Traditionally, biosensors are designed to detect one specific analyte. Nevertheless, disease progression is regulated in a highly interactive way by different classes of biomolecules like proteins and nucleic acids. Therefore, a more comprehensive analysis of biomarkers from a single sample is of utmost importance to further improve both, the accuracy of diagnosis as well as the therapeutic success. This review summarizes fundamentals like biorecognition and sensing strategies for the simultaneous detection of proteins and nucleic acids and discusses challenges related to multianalyte biosensor development. We present an overview of the current state of biosensors for the combined detection of protein and nucleic acid biomarkers associated with widespread diseases, among them cancer and infectious diseases. Furthermore, we outline the multianalyte analysis in the rapidly evolving field of single-cell multiomics, to stress its significance for the future discovery and validation of biomarkers. Finally, we provide a critical perspective on the performance and translation potential of multianalyte biosensors for medical diagnostics.

The rapid detection of pathogens in infected wounds can significantly improve the clinical outcome. Wound exudate, which can be collected in a non-invasive way, offers an attractive sample material for the detection of pathogens at the point-of-care (POC). Here, we report the development of a nucleic acid lateral flow immunoassay for direct detection of isothermally amplified DNA combined with fast sample preparation. The streamlined protocol was evaluated using human wound exudate spiked with the opportunistic pathogen Pseudomonas aeruginosa that cause severe health issues upon wound colonization. A detection limit of 2.1 × 10⁵ CFU per mL of wound fluid was achieved, and no cross-reaction with other pathogens was observed. Furthermore, we integrated an internal amplification control that excludes false negative results and, in combination with the flow control, ensures the validity of the test result. The paper-based approach with only three simple hands-on steps has a turn-around time of less than 30 min and covers the complete analytical process chain from sample to answer. This newly developed workflow for wound fluid diagnostics has tremendous potential for reliable pathogen POC testing and subsequent target-oriented therapy.

Significance: The topology of membrane proteins is defined by the successive integration of α-helical transmembrane domains at the Sec61 translocon. For each polypeptide segment of ∼20 residues entering the translocon, their combined hydrophobicities were previously shown to define membrane integration. Here, we discovered that different sequences preceding a potential transmembrane domain substantially affect the hydrophobicity threshold. Sequences that are rapidly folding, intrinsically disordered, very short, or strongly binding chaperones allow efficient integration at low hydrophobicity. Folding deficient mutant domains and artificial sequences not binding chaperones interfered with membrane integration likely by remaining partially unfolded and exposing hydrophobic surfaces that compete with the translocon for the emerging transmembrane segment, reducing integration efficiency. Rapid folding or strong chaperone binding thus promote efficient integration.

Food allergies are a severe burden for affected individuals and healthcare systems. To tackle the need for simple food allergen detection, we developed a system for the detection of the soy protein glycinin via a centrifugal microfluidics-assisted lateral flow immunoassay (LFIA). Glycinin is a complex allergen requiring extensive sample preparation. The presented workflow includes a manual denaturing extraction, followed by automated centrifugal microfluidic desalting, metering and detection via LFIA. The functionality of the microfluidic cassettes was tested on prototypes produced via microthermoforming before an injection molding tool was designed, which added a cylindrical lens to improve the readout. Overall, this system aims to aid in food allergen detection with high sensitivity and minimized manual steps.

The development of new multianalyte biosensors that can detect multiple classes of biomolecules is highly desirable and will greatly improve medical diagnostics. In the field of infectious diseases, for example, it is beneficial to detect pathogens via nucleic acid analysis together with host immune response markers. In this work, we present a multianalyte-compatible lysis using antimicrobial peptides (AMPs). This strategy enables the simultaneous detection of bacterial DNA and inflammatory biomarkers via multianalyte lateral flow immunoassay (LFIA).