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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.

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.

The pyrophosphate ion (P 2 O 7 4- , PPi) plays a critical role in various biological processes and acts as an essential indicator for physiological mechanism investigations and disease control monitoring. However, most of the currently available approaches for PPi species detection for practical usage still lack appropriate indicator generation, straightforward detection requirements, and operation convenience. In this study, a highly sensitive and selective PPi detection approach via the use of nanozymatic carbon dots (CDs) is introduced. This strategy eliminates the common need for metal ions in the detection process, where a direct indicator-PPi interaction is adopted to provide straightforward signal reports, and importantly, through a green indicator preparation. The preparation of this nanozymatic CDs' indicator utilizes an aqueous solution refluxing, employing galactose and histidine as the precursor materials. The mild conditions of the solution refluxing produce fluorescent CDs exhibiting peroxidase-mimic properties, which can catalyze the o -phenylenediamine oxidation under the presence of H 2 O 2 . The introduction of PPi species, interestingly, inhibits this process very efficiently, the extent of which can be colorimetrically monitored by the generated yellow product 2,3-diaminophenazine. Spectroscopic results point to CD surface functional groups' selective binding toward PPi species, which severely interferes with the electron transfer process in the enzymatic catalysis. Relying on this CD peroxidase-mimetic property inhibition, sensitive and selective recognition of PPi reaches a detection limit of 4.29 nM, enabling practical usage in complex matrixes. Owing to the superior compatibility and high stability of nanozymatic CDs, they can also be inkjet-printed on paper-based devices to create a portable and convenient platform for PPi detection. Both the solution and the paper-device-based selective recognitions confirm this unique and robust metal-free inhibitive PPi detection, which is supported by a convenient green preparation of nanozymatic CDs.

This paper introduces the concept of "Drawing-PADs" (Drawing paper-based microfluidic analytical devices) allowing to intuitively evaluate the urinary albumin (Alb) index, a clinically important parameter used for the early detection of renal deficiencies related to diabetes, among others. To enable regular monitoring of the Alb index, a simple examination method suitable for self-diagnosis is highly desirable. The Drawing-PADs rely on the simultaneous naked eye detection of Alb and creatinine (Cre) on a single device according to the distance-based microfluidic PAD (μPAD) approach. The Alb index is visualized by simply drawing a straight line connecting the top of two color-changed assay channel sections (Alb and Cre channels), followed by visually confirming the position of the intercept of the drawn straight line. The semiquantitative Alb index evaluation performed with Drawing-PADs does not require any equipment such as a camera, software, or a color reference chart. The obtained results are independent of the sample volume and are not influenced by changes in the absolute Alb and Cre concentrations caused by urine excretion variations, making spot urine assays possible. Classification of Alb index values according to clinically relevant criteria (normoalbuminuria, microalbuminuria, and macroalbuminuria) is readily achieved within 15 min and has been validated for 15 human urine samples including diabetic patients and healthy volunteers.