free-flow zone electrophoresis.

Label-free fluorescence detection of aromatic compounds in chip electrophoresis applying two-photon excitation and time-correlated single-photon counting.

In this study, we introduce time-resolved fluorescence detection with two-photon excitation at 532 nm for label-free analyte determination in microchip electrophoresis. In the developed method, information about analyte fluorescence lifetimes is collected by time-correlated single-photon counting, improving reliable peak assignment in electrophoretic separations. The determined limits of detection for serotonin, propranolol, and tryptophan were 51, 37, and 280 nM, respectively, using microfluidic chips made of fused silica. Applying two-photon excitation microchip separations and label-free detection could also be performed in borosilicate glass chips demonstrating the potential for label-free fluorescence detection in non-UV-transparent devices. Microchip electrophoresis with two-photon excited fluorescence detection was then applied for analyses of active compounds in plant extracts. Harmala alkaloids present in methanolic plant extracts from Peganum harmala could be separated within seconds and detected with on-the-fly determination of fluorescence lifetimes.

Micro flow reactor chips with integrated luminescent chemosensors for spatially resolved on-line chemical reaction monitoring.

Real-time chemical reaction monitoring in microfluidic environments is demonstrated using luminescent chemical sensors integrated in PDMS/glass-based microscale reactors. A fabrication procedure is presented that allows for straightforward integration of thin polymer layers with optical sensing functionality in microchannels of glass-PDMS chips of only 150 µm width and of 10 to 35 µm height. Sensor layers consisting of polystyrene and an oxygen-sensitive platinum porphyrin probe with film thicknesses of about 0.5 to 4 µm were generated by combining spin coating and abrasion techniques. Optimal coating procedures were developed and evaluated. The chip-integrated sensor layers were calibrated and investigated with respect to stability, reproducibility and response times. These microchips allowed observation of dissolved oxygen concentration in the range of 0 to over 40 mg L(-1) with a detection limit of 368 µg L(-1). The sensor layers were then used for observation of a model reaction, the oxidation of sulphite to sulphate in a microfluidic chemical reactor and could observe sulphite concentrations of less than 200 µM. Real-time on-line monitoring of this chemical reaction was realized at a fluorescence microscope setup with 405 nm LED excitation and CCD camera detection.