Published papers

Firefly bioluminescence is broadly applied as a noninvasive imaging modality in the biomedical research field. One limitation in firefly bioluminescence imaging is the limited variety of luciferins emitting in the near-infrared (NIR) region (650-900 nm), where tissue penetration is high. Herein, we describe a series of structure-inherent NIR emitting firefly luciferin analogues, NIRLucs, designed through a ring fusion strategy. This strategy resulted in pH-independent structure-inherent NIR emission with a native firefly luciferase, which was theoretically supported by quantum chemical calculations of the oxidized form of each luciferin. When applied to cells, NIRLucs displayed dose-independent improved NIR emission even at low concentrations where the native d-luciferin substrate does not emit. Additionally, excellent blood retention and brighter photon flux (7-fold overall, 16-fold in the NIR spectral range) than in the case of d-luciferin have been observed with one of the NIRLucs in mice bearing subcutaneous tumors. We believe that these synthetic luciferins provide a solution to the longstanding limitation in the variety of NIR emitting luciferins and pave the way to the further development of NIR bioluminescence imaging platforms.

This work describes the generation of plasticized poly(vinyl chloride)-based ion-selective optode (ISO) nanoparticles by a piezoelectric inkjet dispensing device. The monodisperse inkjet-generated particles (polydispersity index: 0.177) with an average hydrodynamic diameter of 202 nm are suitable for the highly reproducible deposition onto cellulosic filter paper substrates, resulting in paper-based analytical devices (PADs) for colorimetric cation detection, as demonstrated on the example of potassium-responsive ISOs. In contrast to the deposition of ISO bulk membranes from organic solutions requiring specialized printing equipment, the ISO nanoparticles can be inkjet-deposited from aqueous dispersions by conventional office inkjet printing. The obtained ISO PADs showed highly reproducible (relative standard deviations of the hue mean values in the range of 0.06-0.83% for three independently fabricated batches) and immediate (t95% ≤ 1 min) response to aqueous K+ solutions buffered at pH 7.4, with high selectivity over Li+, Na+, Ca2+, and Mg2+ (log KK+,Mz+opt ≤ -5.2 (Li+); -3.6 (Na+); < -5.0 (Ca2+); < -5.2 (Mg2+)). In addition, causes for the distinctively different response observed for ISO nanoparticles in the liquid phase and on paper substrates were considered. For this purpose, several parameters related to the cation exchange reaction mechanism of ISOs were experimentally evaluated, allowing to semiquantitatively discuss the paper substrate-specific response of ISOs.

We present two diagnostic platforms, a microfluidic thread-based and a conventional immunochromatographic strip approach, for single-step lateral flow immunoassays (LFIAs) with bioluminescence (BL) signaling requiring only sample application and detection of emission color changes by a digital camera. Both rely on bioluminescence resonance energy transfer (BRET)-switching proteins. The detection of three clinically relevant targets is demonstrated: Cetuximab as example of an antibody-based anticancer drug requiring monitoring of blood drug levels in patients, and HIV1-p17 antibodies and antigens as examples of infectious disease markers.

This highlight review overviews recent progress in fluorescence imaging technology for the alkaline-earth metal ions calcium, and magnesium, by small molecule-based fluorescent probes. Fluorescent probes for Ca2+ using 1,2-bis-(o-aminophenoxy)- ethane-N,N,N′,N′-tetraacetic acid (BAPTA) as a recognition unit are introduced. Focus is set on the fluorescence characteristics and intracellular dynamics of Ca2+ fluorescent probes depending on the differences in fluorophore units. In addition, this review comprehensively covers Mg2+ fluorescent probes from Mg2+ selective ligands and fluorophore units to their application. In particular, the influence of the molecular structure of the fluorescent probes on organelle localization and fluorescence wavelength for imaging intracellular signals in organelles and intercellular multiple signal interactions are summarized, providing a strategy for probe design for chemists, and for selecting the appropriate imaging technique for biologists.

Three benzothiazole-derived heptamethine cyanine dyes were prepared via nucleophilic substitution at meso-position along the polymethine backbone. The synthetic route to the title polymethines includes a total of four steps starting from inexpensive commercially available materials. Structural elucidation of the newly synthesized chromophores was achieved using 1H-NMR, 13C-NMR and high-resolution mass spectrometry (HRMS) in ESI mode. Current functionalized amino-derived NIR dyes exhibit significant Stokes shift (Δν up to 1835 cm−1, 108 nm) compared to the parent unsubstituted chromophore (Δν = 303 cm−1, 23 nm). The probability of solvatochromism was determined from the correlation between the Stokes shift against the Dimroth-Reichardt ET (30) and the Lippert–Mataga function (ΔfLM) of orientation polarizability. Based on the experimental findings, this large Stokes shift presumably reflects a solvatochromic intramolecular charge transfer (ICT) across the molecule, which is induced by the introduced secondary amine.

Competitive lateral flow immunochromatographic assays (LFIAs) enabling naked-eye semi-quantitative result readout in the form of “text” have been developed. User operation is limited to sample application, followed by direct reading of assay results written in text representing the target concentration. LFIA strips enabled the detection of 8-hydroxy-2’-deoxyguanosine (8-OHdG) at concentrations down to 5 ng/mL, which is comparable to existing immunochromatography using external readout equipment. The system is readily adaptable to other analytical targets and allows for high flexibility in text design and detectable analyte concentrations. The simple assay handling and intuitive data interpretation offer great potential for point-of-care testing applications.

This work describes a colorimetric signaling approach for competitive lateral flow immunoassays (LFIAs) enabling sensitive and semiquantitative direct visual result readout in the form of "text", demonstrated on the example of 8-hydroxy-2′-deoxyguanosine (8-OHdG) detection. The distinctive feature of the developed text-displaying LFIA (TD-LFIA) is the test zone system consisting of a combination of two types of inkjet-deposited capture molecules referred to as "mask antigen"and "text antibody", allowing for sensitive turn-on signaling as opposed to the inverse response of conventional competitive LFIAs. The user operation is limited to sample application, followed by direct reading of assay results written in text after approximately 10 min. TD-LFIAs enabled the visual detection of 8-OHdG at concentrations down to 3 ng/mL, which is a 2-3 orders of magnitude lower visual detection limit than that achieved with the corresponding conventional design and is comparable to the existing LFIAs relying on external signal readout equipment. Highly reproducible observer-independent assay performance was confirmed, and the result interpretation is not influenced by sample color and readout timing. Making use of customizable threshold settings for text appearance, a device for semiquantitative assays was developed and successfully applied to the detection of 8-OHdG at four concentration levels (trace, low, medium, and high) in 54 human urine samples within the clinically relevant concentration range. The sensitive and intuitive signaling method of the developed system offers great potential for an alternative competitive LFIA platform suitable for real-world point-of-care testing applications.

Bioluminescent protein integrating microfluidic thread-based analytical devices (µTADs) for point-of-care testing (POCT) of antibodies in whole blood have been developed. Devices consist of a blood separation membrane and a plastic film layer with sewn-in cotton thread, in which proteins and luciferin for signal generation are pre-deposited. Colorimetry-based response curves for three model antibodies spiked into porcine whole blood have been obtained. Stable signals from 6-18 min after sample application and no cross-talk when assaying multiple different antibodies are observed. The µTADs offer quantitative simultaneous detection of multiple antibodies in a small volume of whole blood (8 µL).

Antibodies are important biomarkers in clinical diagnostics in addition to being increasingly used for therapeutic purposes. Although numerous methods for their detection and quantification exist, they predominantly require benchtop instruments operated by specialists. To enable the detection of antibodies at point-of-care (POC), the development of simple and rapid assay methods independent of laboratory equipment is of high relevance. In this study, we demonstrate microfluidic thread-based analytical devices (μTADs) as a new platform for antibody detection by means of bioluminescence resonance energy-transfer (BRET) switching sensor proteins. The devices consist of vertically assembled layers including a blood separation membrane and a plastic film with a sewn-in cotton thread, onto which the BRET sensor proteins together with the substrate furimazine have been predeposited. In contrast to intensity-based signaling, the BRET mechanism enables time-independent, ratiometric readout of bioluminescence signals with a digital camera in a darkroom or a smartphone camera with a 3D-printed lens adapter. The device design allows spatially separated deposition of multiple bioluminescent proteins on a single sewn thread, enabling quantification of multiple antibodies in 5 μL of whole blood within 5 min. The bioluminescence response is independent of the applied sample volume within the range of 5-15 μL. Therefore, μTADs in combination with BRET-based sensor proteins represent user-friendly analytical tools for POC quantification of antibodies without any laboratory equipment in a finger prick (5 μL) of whole blood.

Microfluidic paper-based analytical devices (μPADs) have attracted great attention as potential candidates for point-of-care testing (POCT). Nevertheless, only a limited number of μPADs expected to satisfy the standard of Clinical Laboratory Improvement Amendments (CLIA) waived tests as issued by the US Food and Drug Administration (FDA) have been reported. This work introduces a “traffic light type μPAD”, enabling highly intuitive semi-quantitative equipment-free naked-eye readout with no need for calibration, subjective interpretation or calculation. Assay results are displayed as traffic light colours reporting 5 analyte concentration levels (green/green & yellow/yellow/yellow & red/red). The device has been designed to never display all three colours simultaneously, eliminating any risk for misinterpretation. The mechanism relies on the modulation of sample flow through a network of paperfluidic channels modified with a hydrophobic to hydrophilic phase-switching substance responsive to H2O2. User operation is limited to sample application, followed by observing a clear and time-independent traffic light signal after approximately 10-30 min. Multiple factors influencing the H2O2 concentration-dependent appearance of a specific traffic light signal were studied. Making use of the possibilities for customising the concentration threshold levels for traffic light colour appearance, quantification of glucose at 5 levels in a clinically relevant concentration range was demonstrated in artificial urine as a model proof-of-concept. This platform is expected to offer the possibility for the future detection of other important metabolites.

This work reports an intuitive signaling approach enabling naked-eye semi-quantitative result readout in the form of a “traffic-light” for microfluidic paper-based analytical devices (µPADs). The detection of hydrogen peroxide has been demonstrated with the aim of quantifying metabolites relevant as biomarkers of various lifestyle-related diseases. User operation is limited to simple sample application, followed by observing the traffic-light signal after approximately 10 min. Due to its intuitiveness with very low probability of result misinterpretation, this system is expected to offer great potential for home-based self-diagnostic point-of-care testing (POCT) applications.

Bioluminescence (BL) imaging, which utilizes light emitted through the enzymatic reaction of luciferase oxidizing its substrate luciferin, enables sensitive and noninvasive monitoring of life phenomena. Herein, we developed a series of caged furimazine (FMZ) derivatives by introducing a protective group at the C-3 position and a hydroxy group at the C-6 phenyl ring to realize long-term live-cell BL imaging based on the NanoLuc (NLuc)/NanoKAZ (NKAZ)-FMZ system. The membrane permeability and cytotoxicity of the substrates were evaluated and related to their hydrophobicity. Among the series, the derivative with the bulkiest protective group (adamantanecarbonyl group) and a hydroxy substituent (named Ad-FMZ-OH) showed significantly prolonged and constant BL signal in cells expressing NLuc compared to the native FMZ substrate. This derivative enabled continuous BL imaging at the single-cell level for 24 h. Furthermore, we applied Ad-FMZ-OH to BL imaging of myocyte fusion and succeeded in the consecutive and sensitive monitoring at a single-cell level over a day. In summary, NLuc/NKAZ-caged FMZ derivatives have the potential to be applied to live-cell BL imaging of various life phenomena that require long-term observation.

This work presents the development and application of a novel analytical approach for the determination of acid and base concentrations by titration using a microfluidic thread-based analytical device (μTAD). This approach proved to be a simple to fabricate and to use, high precision, and cost-efficient means of acid-base quantification. The μTAD was fabricated by immobilizing the untreated cotton threads onto a wood frame, followed by pre-coating with an indicator (20 μL) and a primary standard solution (3 μL), and was tested using real samples including drug, food, and household products where 3 μL of each sample was dropped onto the center of a thread. Afterward, the distance of color change on the thread, easily observed and measured using the naked eye and a ruler, was used for analysis. The analysis using the μTAD, completed within 2 minutes and validated by the conventional titration, showed high accuracy and precision (RSD < 12.9%), good linearity ranges and low limit of quantification. The fabricated μTAD also remained stable for an extended period of time (>2 weeks under various storage conditions).

A paper-based test kit for selective and sensitive determination of cyanide was successfully developed using a Ag3PO4/Ag nanocomposite for catalytic colorimetry. The Ag3PO4/Ag nanocomposite synthesized via a one-step chemical bath method could instantly oxidize the chromogenic substrate ABTS for the color development under acidic conditions without the requirement of H2O2. During the detection step, cyanide in the sample was converted into gaseous hydrogen cyanide (HCN) using sulfuric acid. The HCN species then reacted with the generated Ag+ species via coordinate covalent bonding to form silver cyanide complex ions [Ag (CN2)]-. The amount of Ag+ species used for oxidizing ABTS subsequently decreased and so did the color intensity on the paper-based test kit. Specifically, the color intensity decreased with increasing cyanide concentrations in the range of 2.50–20.0 mg L−1. The limit of detection (LOD) and limit of quantification (LOQ) were 0.48 mg L−1 and 1.59 mg L−1, respectively. The developed test kit showed relatively high selectivity towards cyanide and high tolerance towards other cations and anions present in real samples. When applied to real samples including fruit juices and food products, the performance of the rapid and low-cost test kit was well validated by a conventional precipitation titration method.

The wide spread of smartphones and QR codes for various end-user applications has had an impact beyond traditional fields of use, recently also reaching point-of-care testing (POCT). This work presents the integration of QR code recognition into paper-based analytical devices (PADs) with "distance-based"colorimetric signalling, resulting in semiquantitative readout fully relying on straightforward barcode reader solutions. PADs consist of an array of QR codes arranged in series inside a paperfluidic channel. A mask dye concept has been developed, which enables utilisation of colour changing indicators by initially hiding QR codes. The colour change of the indicator induced by the presence of an analyte of interest results in gradual unmasking of QR codes, which become recognisable by the smartphone barcode reader app. To reproducibly fabricate devices, all fabrication steps were performed by commercial desktop solid ink and inkjet printing. The QR code masking function was optimised by controlling the amount of printed mask dye through adjusting the opacity of printing patterns during the inkjet deposition process. For proof-of-concept, a model assay in the form of colorimetric copper ion (Cu2+) detection in the concentration range of 0.4 mM to 3.2 mM was evaluated. Consistent results independent of the smartphone model and environmental light condition were achieved with a free barcode reader app. To the best of our knowledge, this work is the first demonstration of a semiquantitative assay approach fully relying on QR code readout without digital colour analysis, customised app or hardware modification.

Although the field of antibody drugs has grown larger, the antibody production still faces several challenges. Effective antibody purification is required, but the conventional purification method for antibodies is cost intensive and often causes aggregation problems, indicating the need for new alternative antibody purification methods. In the present study, a constant temperature antibody purification system for use with a thermo-responsive polymer column was developed based on switching of anion species in eluents. By adjusting the temperature for each antibody, the developed column enabled separation of the therapeutic monoclonal antibodies, rituximab and trastuzumab, from contaminants without changing salt concentration or pH of the eluents. The thermo-responsive hydrogel-modified column packing material was synthesized by introducing n-butyl methacrylate, acrylic acid, N,N′-methylenebisacrylamide and N-isopropylacrylamide to the surface of silica beads with an initiator by a graft-from approach. Elution behavior of antibodies with three types of anions, such as citrate, phosphate, and chloride were tested under three different temperature conditions. It was demonstrated that the thermo-responsive hydrogel grafted column showed a switchable antibody retention behavior at constant temperature and salt concentration, with antibody adsorption by NaCl eluent and desorption by citric acid buffer eluent.

This report suggests utilizing “cellophane” as a new substrate material for point-of-care testing (POCT) devices. Cellophane-based microfluidic devices were fabricated by a print, cut and lamination (PCL) methodology and sample liquid was transported into four assay areas without external pumping. Furthermore, making use of the transparency of cellophane, background-tinted devices were fabricated. Based on a blue background-tinted cellophane device, the dynamic range of a hue-based H2O2 assay was 46-fold increased compared to performing the same assay on a white background device. Finally, turbidity testing for albumin was implemented on black background-tinted devices with a visual detection limit of 15 mg/dL.

Distance-based readout is one of the most user-friendly and simple colorimetric signaling methods widely applied for paper-based analytical devices (PADs). This work presents the integration of distance readout PADs into a centrifugal platform enabling affordable, rapid, and sample volume-independent colorimetric assays. Centrifugally assisted distance-based PADs (CD-PADs) eliminate the requirement to use micropipets for sample introduction and reduce the overall time for distance-based assays. All device fabrication steps were performed through computer-controlled print, cut, and laminate (PCL) techniques oriented toward mass production. The inexpensive centrifugal platform was built on a recycled DVD player combined with an open source microcomputer (Arduino). Assay protocols, including rotational velocity and rotation time, were optimized to obtain a maximum dynamic range and reproducible results for sample volume metering (coefficient of variation 3.62%). A colorimetric Ni2+ assay chosen to demonstrate measurements on CD-PADs allowed the detection of nickel ions (Ni2+) with naked-eye interpretation within 1.5 min and a limit of detection (LOD) of 44.1 ng of Ni2+, which to the best of our knowledge is the lowest value reported for a distance-based Ni2+ assay.

Although microfluidic paper-based analytical devices (μPADs) get a lot of attention in the scientific literature, they rarely reach the level of commercialization. One possible reason for this is a lack of application of machine learning techniques supporting the design, optimization and fabrication of such devices. This work demonstrates the potential of two chemometric techniques including design of experiments (DoE) and digital image processing to support the production of μPADs. On the example of a simple colorimetric assay for isoniazid relying on the protonation equilibrium of methyl orange, the experimental conditions were optimized using a D-optimal design (DO) and the impact of multiple factors on the μPAD response was investigated. In addition, this work demonstrates the impact of automatic image processing on accelerating color value analysis and on minimizing errors caused by manual detection area selection. The employed algorithm is based on morphological recognition and allows the analysis of RGB (red, green, and blue) values in a repeatable way. In our belief, DoE and digital image processing methodologies are keys to overcome some of the remaining weaknesses in μPAD development to facilitate their future market entry.

This manuscript reports on a simple paper-based bienzymatic colorimetric assay for sarcosine as an important urinary biomarker of prostate cancer. All required assay reagents are pre-deposited on hydrophilic filter paper spots surrounded by a hydrophobic barrier. Sarcosine in the sample solution is selectively oxidized in the presence of sarcosine oxidase (SOx), resulting in the formation of hydrogen peroxide, which is subsequently detected through the horseradish peroxidase (HRP)–catalyzed conversion of the colorless indicator 3,3’,5,5’-tetramethylbenzidine (TMB) into its blue-colored oxidation product. By the modification of the paper with positively charged poly(allylamine hydrochloride) (PAH), a linear response to sarcosine between 0 and 10 μM and a significant lowering of the limit of detection (LOD) (0.6 μM) compared to the unmodified paper substrate (12.6 μM) has been achieved. The improvement of the LOD was attributed to the fact that the presence of the polymer limits the enzyme-driven colorimetric reaction to the surface of the paper substrate, resulting in stronger color development. In experiments in artificial urine matrix, the bicarbonate anion was identified as an inhibitor of the colorimetric reaction. This inhibition was successfully eliminated through on-device sample pH adjustments with pH-buffer components pre-deposited onto assay devices. The LOD for sarcosine achieved in artificial urine matrix (2.5 μM) is below the 5 μM threshold value for this urinary biomarker required for diagnostic purposes. Finally, good selectivity over all 20 natural amino acids and satisfactory long-term storage stability of reagent-modified paper substrates at − 20 °C for a period of 50 days were confirmed. Graphical abstract: [Figure not available: see fulltext.]